Novel rna-guided nucleases and proteins for polymerase editing

Abstract

Compositions and methods for binding target sequences of interest are provided. These compositions can be used to cleave or modify target sequences of interest, visualize target sequences of interest, and modify the expression of sequences of interest. The compositions comprise RNA-guided nuclease (RGN) peptides, polymerase editors (PEs), guide RNA, and polynucleotides encoding them. Vectors and host cells containing these polynucleotides are also provided. RGN systems for binding and modifying target sequences of interest are also provided, wherein the RGN system comprises an RNA-guided nuclease peptide and one or more guide RNAs; and PE systems for modifying target sequences of interest, wherein the PE system comprises a polymerase, an RGN peptide, and one or more polymerases that edit the guide RNA.

Description

[0001] Cross-reference of related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 552,421, filed February 12, 2024, and U.S. Provisional Application No. 63 / 559,159, filed February 28, 2024, each of which is incorporated herein by reference in its entirety.

[0003] Reference to the sequence list submitted electronically as an XML file

[0004] This application contains a sequence list, which has been submitted in XML format and incorporated herein by reference in its entirety. The XML copy was created on February 11, 2025, named L103438_1390WO_0330_1_SL, and is 4,005,847 bytes in size. Technical Field

[0005] This invention relates to the fields of molecular biology and gene editing.

[0006] Existing technology

[0007] Targeted genome editing or modification is rapidly becoming an important tool in basic and applied research. Initial approaches involve engineering nucleases (such as macronucleases, zinc finger fusion proteins, or TALENs), requiring the use of engineered, programmable, sequence-specific DNA-binding domains to generate chimeric nucleases. RNA-guided nucleases (RGNs), such as CRISPR-Cas bacterial system-associated (Cas) proteins with clustered regularly spaced short palindromic repeats, allow targeting of specific sequences by complexing the nuclease with guide RNA that specifically hybridizes to the target sequence. For each target sequence, generating target-specific guide RNA is less costly and more efficient than generating chimeric nucleases. These RNA-guided nucleases can be used to edit the genome, where appropriate, by introducing sequence-specific double-strand breaks that are repaired via error-prone non-homologous end joining (NHEJ) to introduce mutations at specific genomic locations. Alternatively, heterologous DNA can be introduced into genomic sites via homologous directed repair. RNA-guided nucleases (RGNs) can also be used for base editing when fused with deaminases or for lead editing when fused with reverse transcriptases.

[0008] Leader editing is a general and precise genome editing method that uses an RNA-guided DNA-binding protein (such as a reverse transcriptase) that works in cooperation with reverse transcriptase to directly insert new genetic information into a designated DNA site (described, for example, US 11,447,770B1; WO2021072328; WO2021226558; WO2020156575; WO2021042047; US11193123; each incorporated herein by reference in its entirety). The RGN used in leader editing systems consists of a nicking enzyme and a polymerase, and the system is programmed with a leader-guided RNA containing a primer-binding site (PBS) and a DNA synthesis template that serves as the template for the replacement strand to be edited. The leader editor makes a nick upstream of the sequence to be edited and upstream of the PAM in the non-target strand, thereby creating a 3' overhang on the non-target strand. The PBS is complementary to the 3' protrusion of the non-target strand, and hybridization of the PBS with the 3' protrusion of the non-target strand allows for the polymerization of the edited replacement strand using a DNA synthesis template. Summary of the Invention

[0009] Compositions and methods are provided for binding to and modifying target sequences of interest in target polynucleotides. These compositions can be used to cleave or modify target polynucleotides of interest, detect target sequences of interest, and modify the expression of genes of interest containing the target sequences. The compositions comprise an RNA-guided nuclease (RGN) polypeptide; a polymerase editor (PE); a guide RNA (gRNA); a polynucleotide encoding each of the above; a vector and host cell containing the polynucleotide; and a pharmaceutical composition containing each of the above. An RGN system and ribonucleoprotein complex for binding target sequences of interest are also provided, wherein the RGN system and ribonucleoprotein complex comprise an RNA-guided nuclease polypeptide and one or more guide RNAs. A polymerase editor (PE) system is also provided, comprising one or more polymerase-edited guide RNAs (PEgRNA), a polymerase, and an RGN polypeptide. Therefore, the methods disclosed herein are for binding to target sequences of interest in target polynucleotides, and in some embodiments, the target polynucleotide of interest is cleaved or modified. The target polynucleotide of interest may be modified, for example, by non-homologous end joining, homology-directed repair through an introduced donor sequence, base editing, or polymerase editing. Detailed Implementation

[0010] Thanks to the teachings presented in the foregoing description and related figures, those skilled in the art will conceive of many modifications and other embodiments of the invention set forth herein. Therefore, it should be understood that the invention is not limited to the specific embodiments disclosed, and modifications and other embodiments are intended to be included within the scope of the appended embodiments. Although specific terminology is used herein, it is used in a general and descriptive sense only and not for limiting purposes.

[0011] I. Overview

[0012] This document provides an RNA-guided nuclease (RGN) variant of APG07433.1 RGN disclosed in International Application Publication No. WO 2019 / 236566 (which is incorporated herein by reference in its entirety), capable of recognizing a protospacer adjacent motif (PAM) at the 3' end of the target sequence having the nucleotide sequences nnnnCY, nnnnCT, nnnnC, or nnnnCM, rather than the parental APG07433.1 PAM having the nucleotide sequence nnnnCC. One such APG07433.1 variant is LPG10221, as shown in SEQ ID NO: 1, which contains five mutations within the PAM interaction domain of APG07433.1; specifically, D1020S, S1021G, D1022N, R1023K, and N1025E; and recognizes a PAM having the nucleotide sequence nnnnCY. SEQ ID NO: 579 shows LPG10209 with an R1023Q mutation and recognizes a PAM with the nucleotide sequence nnnnC. SEQ ID NO: 584 shows LPG10210 with S1021T and R1023N mutations and recognizes a PAM with the nucleotide sequence nnnnCM (where M is C or A). SEQ ID NO: 583 shows LPG10212 with S1021N and R1023N mutations and recognizes a PAM with the nucleotide sequence nnnnC. SEQ ID NO: 591 shows LPG10213 containing four mutations; specifically, S975Q, D1020A, D1022G, and R1023Q; and recognizing a PAM with the nucleotide sequence nnnnCT.

[0013] The RGNs disclosed herein can alter gene expression by modifying target polynucleotides containing a target sequence. In certain embodiments, the RGN is directed to the target sequence (e.g., a target DNA sequence) via a guide RNA (gRNA) that is part of the CRISPR RGN system. The RGN is considered "RNA-guided" because the guide RNA forms a complex with the RGN to direct the RGN to bind to the target sequence, and in some embodiments, double-strand breaks are introduced into the target sequence (e.g., the target DNA sequence), while in other embodiments, single-strand breaks ("cuts") are introduced into the target sequence (e.g., the target DNA sequence). After the target sequence is cleaved, the breaks can be repaired so that the sequence of the target polynucleotide is modified during the repair process. Therefore, this document provides methods for modifying target polynucleotides in host cells using RGNs. For example, RGNs can be used to modify target sequences at genomic loci in eukaryotic or prokaryotic cells.

[0014] This document also provides polymerase editors, polymerase editor systems, and methods for editing target DNA molecules using them, wherein these polymerase editor systems comprise: a DNA polymerase; and novel RGN LPG10221 (as shown in SEQ ID NO: 1) or its active variants or fragments as described herein, novel RGN LPG10209 (as shown in SEQ ID NO: 579) or its active variants or fragments as described herein, novel RGN LPG10210 (as shown in SEQ ID NO: 584) or its active variants or fragments as described herein, novel RGN LPG10212 (as shown in SEQ ID NO: 583) or its active variants or fragments as described herein, novel RGN LPG10213 (as shown in SEQ ID NO: 591) or its active variants or fragments, and RGN APG05586 (as shown in SEQ ID NO: 2 and previously published in International Application No. WO). The active variants or fragments thereof described in SEQ ID NO: 2021 / 217002 (which is incorporated herein by reference in its entirety), RGN APG07433.1 (as shown in SEQ ID NO: 7 and previously described in International Application Publication No. 2021 / 217002, which is incorporated herein by reference in its entirety), RGN APG01604 (as shown in SEQ ID NO: 565 and previously described in International Application Publication No. 2021 / 217002, which is incorporated herein by reference in its entirety), or RGN LPG10145 (as shown in SEQ ID NO: 566 and previously described in International Application Publication No. 2023 / 139557, which is incorporated herein by reference in its entirety), such as SEQ ID NO: The RGN LPG10145 variants shown in 1165-1179 and previously described in U.S. Provisional Application No. 63 / 516,127, filed July 27, 2023 (which is incorporated herein by reference in its entirety).

[0015] As used herein, "polymerase editor" or "PE" refers to one or more proteins comprising an RGN and a polymerase, which, together with a polymerase editing guide RNA (PEgRNA), enables the editing of double-stranded polynucleotides by replacing the target sequence using a DNA synthesis template as a template for the polymerase. The PEgRNA comprises an extension arm containing a primer binding site (PBS) and a DNA synthesis template containing the DNA to be edited. In some embodiments, the RGN and polymerase are operatively ligated (by fusion or insertion). In other embodiments, the RGN and polymerase are not operatively ligated. In one particular embodiment, the RGN and polymerase are two separate polypeptides. In some embodiments, the polymerase editor does not require the introduction of double-strand breaks, but instead utilizes an RGN nickase that nicks the non-target strand upstream of the sequence to be edited and upstream of the PAM, thereby creating a 3' overhang on the non-target strand. The PBS of the PEgRNA is complementary to the 3' overhang of the non-target strand, and hybridization of the PBS with the 3' overhang of the non-target strand allows the polymerase to polymerize the replacement strand containing the edit using the DNA synthesis template. Those polymerase editors that use reverse transcriptase as a polymerase are referred to in this paper as "RT editors" or "RTEs".

[0016] II. Polymerase Editor

[0017] The polymerase editor disclosed in this invention comprises a polymerase (e.g., reverse transcriptase) and an RGN polypeptide. The polymerase (e.g., reverse transcriptase) includes (but is not limited to) the polymerase (e.g., reverse transcriptase) described herein and its variants or fragments, including (but not limited to) reverse transcriptases having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identity with any one of SEQ ID NO: 174, 175, and 2127-2131. RGNs may include (but are not limited to) the RGNs described herein and their variants or fragments, including (but not limited to) RGNs having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identity with SEQ ID NO: 1, 2, 7, 565, 566, 579, 583, 584, 591 or 1165-1179.

[0018] A. RNA-guided nuclease

[0019] The polymerase editors (PEs) or PE systems disclosed herein include LPG10221, LPG10209, LPG10210, LPG10212, and LPG10213 (all described in more detail herein), APG07433.1, APG05586, LPG10145, and APG01604 RGN, or active variants or fragments thereof that retain the ability to bind to a target sequence in an RNA-guided, sequence-specific manner. In some of these embodiments, active fragments or variants of LPG10221, LPG10209, LPG10210, LPG10212, LPG10213, APG07433.1, APG05586, LPG10145, and APG01604 RGN are capable of cleaving single strands of a double-stranded target sequence. In some embodiments, the active variants of LPG10221, LPG10209, LPG10210, LPG10212, LPG10213APG05586, APG07433.1, LPG10145, and APG01604 RGN comprise an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity with the amino acid sequences shown in SEQ ID NO: 1, 2, 7, 565, 566, 579, 584, 599, or 1165-1179, respectively.

[0020] In some embodiments, the PE or PE system comprises an active variant of LPG10221 RGN containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and retaining RNA-guided sequence-specific binding activity, and in some embodiments, retaining RNA-guided sequence-specific cleavage activity. In some embodiments, the active variant of LPG10221 RGN contains an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and retaining RNA-guided sequence-specific binding activity, and in some embodiments, retaining RNA-guided sequence-specific cleavage activity. In some embodiments, the active variant of LPG10221 RGN contains an amino acid sequence having at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and retaining RNA-guided sequence-specific binding activity, and in some embodiments, retaining RNA-guided sequence-specific cleavage activity. In some embodiments, the active variant of LPG10221 RGN comprises an amino acid sequence having at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and retains RNA-guided sequence-specific binding activity, and in some embodiments, retains RNA-guided sequence-specific nicking activity.

[0021] In some embodiments, the PE or PE system comprises an active variant of LPG10209 RGN containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 579, and retaining RNA-guided sequence-specific binding activity, and in some embodiments, retaining RNA-guided sequence-specific cleavage activity. In some embodiments, the active variant of LPG10209 RGN contains an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 579, and retains RNA-guided sequence-specific binding activity, and in some embodiments, retains RNA-guided sequence-specific cleavage activity. In some embodiments, the active variant of LPG10209 RGN contains an amino acid sequence having at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 579, and retains RNA-guided sequence-specific binding activity, and in some embodiments, retains RNA-guided sequence-specific cleavage activity. In some embodiments, the active variant of LPG10209 RGN contains an amino acid sequence having at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 579, and retains RNA-guided sequence-specific binding activity, and in some embodiments, retains RNA-guided sequence-specific nicking activity.

[0022] In some embodiments, the PE or PE system comprises an active variant of LPG10210 RGN containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 584, and retaining RNA-guided sequence-specific binding activity, and in some embodiments, retaining RNA-guided sequence-specific cleavage activity. In some embodiments, the active variant of LPG10210 RGN contains an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 584, and retaining RNA-guided sequence-specific binding activity, and in some embodiments, retaining RNA-guided sequence-specific cleavage activity. In some embodiments, the active variant of LPG10210 RGN contains an amino acid sequence having at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 584, and retaining RNA-guided sequence-specific binding activity, and in some embodiments, retaining RNA-guided sequence-specific cleavage activity. In some embodiments, the active variant of LPG10210 RGN comprises an amino acid sequence having at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 584, and retains RNA-guided sequence-specific binding activity, and in some embodiments, retains RNA-guided sequence-specific nicking activity.

[0023] In some embodiments, the PE or PE system comprises an active variant of LPG10212 RGN containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 583, and retaining RNA-guided sequence-specific binding activity, and in some embodiments, retaining RNA-guided sequence-specific cleavage activity. In some embodiments, the active variant of LPG10212 RGN contains an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 583, and retaining RNA-guided sequence-specific binding activity, and in some embodiments, retaining RNA-guided sequence-specific cleavage activity. In some embodiments, the active variant of LPG10212 RGN contains an amino acid sequence having at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 583, and retaining RNA-guided sequence-specific binding activity, and in some embodiments, retaining RNA-guided sequence-specific cleavage activity. In some embodiments, the active variant of LPG10212 RGN comprises an amino acid sequence having at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 583, and retains RNA-guided sequence-specific binding activity, and in some embodiments, retains RNA-guided sequence-specific nicking activity.

[0024] In some embodiments, the PE or PE system comprises an active variant of LPG10213 RGN containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 591, and retaining RNA-guided sequence-specific binding activity, and in some embodiments, retaining RNA-guided sequence-specific cleavage activity. In some embodiments, the active variant of LPG10213 RGN contains an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 591, and retains RNA-guided sequence-specific binding activity, and in some embodiments, retains RNA-guided sequence-specific cleavage activity. In some embodiments, the active variant of LPG10213 RGN contains an amino acid sequence having at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 591, and retains RNA-guided sequence-specific binding activity, and in some embodiments, retains RNA-guided sequence-specific cleavage activity. In some embodiments, the active variant of LPG10213 RGN comprises an amino acid sequence having at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 591, and retains RNA-guided sequence-specific binding activity, and in some embodiments, retains RNA-guided sequence-specific nicking activity.

[0025] In some embodiments, the PE or PE system comprises an active variant of APG05586 RGN, which contains an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 2, and retains RNA-guided sequence-specific binding activity, and in some embodiments, retains RNA-guided sequence-specific cleavage activity. In some embodiments, the active variant of APG05586 RGN comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 2, and retains RNA-guided sequence-specific binding activity, and in some embodiments, retains RNA-guided sequence-specific cleavage activity. In some embodiments, the active variant of APG05586 RGN comprises an amino acid sequence having at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 2, and retains RNA-guided sequence-specific binding activity, and in some embodiments, retains RNA-guided sequence-specific cleavage activity. In some embodiments, the active variant of APG05586 RGN contains an amino acid sequence that has at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 2, and retains RNA-guided sequence-specific binding activity, and in some embodiments, retains RNA-guided sequence-specific nicking activity.

[0026] In some embodiments, the PE or PE system comprises an active variant of APG07433.1 RGN, which contains an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and retains RNA-guided sequence-specific binding activity, and in some embodiments, retains RNA-guided sequence-specific cleavage activity. In some embodiments, the active variant of APG07433.1 RGN comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and retains RNA-guided sequence-specific binding activity, and in some embodiments, retains RNA-guided sequence-specific cleavage activity. In some embodiments, the active variant of APG07433.1 RGN comprises an amino acid sequence having at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and retains RNA-guided sequence-specific binding activity, and in some embodiments, retains RNA-guided sequence-specific cleavage activity. In some embodiments, the active variant of APG07433.1 RGN contains an amino acid sequence that has at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and retains RNA-guided sequence-specific binding activity, and in some embodiments, retains RNA-guided sequence-specific nicking activity.

[0027] In some embodiments, the PE or PE system comprises an active variant of LPG10145 RGN containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 566 or 1165-1179, and retaining RNA-guided sequence-specific binding activity, and in some embodiments, retaining RNA-guided sequence-specific cleavage activity. In some embodiments, the active variant of LPG10145 RGN contains an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 566 or 1165-1179, and retaining RNA-guided sequence-specific binding activity, and in some embodiments, retaining RNA-guided sequence-specific cleavage activity. In some embodiments, the active variant of LPG10145 RGN contains an amino acid sequence having at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 566 or 1165-1179, and retaining RNA-guided sequence-specific binding activity, and in some embodiments, retaining RNA-guided sequence-specific cleavage activity. In some embodiments, the active variant of LPG10145 RGN contains an amino acid sequence having at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 566 or 1165-1179, and retains RNA-guided sequence-specific binding activity, and in some embodiments, retains RNA-guided sequence-specific nicking activity.

[0028] In embodiments where the PE or PE system comprises an RGN having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 566, it comprises at least one of the following amino acid residues:

[0029] a) R at position 55 corresponding to amino acid position 55 in SEQ ID NO: 566;

[0030] b) R at position 647 corresponding to amino acid position in SEQ ID NO: 566;

[0031] c) R at position 778 corresponding to amino acid position in SEQ ID NO: 566;

[0032] d) R at position 822 corresponding to amino acid position in SEQ ID NO: 566;

[0033] e) R at position 856 corresponding to amino acid position in SEQ ID NO: 566; and

[0034] f) R at position 969 corresponding to amino acid position in SEQ ID NO: 566.

[0035] In embodiments where the PE or PE system comprises an RGN having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1165, it comprises:

[0036] a) R at position 55 corresponding to amino acid position in SEQ ID NO: 1165;

[0037] b) R at position 647 corresponding to amino acid position in SEQ ID NO: 1165;

[0038] c) R at position 778 corresponding to amino acid position in SEQ ID NO: 1165; and

[0039] d) R at position 969 corresponding to amino acid position in SEQ ID NO: 1165.

[0040] In embodiments where the PE or PE system comprises an RGN having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1166, it comprises:

[0041] a) R at position 647 corresponding to amino acid position in SEQ ID NO: 1166;

[0042] b) R at position 778 corresponding to amino acid position in SEQ ID NO: 1166;

[0043] c) R at position 856 corresponding to amino acid position in SEQ ID NO: 1166; and

[0044] d) R at position 969 corresponding to amino acid position in SEQ ID NO: 1166.

[0045] In embodiments where the PE or PE system comprises an RGN having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1167, it comprises:

[0046] a) R at position 55 corresponding to amino acid position in SEQ ID NO: 1167;

[0047] b) R at position 778 corresponding to amino acid position in SEQ ID NO: 1167;

[0048] c) R at position 856 corresponding to amino acid position in SEQ ID NO: 1167; and

[0049] d) R at position 969 corresponding to amino acid position in SEQ ID NO: 1167.

[0050] In embodiments where the PE or PE system comprises an RGN having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1168, it comprises:

[0051] a) R at position 778 corresponding to amino acid position in SEQ ID NO: 1168;

[0052] b) R at position 856 corresponding to amino acid position in SEQ ID NO: 1168; and

[0053] c) R at position 969 corresponding to amino acid position in SEQ ID NO: 1168.

[0054] In embodiments where the PE or PE system comprises an RGN having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1169, it comprises:

[0055] a) R at position 55 corresponding to amino acid position in SEQ ID NO: 1169;

[0056] b) R at position 778 corresponding to amino acid position in SEQ ID NO: 1169; and

[0057] c) R at position 969 corresponding to amino acid position in SEQ ID NO: 1169.

[0058] In embodiments where the PE or PE system comprises an RGN having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1170, it comprises:

[0059] a) R at position 778 corresponding to amino acid position in SEQ ID NO: 1170; and

[0060] b) R at position 969 corresponding to amino acid position in SEQ ID NO: 1170.

[0061] In embodiments where the PE or PE system comprises an RGN having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1171, it comprises:

[0062] a) R at position 778 corresponding to amino acid position in SEQ ID NO: 1171; and

[0063] b) R at position 856 corresponding to amino acid position in SEQ ID NO: 1171.

[0064] In embodiments where the PE or PE system comprises an RGN having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1172, it comprises:

[0065] a) R at position 55 corresponding to amino acid position in SEQ ID NO: 1172;

[0066] b) R at position 778 corresponding to amino acid position in SEQ ID NO: 1172; and

[0067] c) R at position 822 corresponding to amino acid position in SEQ ID NO: 1172.

[0068] In embodiments where the PE or PE system comprises an RGN having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1173, it comprises:

[0069] a) R at position 55 corresponding to amino acid position in SEQ ID NO: 1173;

[0070] b) R at position 778 corresponding to amino acid position in SEQ ID NO: 1173; and

[0071] c) R at position 822 corresponding to amino acid position in SEQ ID NO: 1173.

[0072] In embodiments where the PE or PE system comprises an RGN having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1174, it comprises:

[0073] a) R at position 55 corresponding to amino acid position in SEQ ID NO: 1174;

[0074] b) R at position 778 corresponding to amino acid position in SEQ ID NO: 1174;

[0075] c) R at position 822 corresponding to amino acid position in SEQ ID NO: 1174; and

[0076] d) R at position 969 corresponding to amino acid position in SEQ ID NO: 1174.

[0077] In embodiments where the PE or PE system comprises an RGN having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1175, it comprises:

[0078] a) R at position 55 corresponding to amino acid position in SEQ ID NO: 1175;

[0079] b) R at position 647 corresponding to amino acid position in SEQ ID NO: 1175;

[0080] c) R at position 778 corresponding to amino acid position in SEQ ID NO: 1175; and

[0081] d) R at position 822 corresponding to amino acid position in SEQ ID NO: 1175.

[0082] In embodiments where the PE or PE system comprises an RGN having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1176, it comprises:

[0083] a) R at position 55 corresponding to amino acid position in SEQ ID NO: 1175;

[0084] b) R at position 674 corresponding to amino acid position in SEQ ID NO: 1175;

[0085] c) R at position 778 corresponding to amino acid position in SEQ ID NO: 1175;

[0086] d) R at position 822 corresponding to amino acid position in SEQ ID NO: 1175; and

[0087] e) R at position 969 corresponding to amino acid position in SEQ ID NO: 1175.

[0088] In embodiments where the PE or PE system comprises an RGN having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1177, it comprises:

[0089] a) R at position 647 corresponding to amino acid position in SEQ ID NO: 1177;

[0090] b) R at position 778 corresponding to amino acid position in SEQ ID NO: 1177;

[0091] c) R at position 822 corresponding to amino acid position in SEQ ID NO: 1177; and

[0092] d) R at position 969 corresponding to amino acid position in SEQ ID NO: 1177.

[0093] In embodiments where the PE or PE system comprises an RGN having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1178, it comprises:

[0094] a) R at position 647 corresponding to amino acid position in SEQ ID NO: 1178;

[0095] b) R at position 778 corresponding to amino acid position 778 in SEQ ID NO: 1178; and

[0096] c) R at position 822 corresponding to amino acid position in SEQ ID NO: 1178.

[0097] In embodiments where the PE or PE system comprises an RGN having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1179, it comprises:

[0098] a) R at position 55 corresponding to amino acid position in SEQ ID NO: 1179;

[0099] b) R at position 647 corresponding to amino acid position in SEQ ID NO: 1179;

[0100] c) R at position 778 corresponding to amino acid position in SEQ ID NO: 1179;

[0101] d) R at position 822 corresponding to amino acid position in SEQ ID NO: 1179; and

[0102] e) R at position 856 corresponding to amino acid position in SEQ ID NO: 1179.

[0103] In some embodiments, the PE or PE system comprises an active variant of APG01604 RGN, which contains an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 565, and retains RNA-guided sequence-specific binding activity, and in some embodiments, retains RNA-guided sequence-specific cleavage activity. In some embodiments, the active variant of APG01604 RGN comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 565, and retains RNA-guided sequence-specific binding activity, and in some embodiments, retains RNA-guided sequence-specific cleavage activity. In some embodiments, the active variant of APG01604 RGN comprises an amino acid sequence having at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO: 565, and retains RNA-guided sequence-specific binding activity, and in some embodiments, retains RNA-guided sequence-specific cleavage activity. In some embodiments, the active variant of APG01604 RGN contains an amino acid sequence having at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 565, and retains RNA-guided sequence-specific binding activity, and in some embodiments, retains RNA-guided sequence-specific nicking activity.

[0104] The PE or PE system may contain an active fragment of LPG10221, APG07433.1, APG05586, APG01604, LPG10145, LPG10209, LPG10212, LPG10210 or LPG10213 RGN, which contains at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 or more linked amino acid residues of the amino acid sequence shown in SEQ ID NO: 1, 2, 7, 565, 566, 579, 583, 584, 591 or 1165-1179, respectively.

[0105] The PE or PE system disclosed herein comprises an RGN that binds to a target sequence, such as those RGNs disclosed herein. In some embodiments, the PE or PE system comprises an RGN or an active variant or fragment thereof that recognizes a protospacer adjacent motif (PAM) (where N is A, C, T / U, or G and Y is C or T / U) of a shared nucleotide sequence with NNNCY at the 3' end of the target sequence on a non-target strand. In some embodiments, the PE or PE system comprises an RGN (LPG10221) having the amino acid sequence shown in SEQ ID NO: 1, which recognizes a PAM of a shared nucleotide sequence with NNNCY at the 3' end of the target sequence on a non-target strand.

[0106] The PE or PE system disclosed herein comprises an RGN that binds to a target sequence, such as those RGNs disclosed herein. In some embodiments, the PE or PE system comprises an RGN or an active variant or fragment thereof that recognizes a protospacer adjacent motif (PAM) (where N is A, C, T / U, or G) of a shared nucleotide sequence with NNNNC at the 3' end of the target sequence on a non-target strand. In some embodiments, the PE or PE system comprises an RGN (LPG10209) having the amino acid sequence shown in SEQ ID NO: 579 or an active variant or fragment thereof that recognizes a PAM of a shared nucleotide sequence with NNNNC at the 3' end of the target sequence on a non-target strand. In some embodiments, the PE or PE system comprises an RGN (LPG10212) having the amino acid sequence shown in SEQ ID NO: 583 or an active variant or fragment thereof that recognizes a PAM of a shared nucleotide sequence with NNNNC at the 3' end of the target sequence on a non-target strand.

[0107] The PE or PE system disclosed herein comprises an RGN that binds to a target sequence, such as those RGNs disclosed herein. In some embodiments, the PE or PE system comprises an RGN or an active variant or fragment thereof that recognizes a protospacer adjacent motif (PAM) (where N is A, C, T / U, or G and M is C or A) of a shared nucleotide sequence at the 3' end of the target sequence on a non-target strand, having the amino acid sequence shown in SEQ ID NO: 584. In some embodiments, the PE or PE system comprises an RGN (LPG10210) or an active variant or fragment thereof having the amino acid sequence shown in SEQ ID NO: 584 that recognizes a PAM of a shared nucleotide sequence at the 3' end of the target sequence on a non-target strand, having the shared nucleotide sequence, having the shared nucleotide sequence, having the shared nucleotide sequence, having the shared nucleotide sequence, having the shared nucleotide sequence, having the shared nucleotide sequence, having the shared nucleotide sequence, having the shared nucleotide sequence, having the shared nucleotide sequence, on a non-target strand ... having the shared nucleotide sequence, on a non-target strand, having the shared nucleotide sequence, having the shared nucleotide sequence, having the shared nucleotide

[0108] The PE or PE system disclosed herein comprises an RGN that binds to a target sequence, such as those RGNs disclosed herein. In some embodiments, the PE or PE system comprises an RGN or an active variant or fragment thereof that recognizes a protospacer adjacent motif (PAM) (where N is A, C, T / U, or G) of a shared nucleotide sequence with NNNNCT at the 3' end of the target sequence on a non-target strand. In some embodiments, the PE or PE system comprises an RGN (LPG10213) having the amino acid sequence shown in SEQ ID NO: 591, which recognizes a PAM of a shared nucleotide sequence with NNNNCT at the 3' end of the target sequence on a non-target strand.

[0109] The PE or PE system disclosed herein comprises an RGN that binds to a target sequence, such as those RGNs disclosed herein. In some embodiments, the PE or PE system comprises an RGN or an active variant or fragment thereof that recognizes a protospacer adjacent motif (PAM) of a shared nucleotide sequence with NNRYA at the 3' end of the target sequence on a non-target strand (where N is A, C, T / U, or G, R is G or A, and Y is C or T / U). In some embodiments, the PE or PE system comprises an RGN (APG05586) having the amino acid sequence shown in SEQ ID NO: 2, or an active variant or fragment thereof, that recognizes a PAM of a shared nucleotide sequence with NNRYA at the 3' end of the target sequence on a non-target strand.

[0110] The PE or PE system disclosed herein comprises an RGN that binds to a target sequence, such as those RGNs disclosed herein. In some embodiments, the PE or PE system comprises an RGN or an active variant or fragment thereof that recognizes a protospacer adjacent motif (PAM) of a shared nucleotide sequence with NNNNCC at the 3' end of the target sequence on a non-target strand (where N is A, C, T / U, or G, R is G or A, and Y is C or T / U). In some embodiments, the PE or PE system comprises an RGN (APG07743.1) having the amino acid sequence shown in SEQ ID NO: 7, which recognizes a PAM of a shared nucleotide sequence with NNNNCC at the 3' end of the target sequence on a non-target strand.

[0111] The PE or PE system disclosed herein comprises an RGN that binds to a target sequence, such as those RGNs disclosed herein. In some embodiments, the PE or PE system comprises an RGN or an active variant or fragment thereof that recognizes a protospacer adjacent motif (PAM) of a shared nucleotide sequence with NNGRR at the 3' end of the target sequence on a non-target strand (where N is A, C, T / U, or G, R is G or A, and R is A or G). In some embodiments, the PE or PE system comprises an RGN (APG01604) having the amino acid sequence shown in SEQ ID NO: 565, or an active variant or fragment thereof, that recognizes a PAM of a shared nucleotide sequence with NNGRR at the 3' end of the target sequence on a non-target strand.

[0112] The PE or PE system disclosed herein comprises an RGN that binds to a target sequence, such as those RGNs disclosed herein. In some embodiments, the PE or PE system comprises an RGN or an active variant or fragment thereof that recognizes a protospacer adjacent motif (PAM) (where N is A, C, T / U, or G, and R is G or A) of a shared nucleotide sequence with NNGG at the 3' end of the target sequence on a non-target strand. In some embodiments, the PE or PE system comprises an RGN or an active variant or fragment thereof having the amino acid sequence shown in SEQ ID NO: 566 (LPG10145) or 1165-1179 that recognizes a PAM of a shared nucleotide sequence with NNGG at the 3' end of the target sequence on a non-target strand.

[0113] As used herein with respect to PAMs that recognize nucleotide motifs, Y refers to cytosine (C) or thymine / uracil (T / U); N refers to adenine (A) or cytosine (C) or thymine / uracil (T / U) or guanine (G); R refers to adenine (A) or guanine (G); V refers to adenine (A) or cytosine (C) or guanine (G); and M refers to adenine (A) or cytosine (C). In some embodiments, the active fragment or variant of the RGN that recognizes such PAM sequences can bind to and, in some embodiments, cleave or cleave the target sequence.

[0114] The PE or PE system disclosed herein may comprise an RGN or an active variant or fragment thereof, having a PAM interaction (PI) domain that facilitates the recognition of PAM sites in target polynucleotides. The PI domain may contain 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 or more amino acid residues. In some embodiments, the PI domain of the RGN or an active variant or fragment thereof disclosed herein is located within the carboxyl (C) terminal region of the RGN. The C-terminal region of the PI domain of the RGN or its active variants or fragments disclosed herein may include 151, 150, 140, 135, 132, 130, 125, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 amino acid residues of the RGN. In some embodiments, the PI domain of the RGN or its active variants or fragments disclosed herein is located within or includes amino acid residues 939-1071 or 999-1150 of the RGN. In some embodiments, the PI domain of an RGN having the amino acid sequence shown in SEQ ID NO: 1, 7, 565, 566, 579, 583, 584, 591 or 1165-1179, or an active variant or fragment thereof, is within or includes amino acid residues 939-1071 of the RGN. In some embodiments, the PI domain of an RGN having at least 90% sequence identity with any one of SEQ ID NO: 1, 7, 579, 583, 584 or 591 has the amino acid sequence shown in SEQ ID NO: 575. In some embodiments, the PI domain of an RGN having the amino acid sequence shown in SEQ ID NO: 2, or an active variant or fragment thereof, is within or includes amino acid residues 999-1150 of the RGN. In some embodiments, the PI domain of an RGN having at least 90% sequence identity with SEQ ID NO: 2 has the amino acid sequence shown in SEQ ID NO: 1186. In some embodiments, the PI domain of the RGN having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 565 has the amino acid sequence shown in SEQ ID NO: 1187.In some embodiments, the PI domain of the RGN having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 566 or 1165-1179 has the amino acid sequence shown in SEQ ID NO: 1188.

[0115] The PE or PE system provided herein may include an RGN containing at least one nuclease domain (e.g., a DNase or RNase domain) and at least one RNA recognition and / or RNA binding domain for interacting with guide RNA. In some embodiments, the RGN of the PE or PE system contains only one active nuclease domain and thus acts as a nicking enzyme. In some embodiments, the active RGN nuclease domain in the RGN nicking enzyme is a RuvC domain. In some embodiments, the RGN contains an inactive HNH nuclease domain. Other domains that may be found in the RGN include (but are not limited to): a DNA-binding domain, a helicase domain, a protein-protein interaction domain, and a dimerizing domain. In some embodiments, the RGN may contain at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity with one or more of the DNA-binding domain, helicase domain, protein-protein interaction domain, and dimerizing domain.

[0116] In various embodiments, the target sequence binds to the RGN of the PE or PE system provided herein. In those cases where the target sequence is double-stranded (e.g., double-stranded DNA), the target strand of the target sequence hybridizes with the guide RNA associated with the RGN. If the polypeptide has nuclease activity, the target strand and / or non-target strand of the target sequence (e.g., the target DNA sequence) can subsequently be cleaved by the RGN. The term "cleave" or "cleavage" refers to the hydrolysis of at least one phosphodiester bond within the backbone of one or both strands of the double-stranded target sequence (e.g., the target DNA sequence), which can cause single-strand or double-strand breaks within the target sequence. The RGN can cleave nucleotides within the polynucleotide, acting as an endonuclease, or can be an exonuclease, removing consecutive nucleotides from the ends (5' and / or 3' ends) of the polynucleotide. In some embodiments, the RGN can cleave nucleotides of the target polynucleotide at any position within the polynucleotide and thus acts as both an endonuclease and an exonuclease. Cleavage of the target polynucleotide by the RGN can cause staggered breaks or blunt ends. The staggered cleavage in a polynucleotide produces two sticky ends or overhanging ends, which are formed when a nuclease cleaves the chains of the polynucleotide so that the cuts are not directly opposite each other. For each sticky end of the cleaved polynucleotide, one chain (i.e., the overhanging chain) is longer than the other chain (typically by at least several nucleotides), such that the longer chain has unpaired bases. The longer chain of the overhanging end of the cleaved polynucleotide can have one, two, three, four, five, or more unpaired nucleotides. In some embodiments, the longer chain of the overhanging end of the cleaved polynucleotide may have one unpaired nucleotide. The overhanging end of the cleaved polynucleotide can be a 3' overhang or a 5' overhang. In some embodiments, the overhanging end of the cleaved polynucleotide is a 3' overhang. In some embodiments, the overhanging end of the cleaved polynucleotide is a 5' overhang. In some embodiments, the RGN of this disclosure or its active variants or fragments cleave the target polynucleotide to form staggered cuts, wherein the staggered cuts produce a 3' dangling with an unpaired nucleotide. In contrast, blunt cuts produce two blunt ends such that each blunt end of the cleaved polynucleotide has two chains of equal length—that is, neither chain of the blunt end contains an unpaired base.

[0117] The RGN of the PE and PE system disclosed in this invention may be a wild-type sequence derived from bacterial or archaea species. Alternatively, the RGN may be a variant or fragment of a wild-type polypeptide. For example, the wild-type RGN may be modified to alter nuclease activity or PAM specificity. In some embodiments, the RGN is not naturally occurring.

[0118] In some embodiments, the PE and RGN of the PE system disclosed in this invention act as a cleaving enzyme, cleaving only the single strand of a double-stranded target sequence (e.g., a target DNA sequence). These RGNs have a single-function nuclease domain. In specific embodiments, the cleaving enzyme is capable of cleaving either the target strand (with an active HNH domain) or the non-target strand (with an active RuvC domain) of a double-stranded target sequence (e.g., a target DNA sequence). To achieve cleavage of the single strand of a double-stranded target polynucleotide (e.g., target DNA), at least one inactivating mutation (also referred to herein as a mutation reducing nuclease activity) can be introduced into the nuclease domain of the RGN. For example, to reduce or deactivate the HNH domain of LPG10221, APG07433.1, LPG10209, LPG10210, LPG10212, or LPG10213, one or more of the following catalytic amino acid residues can be mutated to, for example, alanine or another non-conserved amino acid residue: D603, H604, N618, N627. To reduce or deactivate the HNH domain of APG05586, one or more of the following catalytic amino acid residues can be mutated to, for example, alanine or another non-conserved amino acid residue: D600, H601, N615, N624. More than one mutation reducing nuclease activity can be introduced into the nuclease domain of RGN to further reduce or eliminate its activity. Therefore, in some embodiments, the HNH nuclease domain of the RGN of the PE or PE system comprises alanine (or another non-conserved amino acid residue) at position 604 corresponding to SEQ ID NO: 1, 7, 579, 583, 584 or 591 and alanine (or another non-conserved amino acid residue) at position 627 corresponding to SEQ ID NO: 1, 7, 579, 583, 584 or 591; alanine (or another non-conserved amino acid residue) at position 601 corresponding to SEQ ID NO: 2 and alanine (or another non-conserved amino acid residue) at position 624 corresponding to SEQ ID NO: 2; alanine (or another non-conserved amino acid residue) at position 559 corresponding to SEQ ID NO: 565 and alanine (or another non-conserved amino acid residue) at position 582 corresponding to SEQ ID NO: 565; or alanine (or another non-conserved amino acid residue) at position 611 corresponding to SEQ ID NO: 566 or 1165-1179 and alanine (or another non-conserved amino acid residue) at position 627 corresponding to SEQ ID NO: 1, 7, 579, 583, 584 or 591. NO: Alanine (or another non-conserved amino acid residue) at position 634 of 566 or 1165-1179.Non-limiting examples of DNA constructs encoding a PE containing a double-mutated RGN nickase within an HNH domain are shown in SEQ ID NO: 1182 (containing the APG07433.1 HNH double mutant) and 1183 (containing the APG05586 HNH double mutant). In some embodiments, the RGN nickase comprises the sequence shown in SEQ ID NO: 8, 106, 365, 367, 368, 396, 567, 568, 569, or 570, or an active fragment or variant thereof, such as an active fragment or variant having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity with any one of SEQ ID NO: 8, 106, 365, 367, 368, 396, 567, 568, 569, or 570. RGN nickases with inactive HNH domains can be used for polymerase editing and can be fused with polymerases as described herein. In some embodiments, all or part of the HNH domain of the RGN peptide is replaced by a polymerase (e.g., DNA polymerase) to produce a PE.

[0119] In other embodiments, the RGNs of the PE and PE systems disclosed herein are completely devoid of nuclease activity and are referred to herein as nuclease-inactive or nuclease-free. In some embodiments, the nuclease-inactive or nuclease-free form of the enzyme is used to modify nucleic acids, such as for base editing as described herein. Any methods known in this art for introducing mutations into amino acid sequences, such as PCR-mediated mutation induction and site-directed mutagenesis induction, can be used to generate nickase or nuclease-inactive RGNs. See, for example, U.S. Publication No. 2014 / 0068797 and U.S. Patent No. 9,790,490; each of which is incorporated herein by reference in its entirety.

[0120] B. Polymerase

[0121] This document provides polymerases (e.g., DNA polymerases, reverse transcriptases, etc.) that can be used in the described PE or PE system. As used herein, "polymerase" is an enzyme that catalyzes the formation of nucleic acid polymers. The polymerase may be an RNA polymerase (catalyzing RNA polymers) or a DNA polymerase (catalyzing DNA polymers). In some embodiments, the polymerase in the polymerase editor or system is a DNA polymerase. The PE or PE system may contain a DNA-dependent DNA polymerase (using DNA as a template) or an RNA-dependent DNA polymerase (using RNA as a template). In some embodiments, the DNA polymerase in the PE and PE system disclosed herein is an RNA-dependent DNA polymerase (i.e., a reverse transcriptase).

[0122] The reverse transcriptase used in the compositions and methods disclosed in this invention may be any known in the art, including (but not limited to) the reverse transcriptase of Moloney murine leukemia virus (MMLV) with the sequence shown in SEQ ID NO: 174, or an active variant thereof, such as an active variant having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 174. MMLV RT having any of the following mutations may also be used in the PE or PE system disclosed in this invention: D200N, L603W, T330P, T306K, and W313F. Therefore, the PE or PE system may contain an RT having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with SEQ ID NO: 174, wherein the RT contains at least one of the following amino acid residues: a) N at position 200 corresponding to SEQ ID NO: 174; b) W at position 603 corresponding to SEQ ID NO: 174; c) P at position 330 corresponding to SEQ ID NO: 174; d) K at position 306 corresponding to SEQ ID NO: 174; and e) F at position 313 corresponding to SEQ ID NO: 174. A reverse transcriptase may be used, wherein the RNase H domain (which is a progressive 5' and 3' ribonuclease specific to the RNA strand of the RNA-DNA hybrid) is inactive or removed. In some embodiments, the reverse transcriptase does not have an RNase H domain, which is truncated immediately following the amino acid at position 497 in SEQ ID NO: 174. Therefore, the PE and RT of the PE system disclosed in this invention may have at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with SEQ ID NO: 175. The PE and RT of the PE system disclosed in this invention may have at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with any one of SEQ ID NO: 2127-2131. In some embodiments, the PE and RT of the PE system disclosed in this invention have the amino acid sequence of any one of SEQ ID NO: 2127-2131.

[0123] C. Various formats of polymerase editors

[0124] The polymerase editor disclosed in this invention may be provided in the trans form, wherein the polymerase (e.g., reverse transcriptase) and RGN are two separate polypeptides. In some embodiments, the polymerase (e.g., reverse transcriptase) and the RGN polypeptide are transcribed together, with a sequence encoding a self-cleaving peptide (e.g., a 2A peptide, such as P2A) between them, such that translation produces two separate polypeptides. Any self-cleaving peptide known in this art can be used in such embodiments, including (but not limited to) 2A peptides, which are a class of 18-22 amino acid long peptides that can function via ribosome jumping during translation. Non-limiting examples of 2A peptides are T2A, P2A, E2A, and F2A.

[0125] Alternatively, the polymerase editor disclosed in this invention may comprise a polymerase operably fused to an RGN, wherein the polymerase and RGN are fused end-to-end with each other, or wherein the polymerase is inserted into the RGN polypeptide, such as those inlaid base editors described in International Application No. PCT / IB2023 / 061192, filed November 6, 2023 (which is incorporated herein by reference in its entirety). In end-to-end fusion, the polymerase may be fused to the amino terminus or the carboxyl terminus of the RGN.

[0126] In embodiments where a polymerase (e.g., reverse transcriptase) inserts into the RGN polypeptide, the polymerase inserts between surface amino acid residues. The polymerase may insert into or between the adapter domain 2, wedge (WED) domain, RuvC domain, HNH domain, Rec-2 domain, or PAM interaction (PI) domain. In some embodiments, the RuvC domain is a RuvCIII domain. The Rec or recognition domain mediates nucleic acid binding by sensing nucleic acids through multiple Rec domains (e.g., Rec1-3), regulates HNH conformational changes, and locks the catalytic HNH domain to the cleavage site. The wedge domain is responsible for recognizing and guiding the RNA backbone. Non-limiting examples of domains within the RGN include: RuvC-I of amino acid residues 1-54; BH of amino acid residues 55-83; REC1 of amino acid residues 84-244; REC2 of amino acid residues 245-462; RuvC-II of amino acid residues 463-521; L1 of amino acid residues 522-552; HNH of amino acid residues 553-672; L2 of amino acid residues 673-685; RuvC-III of amino acid residues 686-833; WED of amino acid residues 834-938; and PI of amino acid residues 939-1071; all with reference to LPG10221, APG07433.1, LPG10209, LPG10210, LPG10212, or LPG10213, as shown in SEQ ID NO: 1, 7, 579, 584, 583, and 591, respectively. APG05586 (as shown in SEQ ID NO: 2) has the following domains: RuvC-I of amino acid residues 1-33; BH of amino acid residues 34-71; REC1 of amino acid residues 72-232; REC2 of amino acid residues 233-468; RuvC-II of amino acid residues 469-517; L1 of amino acid residues 518-552; HNH of amino acid residues 553-672; L2 of amino acid residues 673-687; RuvC-III of amino acid residues 688-837; WED of amino acid residues 838-998; and PI of amino acid residues 999-1150.LPG10145 (as shown in SEQ ID NO: 566) and its variants (as shown in SEQ ID NO: 1165-1179) have the following domains: RuvC-I of amino acid residues 1-42; BH of amino acid residues 43-79; REC1 of amino acid residues 80-236; REC2 of amino acid residues 237-476; RuvC-II of amino acid residues 477-524; L1 of amino acid residues 525-560; HNH of amino acid residues 561-676; L2 of amino acid residues 677-690; RuvC-III of amino acid residues 691-828; WED of amino acid residues 829-976; and PI of amino acid residues 977-1130. APG01604 (as shown in SEQ ID NO: 565) has the following domains: RuvC-I of amino acid residues 1-40; BH of amino acid residues 41-74; REC1 of amino acid residues 75-223; REC2 of amino acid residues 224-430; RuvC-II of amino acid residues 431-483; L1 of amino acid residues 484-516; HNH of amino acid residues 517-631; L2 of amino acid residues 632-651; RuvC-III of amino acid residues 652-775; WED of amino acid residues 776-909; and PI of amino acid residues 910-1052.

[0127] The PAM interaction domain is a domain that binds to the PAM sequence. The universal domain of an RGN protein can be determined by structural comparison with RGN proteins that have defined domains.

[0128] In embodiments where the PE includes an RGN having at least 90% sequence identity with SEQ ID NO: 1 or 7, the polymerase may be inserted into the RGN immediately following an amino acid position selected from the group consisting of:

[0129] i) The amino acid position corresponding to position 30 of SEQ ID NO: 1, 7, 579, 583, 584 or 591;

[0130] ii) The amino acid position 642 corresponding to SEQ ID NO: 1, 7, 579, 583, 584 or 591;

[0131] iii) The amino acid position corresponding to position 670 of SEQ ID NO: 1, 7, 579, 583, 584 or 591;

[0132] iv) The amino acid position 737 corresponding to SEQ ID NO: 1, 7, 579, 583, 584 or 591;

[0133] v) The amino acid position 772 corresponding to SEQ ID NO: 1, 7, 579, 583, 584 or 591;

[0134] vi) The amino acid position 775 corresponding to SEQ ID NO: 1, 7, 579, 583, 584 or 591;

[0135] vii) The amino acid position 778 corresponding to SEQ ID NO: 1, 7, 579, 583, 584 or 591;

[0136] viii) The amino acid position 802 corresponding to SEQ ID NO: 1, 7, 579, 583, 584 or 591;

[0137] ix) corresponds to the amino acid position 900 of SEQ ID NO: 1, 7, 579, 583, 584 or 591; and

[0138] x) corresponds to the amino acid position 910 of SEQ ID NO: 1, 7, 579, 583, 584 or 591.

[0139] In those embodiments where the PE includes an RGN having at least 90% sequence identity with SEQ ID NO: 2, the polymerase may be inserted into the RGN immediately following an amino acid position selected from the group consisting of:

[0140] i) The amino acid position corresponding to position 342 of SEQ ID NO: 2;

[0141] ii) The amino acid position corresponding to position 518 of SEQ ID NO: 2;

[0142] iii) The amino acid position corresponding to position 630 of SEQ ID NO: 2;

[0143] iv) The amino acid position corresponding to position 661 of SEQ ID NO: 2;

[0144] v) The amino acid position corresponding to position 678 of SEQ ID NO: 2;

[0145] vi) The amino acid position corresponding to position 736 of SEQ ID NO: 2;

[0146] vii) corresponds to the amino acid position 778 of SEQ ID NO: 2;

[0147] viii) corresponds to the amino acid position 788 of SEQ ID NO: 2;

[0148] ix) corresponds to the amino acid position 922 of SEQ ID NO: 2; and

[0149] x) corresponds to the amino acid position 1109 of SEQ ID NO: 2.

[0150] In embodiments where the PE includes an RGN having at least 90% sequence identity with SEQ ID NO: 565, the polymerase may be inserted into the RGN immediately following an amino acid position selected from the group consisting of:

[0151] i) The amino acid position corresponding to position 597 of SEQ ID NO: 565;

[0152] ii) The amino acid position corresponding to position 631 of SEQ ID NO: 565;

[0153] iii) The amino acid position corresponding to position 725 of SEQ ID NO: 565;

[0154] iv) The amino acid position corresponding to position 739 of SEQ ID NO: 565; and

[0155] v) corresponds to the amino acid position 744 of SEQ ID NO: 565.

[0156] In those embodiments where the PE includes an RGN having at least 90% sequence identity with SEQ ID NO: 566 or 1165-1179, the polymerase may be inserted into the RGN immediately following an amino acid position selected from the group consisting of:

[0157] i) The amino acid position corresponding to position 347 of SEQ ID NO: 566 or 1165-1179;

[0158] ii) The amino acid position corresponding to position 524 of SEQ ID NO: 566 or 1165-1179;

[0159] iii) The amino acid position corresponding to position 640 of SEQ ID NO: 566 or 1165-1179;

[0160] iv) The amino acid position corresponding to position 666 in SEQ ID NO: 566 or 1165-1179;

[0161] v) The amino acid position corresponding to position 680 of SEQ ID NO: 566 or 1165-1179;

[0162] vi) The amino acid position corresponding to position 740 of SEQ ID NO: 566 or 1165-1179;

[0163] vii) corresponds to the amino acid position 785 of SEQ ID NO: 566 or 1165-1179;

[0164] viii) The amino acid position corresponding to position 910 in SEQ ID NO: 566 or 1165-1179; and

[0165] (ix) corresponds to the amino acid position 1077 of SEQ ID NO: 566 or 1165-1179.

[0166] The polymerase can be directly fused to the RGN peptide, or a linker sequence can ligate the polymerase and the RGN peptide. In embodiments where the polymerase is inserted into the RGN peptide, the linker sequence may be present at one or both ends of the polymerase sequence. Any suitable linker sequence can be used to ligate the polymerase and the RGN peptide (or a fragment thereof), but a suitable linker sequence comprises one or more copies of SGGS (SEQ ID NO: 2126). In some embodiments, the linker sequence comprises one SGGS sequence, two SGGS sequences (SEQ ID NO: 2120), three SGGS sequences, four SGGS sequences, or more SGGS sequences, such that the linker sequence can be 4, 8, 12, or 16 amino acids long. The linker between the polymerase and RGN (or a fragment thereof) can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 or more nucleotides. The adapter sequence for separating the polymerase from RGN (or a fragment thereof) may also include a nuclear localization sequence (NLS), such as (but not limited to) those disclosed elsewhere herein, namely any one of SEQ ID NO: 107, 108, 109, 2086-2101, 2118, and 2119, wherein the NLS can be linked by an adapter (such as SGGS). In some embodiments, the adapter sequence for separating the polymerase from RGN (or a fragment thereof) includes more than one NLS, such as two, three, or more NLS. In some embodiments, the adapter has the formula -(SGGS). x -NLS m-(SGGS) y -NLS n -(SGGS) z - where each of x, y, or z is 0, 1, 2, 3, or 4; and where each of m or n is 0 or 1. In some embodiments, the connector has the formula -(SGGS). x -NLS m -(SGGS) y -NLS n -(SGGS) z - where each of x or z is 0, 1, 2, 3, or 4, and y is 0; and where one of m or n is 0, and the others are 1. In other embodiments, the connector has the formula -(SGGS) x -NLS m -(SGGS) y -NLS n -(SGGS) z - where each of x, y, or z is 0, 1, 2, 3, or 4; and each of m or n is 1, and y is not 0. In some implementations, x, y, or z are not all 0.

[0167] The RGN, polymerase, or PE disclosed in this invention may include at least one nuclear localization signal (NLS) to enhance protein transport to the cell nucleus. The nuclear localization signal is known in this art and generally comprises an extension of a basic amino acid (see, for example, Lange et al., *Journal of Biochemistry*). J. Biol. Chem. (2007) 282:5101-5105). In some embodiments, the RGN, polymerase, or PE contains 2, 3, 4, 5, 6, or more nuclear localization signals. The nuclear localization signals may be heterologous NLS. As used herein, with respect to a polypeptide heterologous to another polypeptide (e.g., polymerase, RGN, PE), "heterologous" means a polypeptide that is not substantially operatively fused to, for example, the polymerase, RGN, or PE described in this invention. Heterologous polypeptides may be derived from foreign species or from the same species. Heterologous polypeptides may be in their native form or substantially modified from their native form at compositional and / or genomic loci through intentional artificial intervention. Heterologous polypeptides may be any polypeptide, including (but not limited to) localization signals, cell-penetrating domains, detectable markers (e.g., fluorescent proteins), or purification tags.

[0168] Non-limiting examples applicable to the nuclear localization signals of RGN, polymerase, or PE disclosed in this invention are the nuclear localization signals of SV40 large T-antigen, nucleoplasmic protein, and c-Myc (see, for example, Ray et al. (2015) Bioconjugated Chemistry). Bioconjug Chem(26(6):1004-7), ADAR2, POLD1 and the NLS disclosed in Table 40. In some embodiments, the RGN, polymerase or PE comprises NLS sequences as shown in SEQ ID NO: 107, 108, 109, 2086-2101, 2118 and / or 2119. The RGN, polymerase or PE may contain one or more NLS sequences at its N-terminus, C-terminus or N-terminus and C-terminus. For example, the RGN, polymerase or PE may contain two NLS sequences in the N-terminal region and four NLS sequences in the C-terminal region. As another example, the RGN, polymerase or PE may contain one NLS sequence in the N-terminal region and one NLS sequence in the C-terminal region. In some embodiments, a peptide linker (e.g. SGGS (SEQ ID NO: 2126), SGGSSGGS (SEQ ID NO: 2120) etc.) may link the NLS to the RGN, polymerase or fusion protein.

[0169] Other known localization signal sequences in this technique of directing peptides to specific subcellular locations can also be used to target RGN, polymerase, or PE, including (but not limited to) plasmid localization sequences, mitochondrial localization sequences, and dual-targeting signal sequences targeting both plasmids and mitochondria (see, for example, Nassoury and Morse (2005), Acta Biochimica et Biophysica Sinica). Biochim Biophys Acta )》1743:5-19; Kunze and Berger (2015) Frontiers in Physiology ( Front Physiol )》dx.doi.org / 10.3389 / fphys.2015.00259; Herrmann and Neupert (2003) "The Life of Ibubu ( IUBMB Life )》55:219-225; Soll (2002) "Contemporary Views on Plant Biology ( Curr Opin Plant Biol )》5:529-535; Carrie and Small (2013) Journal of Biochemistry and Biophysics ( Biochim Biophys Acta (1833:253-259; Carrie et al. (2009)) FEBS J 276:1187-1195; Silva-Filho (2003) "Contemporary Views in Plant Biology" Curr Opin Plant Biol )》6:589-595; Peeters and Small (2001) Journal of Biochemistry and Biophysics ( Biochim Biophys Acta )》1541:54-63; Murcha et al. (2014) Journal of Experimental Botany ( J Exp Bot)》 65:6301-6335; Mackenzie (2005) Journal of Cell Biology ( Trends Cell Biol )》15:548-554; Glaser et al. (1998) Plant Molecular Biology ( Plant Mol Biol )》38:311-338).

[0170] RGN, polymerase, or PE may contain at least one cell-penetrating domain that facilitates cellular uptake of RGN, polymerase, or PE. A cell-penetrating domain is known in this technology and generally comprises an extension of positively charged amino acid residues (i.e., a polycationic cell-penetrating domain), alternating polar and nonpolar amino acid residues (i.e., an amphipathic cell-penetrating domain), or hydrophobic amino acid residues (i.e., a hydrophobic cell-penetrating domain) (see, for example, Milletti F. (2012) Drug Discovery Today). Drug Discov Today (17:850-860). A non-restricted example of a cell-penetrating domain is the trans-activated transcription activator (TAT) from human immunodeficiency virus 1.

[0171] Nuclear localization signals, plastid localization signals, mitochondrial localization signals, dual-targeting localization signals, and / or cell-penetrating domains may be located at the N-terminus (N-terminus), C-terminus (C-terminus), and / or internal location of the RGN, polymerase, or PE.

[0172] RGN, polymerase, or PE may also contain purification tags, which are any molecules that can be used to isolate or fuse proteins from mixtures (e.g., biological samples, culture media). Non-limiting examples of purification tags include biotin, myc, maltose-binding protein (MBP), glutathione S-transferase (GST), and 3X FLAG tags.

[0173] Non-limiting examples of PE include any one of SEQ ID NO: 9-11, 111-130, 137-173, 364, 774-787, 790-814, 817-901, 926, 1143-1155, 1164, 1182-1185, 1189-2085, and 2121-2125, and their active variants and fragments, such as those associated with SEQ ID NO: PEs of any one of 9-11, 111-130, 137-173, 364, 774-787, 790-814, 817-901, 926, 1143-1155, 1164, 1182-1185, 1189-2085, and 2121-2125 having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity. In some embodiments, the PEs of this disclosure include any one of SEQ ID NO: 364, 1208, 1209, 1210, 1221, 1224, 1225, 1226, 1337, 1361, 1371, 1425, 1432, 1443, 1444, 1445, 1447, 1448, 1516, 1577, 1592, 1622, 1640, 1658, 1666, 1682, 1683, 1692, 1694, 1696, and 1718, and their active variants and fragments, such as those associated with SEQ ID NO: PEs of any of the following: 364, 1208, 1209, 1210, 1221, 1224, 1225, 1226, 1337, 1361, 1371, 1425, 1432, 1443, 1444, 1445, 1447, 1448, 1516, 1577, 1592, 1622, 1640, 1658, 1666, 1682, 1683, 1692, 1694, 1696, and 1718, having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity.

[0174] D. Guide RNA

[0175] This disclosure provides guide RNAs and polynucleotides encoding such guide RNAs that enable an associated RNA-guided nuclease (RGN) or a PE containing such an RGN to target a target sequence. The term "guide RNA" refers to a nucleotide sequence that is sufficiently complementary to the target nucleotide sequence to hybridize with the target sequence and directs the associated RGN sequence to specifically bind to the target nucleotide sequence. More specifically, when the target nucleotide sequence is double-stranded, as in the case of DNA, the target nucleotide sequence comprises a target strand and a non-target strand (which contains a PAM sequence). In these embodiments, the guide RNA is sufficiently complementary to the target strand of the double-stranded target sequence (e.g., a target DNA sequence) such that the guide RNA hybridizes with the target strand and directs the associated RGN sequence to specifically bind to the target sequence (e.g., the target DNA sequence). Thus, in some embodiments, the guide RNA includes a spacer region that is sequence-consistent with the non-target strand, except that uracil (U) replaces thymine (T) in the guide RNA. In embodiments using multiplex gene editing and in which multiple guide RNAs are present, each of one or more guide RNAs is sufficiently complementary to the target strand of a particular target sequence and is capable of hybridizing with the target strand of that target sequence. Therefore, the "target sequence" of the guide RNA refers to the target sequence that is sufficiently complementary to the guide RNA and can hybridize with it.

[0176] Each guide RNA of an RGN is one or more RNA molecules (usually one or two) that can bind to the RGN and guide it to bind to a specific target sequence, and in embodiments where the RGN has cleaving enzyme or nuclease activity, it also cleaves the target strand and / or non-target strand. Generally, guide RNAs comprise CRISPR RNA (crRNA) and trans-activated CRISPR RNA (tracrRNA), although some RGNs do not require tracrRNA.

[0177] The guide RNA (e.g., crRNA) disclosed herein may contain at least one chemical modification. At least one chemical modification includes: bridging nucleic acid (BNA) modification; 2'-O-methyl (2'-O-Me) modification; 2'-O-methoxy-ethyl (2'MOE) modification; 2'-fluorine (2'-F) modification; 2'F-4'C α-OMe modification; 2',4'-di-C α-OMe modification; 3'-phosphate 2'-O-methyl (MS) modification; 2'-O-methyl 3'-thiophosphonate acetate (MSP) modification; 2'-O-methyl 3'-phosphonate acetate (MP) modification; and phosphothioate (PS) modification; or combinations thereof. In some embodiments, the BNA contains a 2',4' BNA modification. In some embodiments, the 2',4' BNA modification is selected from the group consisting of locked nucleic acid (LNA) modification, BNA... NC[N-Me] modification, 2'-O,4'-C-extrinsic ethyl-bridged nucleic acid (2',4'-ENA) modification, and S-restricted ethyl (cEt) modification. In some embodiments, the 2',4' BNA is an LNA modification. In some embodiments, the 2',4' BNA is a cEt modification. In some embodiments, at least one chemical modification comprises BNA modification, 2'-O-Me modification, or PS modification. Chemical modifications of spacer regions, crRNA repeat sequences, crRNA, tracrRNA, and guide RNA are described in International Application No. PCT / IB2023 / 058418, filed August 25, 2023 (which is incorporated herein by reference in its entirety).

[0178] This disclosure provides a guide RNA comprising CRISPR RNA (crRNA). The crRNA comprises a spacer region and a CRISPR repeat sequence. The "spacer region" has a nucleotide sequence that directly hybridizes to the target strand of a target sequence of interest (e.g., a target DNA sequence). The spacer region is engineered to be fully or partially complementary to the target strand of the target sequence of interest. In some embodiments, the spacer region may contain about 8 nucleotides to about 30 nucleotides, or more. For example, the length of the spacer region may be about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, or more nucleotides. In some embodiments, the spacer region is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides long. In some embodiments, the spacer region is about 10 to about 26 nucleotides long, or about 12 to about 30 nucleotides long. In some embodiments, the spacer region is about 25 nucleotides long. In some implementations, when optimal alignment is performed using a suitable alignment algorithm, the complementarity between the spacer region and the target strand of the target sequence (e.g., the target DNA sequence) is between 50% and 99% or greater, including (but not limited to) about 50%, about 60%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or greater. In some embodiments, when optimal alignment is performed using a suitable alignment algorithm, the complementarity between the spacer region and the target strand of the target sequence (e.g., the target DNA sequence) is 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more. In some embodiments, the spacer region sequence may be identical to the non-target strand of the target sequence. In some of those embodiments where the target sequence is the target DNA sequence, the spacer region sequence may be identical to the non-target strand of the target DNA sequence, except that thymidine (T) in the non-target strand is replaced with uracil (U) in the spacer region. In certain embodiments, the spacer region is free of secondary structures, which can be predicted using any suitable polynucleotide folding algorithm known in this art, including (but not limited to) mFold (see, for example, Zuker and Stiegler (1981) Nucleic Acid Research (… Nucleic AcidsRes. ( ) 9:133-148) and RNAfold (see, for example, Gruber et al. (2008) Cell ( ) Cell )》 106(1):23-24).

[0179] In addition to the spacer region, the crRNA further comprises a CRISPR RNA (crRNA) repeat sequence. Generally, the crRNA repeat sequence comprises a nucleotide sequence that, alone or together with hybrid tracrRNA, forms a structure recognized by the RGN polypeptide. For LPG10221, LPG10209, LPG10210, LPG10212, LPG10213, APG07433.1, APG05586, APG01604, and LPG10145 guiding crRNAs, the spacer region is located at the 5' of the crRNA repeat sequence. In various embodiments, the crRNA repeat sequence may contain approximately 8 nucleotides to approximately 30 nucleotides or more. For example, the length of the crRNA repeat sequence may be about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30 or more nucleotides. In a particular embodiment, the length of the crRNA repeat sequence is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides. In some embodiments, the crRNA repeat sequence of this disclosure contains a total length of 19 to 40 nucleotides (nt). In some embodiments, the crRNA repeat sequences of this disclosure comprise a total length of up to 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, or 30 nt. In some embodiments, the crRNA repeat sequence is about 19 nt or 21 nt. The crRNA repeat sequences of this disclosure may include common repeat sequences of CRISPR arrays found in the genome of the organism from which the crRNA repeats are obtained.

[0180] In some embodiments, the crRNA repeat sequence comprises the nucleotide sequence shown in SEQ ID NO: 3, 5, 470, 471, 475, 476, 571, 573, or 1180, or an active variant or fragment thereof, which, when included within the guide RNA, directs the associated RGN sequence provided herein to specifically bind to the target sequence of interest. In some embodiments, the active crRNA repeat sequence variant of the wild-type sequence comprises a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity with the nucleotide sequence shown in SEQ ID NO: 3, 5, 470, 471, 475, 476, 571, 573, or 1180. In some embodiments, the active crRNA repeat sequence fragment of the wild-type sequence comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 linked nucleotides of the nucleotide sequence shown in SEQ ID NO: 3, 5, 470, 471, 475, 476, 571, 573, or 1180.

[0181] In embodiments where the RGN has the amino acid sequence shown in SEQ ID NO: 1, 7, 579, 583, 584, or 591, the associated gRNA crRNA repeat sequence may have the nucleotide sequence shown in SEQ ID NO: 3, 470, or 471, or an active variant or fragment thereof. In embodiments where the RGN has the amino acid sequence shown in SEQ ID NO: 2, the associated gRNA crRNA repeat sequence may have the nucleotide sequence shown in SEQ ID NO: 5, 475, or 476, or an active variant or fragment thereof. In embodiments where the RGN has the amino acid sequence shown in SEQ ID NO: 565, the associated gRNA crRNA repeat sequence may have the nucleotide sequence shown in SEQ ID NO: 571, or an active variant or fragment thereof. In embodiments where the RGN has the amino acid sequence shown in SEQ ID NO: 566 or 1165-1179, the associated gRNA crRNA repeat sequence may have the nucleotide sequence shown in SEQ ID NO: 573 or 1180, or an active variant or fragment thereof.

[0182] In some embodiments, the crRNA repeat sequence has the nucleotide sequence shown in SEQ ID NO: 3 or differs from SEQ ID NO: 3 by 1 to 5 nucleotides. In some embodiments, the crRNA repeat sequence has a nucleotide sequence differing from SEQ ID NO: 3 by 5 nucleotides. In some embodiments, the crRNA repeat sequence has a nucleotide sequence differing from SEQ ID NO: 3 by 4 nucleotides. In some embodiments, the crRNA repeat sequence has a nucleotide sequence differing from SEQ ID NO: 3 by 3 nucleotides. In some embodiments, the crRNA repeat sequence has a nucleotide sequence differing from SEQ ID NO: 3 by 2 nucleotides. In some embodiments, the crRNA repeat sequence has a nucleotide sequence differing from SEQ ID NO: 3 by 1 nucleotide. In some embodiments, the crRNA repeat sequence has the nucleotide sequence shown in SEQ ID NO: 3.

[0183] In some embodiments, the crRNA repeat sequence has the nucleotide sequence shown in SEQ ID NO: 5 or differs from SEQ ID NO: 5 by 1 to 5 nucleotides. In some embodiments, the crRNA repeat sequence has a nucleotide sequence differing from SEQ ID NO: 5 by 5 nucleotides. In some embodiments, the crRNA repeat sequence has a nucleotide sequence differing from SEQ ID NO: 5 by 4 nucleotides. In some embodiments, the crRNA repeat sequence has a nucleotide sequence differing from SEQ ID NO: 5 by 3 nucleotides. In some embodiments, the crRNA repeat sequence has a nucleotide sequence differing from SEQ ID NO: 5 by 2 nucleotides. In some embodiments, the crRNA repeat sequence has a nucleotide sequence differing from SEQ ID NO: 5 by 1 nucleotide. In some embodiments, the crRNA repeat sequence has the nucleotide sequence shown in SEQ ID NO: 5.

[0184] In some embodiments, the crRNA repeat sequence has the nucleotide sequence shown in SEQ ID NO: 470 or differs from SEQ ID NO: 470 by 1 to 5 nucleotides. In some embodiments, the crRNA repeat sequence has a nucleotide sequence differing from SEQ ID NO: 470 by 5 nucleotides. In some embodiments, the crRNA repeat sequence has a nucleotide sequence differing from SEQ ID NO: 470 by 4 nucleotides. In some embodiments, the crRNA repeat sequence has a nucleotide sequence differing from SEQ ID NO: 470 by 3 nucleotides. In some embodiments, the crRNA repeat sequence has a nucleotide sequence differing from SEQ ID NO: 470 by 2 nucleotides. In some embodiments, the crRNA repeat sequence has a nucleotide sequence differing from SEQ ID NO: 470 by 1 nucleotide. In some embodiments, the crRNA repeat sequence has the nucleotide sequence shown in SEQ ID NO: 470.

[0185] In some embodiments, the crRNA repeat sequence has the nucleotide sequence shown in SEQ ID NO: 471 or differs from SEQ ID NO: 471 by 1 to 5 nucleotides. In some embodiments, the crRNA repeat sequence has a nucleotide sequence differing from SEQ ID NO: 471 by 5 nucleotides. In some embodiments, the crRNA repeat sequence has a nucleotide sequence differing from SEQ ID NO: 471 by 4 nucleotides. In some embodiments, the crRNA repeat sequence has a nucleotide sequence differing from SEQ ID NO: 471 by 3 nucleotides. In some embodiments, the crRNA repeat sequence has a nucleotide sequence differing from SEQ ID NO: 471 by 2 nucleotides. In some embodiments, the crRNA repeat sequence has a nucleotide sequence differing from SEQ ID NO: 471 by 1 nucleotide. In some embodiments, the crRNA repeat sequence has the nucleotide sequence shown in SEQ ID NO: 471.

[0186] In some embodiments, the crRNA repeat sequence has the nucleotide sequence shown in SEQ ID NO: 475 or differs from SEQ ID NO: 475 by 1 to 5 nucleotides. In some embodiments, the crRNA repeat sequence has a nucleotide sequence differing from SEQ ID NO: 475 by 5 nucleotides. In some embodiments, the crRNA repeat sequence has a nucleotide sequence differing from SEQ ID NO: 475 by 4 nucleotides. In some embodiments, the crRNA repeat sequence has a nucleotide sequence differing from SEQ ID NO: 475 by 3 nucleotides. In some embodiments, the crRNA repeat sequence has a nucleotide sequence differing from SEQ ID NO: 475 by 2 nucleotides. In some embodiments, the crRNA repeat sequence has a nucleotide sequence differing from SEQ ID NO: 475 by 1 nucleotide. In some embodiments, the crRNA repeat sequence has the nucleotide sequence shown in SEQ ID NO: 475.

[0187] In some embodiments, the crRNA repeat sequence has the nucleotide sequence shown in SEQ ID NO: 476 or differs from SEQ ID NO: 476 by 1 to 5 nucleotides. In some embodiments, the crRNA repeat sequence has a nucleotide sequence differing from SEQ ID NO: 476 by 5 nucleotides. In some embodiments, the crRNA repeat sequence has a nucleotide sequence differing from SEQ ID NO: 476 by 4 nucleotides. In some embodiments, the crRNA repeat sequence has a nucleotide sequence differing from SEQ ID NO: 476 by 3 nucleotides. In some embodiments, the crRNA repeat sequence has a nucleotide sequence differing from SEQ ID NO: 476 by 2 nucleotides. In some embodiments, the crRNA repeat sequence has a nucleotide sequence differing from SEQ ID NO: 476 by 1 nucleotide. In some embodiments, the crRNA repeat sequence has the nucleotide sequence shown in SEQ ID NO: 476.

[0188] In some embodiments, the crRNA repeat sequence has the nucleotide sequence shown in SEQ ID NO: 571 or differs from SEQ ID NO: 571 by 1 to 5 nucleotides. In some embodiments, the crRNA repeat sequence has a nucleotide sequence differing from SEQ ID NO: 571 by 5 nucleotides. In some embodiments, the crRNA repeat sequence has a nucleotide sequence differing from SEQ ID NO: 571 by 4 nucleotides. In some embodiments, the crRNA repeat sequence has a nucleotide sequence differing from SEQ ID NO: 571 by 3 nucleotides. In some embodiments, the crRNA repeat sequence has a nucleotide sequence differing from SEQ ID NO: 571 by 2 nucleotides. In some embodiments, the crRNA repeat sequence has a nucleotide sequence differing from SEQ ID NO: 571 by 1 nucleotide. In some embodiments, the crRNA repeat sequence has the nucleotide sequence shown in SEQ ID NO: 571.

[0189] In some embodiments, the crRNA repeat sequence has the nucleotide sequence shown in SEQ ID NO: 573 or differs from SEQ ID NO: 573 by 1 to 5 nucleotides. In some embodiments, the crRNA repeat sequence has a nucleotide sequence differing from SEQ ID NO: 573 by 5 nucleotides. In some embodiments, the crRNA repeat sequence has a nucleotide sequence differing from SEQ ID NO: 573 by 4 nucleotides. In some embodiments, the crRNA repeat sequence has a nucleotide sequence differing from SEQ ID NO: 573 by 3 nucleotides. In some embodiments, the crRNA repeat sequence has a nucleotide sequence differing from SEQ ID NO: 573 by 2 nucleotides. In some embodiments, the crRNA repeat sequence has a nucleotide sequence differing from SEQ ID NO: 573 by 1 nucleotide. In some embodiments, the crRNA repeat sequence has the nucleotide sequence shown in SEQ ID NO: 573.

[0190] In some embodiments, the crRNA repeat sequence has the nucleotide sequence shown in SEQ ID NO: 1180 or differs from SEQ ID NO: 1180 by 1 to 5 nucleotides. In some embodiments, the crRNA repeat sequence has a nucleotide sequence differing from SEQ ID NO: 1180 by 5 nucleotides. In some embodiments, the crRNA repeat sequence has a nucleotide sequence differing from SEQ ID NO: 1180 by 4 nucleotides. In some embodiments, the crRNA repeat sequence has a nucleotide sequence differing from SEQ ID NO: 1180 by 3 nucleotides. In some embodiments, the crRNA repeat sequence has a nucleotide sequence differing from SEQ ID NO: 1180 by 2 nucleotides. In some embodiments, the crRNA repeat sequence has a nucleotide sequence differing from SEQ ID NO: 1180 by 1 nucleotide. In some embodiments, the crRNA repeat sequence has the nucleotide sequence shown in SEQ ID NO: 1180.

[0191] In some implementations, the crRNA is not naturally occurring. In some of these implementations, a particular crRNA repeat sequence is not inherently linked to the engineered spacer region, and the crRNA repeat sequence is considered heterologous to the spacer region. In some implementations, the spacer region is an engineered sequence that is not naturally occurring.

[0192] LPG10221, LPG10209, LPG10210, LPG10212, LPG10213, APG07443.1, APG05586, APG01604, and LPG10145 (and their variants) utilize guide RNA comprising crRNA and trans-activated CRISPR RNA (tracrRNA). The tracrRNA molecule contains a nucleotide sequence (referred to herein as an inverted repeat sequence) containing a region sufficiently complementary to the CRISPR repeat sequence of the crRNA. In some embodiments, the tracrRNA molecule further comprises a region having secondary structures (e.g., stem-loop) or forming secondary structures after hybridization with its corresponding crRNA. In a particular embodiment, the region of the tracrRNA that is fully or partially complementary to the CRISPR repeat sequence is located at the 5' end of the molecule, and the 3' end of the tracrRNA contains secondary structures. This secondary structure region generally contains multiple hairpin structures, including connecting hairpins, which are found to be adjacent to the inverted repeat sequence. The linker forms the core of the interaction between the guide RNA and the RGN, and is located at the intersection of the guide RNA, RGN, and target DNA. The linker hairpin typically has a conserved nucleotide sequence at the base of the hairpin stem; the motif UNANNC is found in many linker hairpins in tracrRNAs. In embodiments, the guide RNA or RGN system of this disclosure uses tracrRNAs containing atypical sequences at the base of the hairpin stem of their linker hairpins, including UNANNG, CNANNC, CNANNU, UNANNU, CNANNG, and CNCNNU. A terminal hairpin is typically present at the 3' end of the tracrRNA; its structure and number can vary, but it typically contains a GC-rich, p-independent transcription termination hairpin, followed by a string of Us at the 3' end. See, for example, Briner et al. (2014) Molecular Cell (… Molecular Cell )》 56:333-339; Briner and Barrangou (2016) Cold Spring Harbor Laboratory Manual ( Cold Spring Harb Protoc (See: doi:10.1101 / pdb.top090902; and U.S. Publication No. 2017 / 0275648, which are incorporated herein by reference in their entirety.)

[0193] In various implementations, the inverted repeat region of the tracrRNA that is wholly or partially complementary to the CRISPR repeat sequence contains about 8 nucleotides to about 30 nucleotides or more. For example, the length of the base-pairing region between the tracrRNA inverted repeat sequence and the CRISPR repeat sequence can be about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30 or more nucleotides. In a particular implementation, the length of the base-pairing region between the tracrRNA inverted repeat sequence and the CRISPR repeat sequence is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides. In some implementations, when optimal alignment is performed using a suitable alignment algorithm, the complementarity between the CRISPR repeat sequence and its corresponding tracrRNA inverted repeat sequence is approximately or greater than approximately 50%, approximately 60%, approximately 70%, approximately 75%, approximately 80%, approximately 81%, approximately 82%, approximately 83%, approximately 84%, approximately 85%, approximately 86%, approximately 87%, approximately 88%, approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or greater. In specific implementations, when optimal alignment is performed using a suitable alignment algorithm, the complementarity between the CRISPR repeat sequence and its corresponding tracrRNA inverted repeat sequence is 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more.

[0194] In various implementations, the entire tracrRNA may contain from about 60 nucleotides to more than about 210 nucleotides. For example, the length of the tracrRNA may be about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210 or more nucleotides. In a specific implementation, the tracrRNA length is 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 150, 160, 170, 180, 190, 200, 210 or more nucleotides. In a specific implementation, the tracrRNA has a length of approximately 57 to approximately 115 nucleotides, including lengths of approximately 57, approximately 58, approximately 59, approximately 60, approximately 61, approximately 62, approximately 63, approximately 64, approximately 65, approximately 66, approximately 67, approximately 68, approximately 69, approximately 70, approximately 71, approximately 72, approximately 73, approximately 74, approximately 75, approximately 76, approximately 77, approximately 78, approximately 79, approximately 80, approximately 81, approximately 82, approximately 83, and approximately 8... 4, approximately 85, approximately 86, approximately 87, approximately 88, approximately 89, approximately 90, approximately 91, approximately 92, approximately 93, approximately 94, approximately 95, approximately 96, approximately 97, approximately 98, approximately 99, approximately 100, approximately 101, approximately 102, approximately 103, approximately 104, approximately 105, approximately 106, approximately 107, approximately 108, approximately 109, approximately 110, approximately 111, approximately 112, approximately 113, approximately 114, and approximately 115 nucleotides.In a specific implementation, the tracrRNA has a length of 59 to 115 nucleotides, including lengths of 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, and 8... 4, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, and 115 nucleotides.

[0195] In certain embodiments, the tracrRNA comprises the nucleotide sequence of SEQ ID NO: 4, 6, 472, 473, 474, 477, 478, 479, or 480, or an active variant or fragment thereof, which, when included within a guide RNA, directs the associated RNA-guided nuclease sequence provided herein to specifically bind to the target DNA sequence of interest. In some embodiments, the active tracrRNA sequence variant comprises a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity with the nucleotide sequence shown in SEQ ID NO: 4, 6, 472, 473, 474, 477, 478, 479, 480, 572, 574, or 1181. In some embodiments, the active tracrRNA sequence fragment comprises at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 or more linked nucleotides of the nucleotide sequence shown in SEQ ID NO: 4, 6, 472, 473, 474, 477, 478, 479, 480, 572, 574, or 1181.

[0196] In embodiments where the RGN has the amino acid sequence shown in SEQ ID NO: 1, 7, 579, 583, 584, or 591, the tracrRNA of the associated gRNA may have the nucleotide sequence shown in SEQ ID NO: 4, 472, 473, or 474, or an active variant or fragment thereof. In embodiments where the RGN has the amino acid sequence shown in SEQ ID NO: 2, the tracrRNA of the associated gRNA may have the nucleotide sequence shown in SEQ ID NO: 6, 477, 478, 479, or 480, or an active variant or fragment thereof. In embodiments where the RGN has the amino acid sequence shown in SEQ ID NO: 565, the tracrRNA of the associated gRNA may have the nucleotide sequence shown in SEQ ID NO: 572, or an active variant or fragment thereof. In embodiments where the RGN has the amino acid sequence shown in SEQ ID NO: 566 or 1165-1179, the tracrRNA of the associated gRNA may have the nucleotide sequence shown in SEQ ID NO: 574 or 1181, or an active variant or fragment thereof.

[0197] The guide RNA can be a single guide RNA or a dual guide RNA system. A single guide RNA comprises a crRNA on a single RNA molecule and, if present, a tracrRNA, while a dual guide RNA system comprises a crRNA and a tracrRNA present on two different RNA molecules, the crRNA and tracrRNA hybridizing to each other by at least a portion of the CRISPR repeat sequence of the crRNA and at least a portion of the tracrRNA, the tracrRNA being fully or partially complementary to the CRISPR repeat sequence of the crRNA. In some embodiments where the guide RNA is a single guide RNA, the crRNA and, if present, the tracrRNA are linked by a linking nucleotide sequence.

[0198] Generally, the linker nucleotide sequence between crRNA and tracrRNA is a nucleotide sequence that does not include complementary bases to avoid the formation of secondary structures within the linker nucleotide sequence or the formation of secondary structures containing the linker nucleotide sequence. In some embodiments, the length of the linker nucleotide sequence between crRNA and tracrRNA is at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12 or more nucleotides. In some embodiments, the length of the linker nucleotide sequence between crRNA and tracrRNA is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more nucleotides. In some embodiments, the length of the linker nucleotide sequence of a single guide RNA is at least 4 nucleotides. In some embodiments, the length of the linker nucleotide sequence of a single guide RNA is 4 nucleotides. In some embodiments, the linker nucleotide sequence is AAAAG.

[0199] In some embodiments, the guide RNA is a single guide RNA (sgRNA) having a backbone sequence (containing a crRNA repeat sequence, a linker nucleotide sequence, and tracrRNA, as appropriate) of any one of SEQ ID NO: 206, 209, 212, 310, 326, 330, 481-483, 1162, and 1163, or an active variant or fragment thereof. In some embodiments, the active sgRNA backbone sequence variant comprises a nucleotide sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity with any one of the nucleotide sequences shown in SEQ ID NO: 206, 209, 212, 310, 326, 330, 481-483, 1162, and 1163. In some embodiments, the active sgRNA backbone sequence fragment comprises at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or 105 linked nucleotides of any one of the nucleotide sequences shown in SEQ ID NO: 206, 209, 212, 310, 326, 330, 481-483, 1162, and 1163.

[0200] In embodiments where the RGN has the amino acid sequence shown in SEQ ID NO: 1, 7, 579, 583, 584, or 591, the sgRNA backbone may have any one of the nucleotide sequences shown in SEQ ID NO: 206 and 481-483, or an active variant or fragment thereof. In embodiments where the RGN has the amino acid sequence shown in SEQ ID NO: 2, the sgRNA backbone may have any one of the nucleotide sequences shown in SEQ ID NO: 209, 212, 310, 326, and 330, or an active variant or fragment thereof. In embodiments where the RGN has the amino acid sequence shown in SEQ ID NO: 565, the sgRNA backbone may have the nucleotide sequence shown in SEQ ID NO: 1162, or an active variant or fragment thereof. In embodiments where the RGN has the amino acid sequence shown in SEQ ID NO: 566 or 1165-1179, the sgRNA backbone may have the nucleotide sequence shown in SEQ ID NO: 1163, or an active variant or fragment thereof.

[0201] When two polynucleotide sequences hybridize with each other under stringent conditions, the two sequences are considered substantially complementary. Similarly, if the guide RNA bound to an RGN under stringent conditions binds to the target sequence, the RGN is considered to bind to the specific target sequence in a sequence-specific manner. “Stringent conditions” or “stringent hybridization conditions” refer to conditions where the degree to which two polynucleotide sequences hybridize with each other is detectably higher than the degree to which they hybridize with other sequences (e.g., at least twice the background). Stringent conditions are sequence-dependent and will vary depending on the circumstances. Typically, stringent conditions will be conditions with a salt concentration of less than about 1.5 M Na ions at pH 7.0 to 8.3, typically about 0.01 to 1.0 M Na ion concentration (or other salts), and a temperature of at least about 30 °C for short sequences (e.g., 10 to 50 nucleotides) and at least about 60 °C for long sequences (e.g., greater than 50 nucleotides). Stringent conditions can also be achieved by adding a destabilizing agent (such as formamide). Exemplary low-toughness conditions include hybridization at 37 °C in a buffer of 30% to 35% formamide, 1 M NaCl, and 1% SDS (sodium dodecyl sulfate), followed by washing at 50 °C to 55 °C in 1X to 2X SSC (20X SSC = 3.0 M NaCl / 0.3 M trisodium citrate). Exemplary medium-toughness conditions include hybridization at 37 °C in a buffer of 40% to 45% formamide, 1.0 M NaCl, and 1% SDS, followed by washing at 55 °C to 60 °C in 0.5X to 1X SSC. Exemplary high-toughness conditions include hybridization at 37 °C in a buffer of 50% formamide, 1 M NaCl, and 1% SDS, followed by washing at 60 °C to 65 °C in 0.1X SSC. The wash buffer may contain approximately 0.1% to approximately 1% SDS, depending on the specific conditions. The hybridization duration is generally less than approximately 24 hours, typically approximately 4 to approximately 12 hours. The washing time will last at least a sufficient duration to achieve equilibrium.

[0202] Tm is the temperature at which 50% of the complementary target sequence hybridizes with the perfectly matched sequence (at defined ionic strength and pH). For DNA-DNA hybrids, Tm can be estimated by the equation of Meinkoth and Wahl (1984): *Analytical Biochemistry* 138:267-284: Tm = 81.5 °C + 16.6 (log M) + 0.41 (GC%) - 0.61 (formamide%) - 500 / L; where M is the molar concentration of monovalent cations, GC% is the percentage of guanine and cytosine nucleotides in the DNA, formamide% is the percentage of formamide in the hybridization solution, and L is the length of the base pair hybrid. Generally, stringent conditions are selected to be approximately 5 °C lower than the thermal melting point (Tm) of a specific sequence and its complement at specified ionic strength and pH. However, severely stringent conditions can be achieved by hybridization and / or washing at temperatures 1, 2, 3, or 4 °C below the specific melting point (Tm); moderately stringent conditions can be achieved by hybridization and / or washing at temperatures 6, 7, 8, 9, or 10 °C below the specific melting point (Tm); and lightly stringent conditions can be achieved by hybridization and / or washing at temperatures 11, 12, 13, 14, 15, or 20 °C below the specific melting point (Tm). Using equations, hybridization and washing compositions, and the desired Tm, those skilled in the art will understand that variations in hybridization stringency and / or washing solutions are described herein. Detailed guidelines for nucleic acid hybridization can be found in Tijssen (1993), *Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes*, Part I, Chapter 2 (Elsevier, New York); and Ausubel et al., eds., *Current Protocols in Molecular Biology* (1995), Chapter 2 (Greene Publishing and Wiley-Interscience, New York). See also Sambrook et al. (1989), *Molecular Cloning: A Laboratory Manual* (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, New York).

[0203] The term "sequence specificity" can also refer to the fact that the affinity of an RGN peptide or PE for the target sequence is greater than its affinity for the randomized background sequence.

[0204] Guide RNA can be synthesized chemically or via in vitro transcription. Assays for determining sequence-specific binding between RGN and guide RNA are known in this technique and include (but are not limited to) in vitro binding assays of expressed RGN to guide RNA, which can be labeled with a detectable marker (e.g., biotin) and used in pull-down assays, wherein the guide RNA:RGN complex is captured via a detectable marker (e.g., streptavidin beads). Control guide RNAs with sequences or structures unrelated to the guide RNA can be used as negative controls for nonspecific binding of RGN to RNA.

[0205] Guide RNA can be introduced into target cells, organelles, or embryos in the form of RNA molecules. In some embodiments, the nucleotide sequence encoding the guide RNA is introduced into the target cells, organelles, or embryos. In some embodiments, the nucleotide sequence encoding the guide RNA is operatively linked to a promoter (e.g., the RNA polymerase III promoter). The promoter can be a native promoter or heterologous to the nucleotide sequence encoding the guide RNA. In some embodiments, the guide RNA can be introduced into target cells, organelles, or embryos as a ribonucleoprotein complex as described herein, wherein the guide RNA is bound to an RGN polypeptide.

[0206] Guide RNA directs the associated RGN to a specific target nucleotide sequence of interest through hybridization with the target sequence of interest. The target sequence can be bound to (and cleaved in some embodiments) by the RGN in vitro or in cells. The target sequence is within the target polynucleotide and may comprise DNA, RNA, or a combination of both, and may be single-stranded or double-stranded. The target sequence may be genomic DNA (i.e., chromosomal DNA), plasmid DNA, or RNA molecules (e.g., messenger RNA, ribosomal RNA, transfer RNA, microRNA, small interfering RNA). In those embodiments where the target sequence is a chromosomal sequence, the chromosomal sequence may be a nuclear, plasmid, or mitochondrial chromosomal sequence. In the compositions and methods disclosed in this invention, the target sequence is within a double-stranded target polynucleotide (e.g., a target DNA sequence). In some embodiments, the target sequence is unique within the target genome. In some embodiments, the target sequence is double-stranded and comprises a target strand and a non-target strand.

[0207] The target sequence is adjacent to the original spacer sequence adjacent to the adjacent motif (PAM), and the non-target strand of the target sequence is the strand containing the PAM. The PAM is immediately adjacent to the target sequence and typically contains N, where N represents any nucleotide. In some embodiments, the PAM contains about 1 to about 10 Ns, including about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 Ns. In specific embodiments, the PAM contains 1 to 10 Ns, including 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 Ns. Generally, the PAM can be at the 5' or 3' of the target sequence on the non-target strand. The PAM of the RGN disclosed in this invention is immediately adjacent to the 3' of the target sequence on the non-target strand. Generally, the PAM is a common sequence of about 3-4 nucleotides, but in specific embodiments, its length can be 2, 3, 4, 5, 6, 7, 8, 9, or more nucleotides.

[0208] In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 1, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 5' end of the PAM sequence shown in NNNNCY. In some embodiments, when bound to a guide RNA comprising a crRNA repeat sequence or an active variant or fragment thereof comprising any one of SEQ ID NO: 3, 470, and 471 and a tracrRNA sequence or an active variant or fragment thereof comprising any one of SEQ ID NO: 4, 472, 473, and 474, an RGN having the amino acid sequence shown in SEQ ID NO: 1, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 5' end of the PAM sequence shown in NNNNCY.

[0209] In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 2, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 5' end of the PAM sequence shown in NNRYA. In some embodiments, when bound to a guide RNA comprising a crRNA repeat sequence or an active variant or fragment thereof comprising any one of SEQ ID NO: 5, 475, and 476 and a tracrRNA sequence or an active variant or fragment thereof comprising any one of SEQ ID NO: 6, 477, 478, 479, and 480, an RGN having the amino acid sequence shown in SEQ ID NO: 2, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 5' end of the PAM sequence shown in NNRYA.

[0210] In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 7, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 5' end of the PAM sequence shown in NNNNCC. In some embodiments, when bound to a guide RNA comprising a crRNA repeat sequence or an active variant or fragment thereof comprising any one of SEQ ID NO: 3, 470, and 471 and a tracrRNA sequence or an active variant or fragment thereof comprising any one of SEQ ID NO: 4, 472, 473, and 474, an RGN having the amino acid sequence shown in SEQ ID NO: 7, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 5' end of the PAM sequence shown in NNNNCC.

[0211] In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 579, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 5' end of the PAM sequence shown in NNNNC. In some embodiments, when bound to a guide RNA comprising a crRNA repeat sequence or an active variant or fragment thereof comprising any one of SEQ ID NO: 3, 470, and 471 and a tracrRNA sequence or an active variant or fragment thereof comprising any one of SEQ ID NO: 4, 472, 473, and 474, an RGN having the amino acid sequence shown in SEQ ID NO: 579, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 5' end of the PAM sequence shown in NNNNC.

[0212] In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 583, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 5' end of the PAM sequence shown in NNNNC. In some embodiments, when bound to a guide RNA comprising a crRNA repeat sequence or an active variant or fragment thereof comprising any one of SEQ ID NO: 3, 470, and 471 and a tracrRNA sequence or an active variant or fragment thereof comprising any one of SEQ ID NO: 4, 472, 473, and 474, an RGN having the amino acid sequence shown in SEQ ID NO: 583, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 5' end of the PAM sequence shown in NNNNC.

[0213] In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 584, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 5' end of the PAM sequence shown in NNNNCM. In some embodiments, when bound to a guide RNA comprising a crRNA repeat sequence or an active variant or fragment thereof comprising any one of SEQ ID NO: 3, 470, and 471 and a tracrRNA sequence or an active variant or fragment thereof comprising any one of SEQ ID NO: 4, 472, 473, and 474, an RGN having the amino acid sequence shown in SEQ ID NO: 584, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 5' end of the PAM sequence shown in NNNNCM.

[0214] In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 591, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 5' end of the PAM sequence shown in NNNNCT. In some embodiments, when bound to a guide RNA comprising a crRNA repeat sequence or an active variant or fragment thereof comprising any one of SEQ ID NO: 3, 470, and 471 and a tracrRNA sequence or an active variant or fragment thereof comprising any one of SEQ ID NO: 4, 472, 473, and 474, an RGN having the amino acid sequence shown in SEQ ID NO: 591, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 5' end of the PAM sequence shown in NNNNCT.

[0215] In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 565, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 5' end of the PAM sequence shown in NNGRR. In some embodiments, when bound to a guide RNA comprising a crRNA repeat sequence or an active variant or fragment thereof of SEQ ID NO: 571 and a tracrRNA sequence or an active variant or fragment thereof of SEQ ID NO: 572, an RGN having the amino acid sequence shown in SEQ ID NO: 565, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 5' end of the PAM sequence shown in NNGRR.

[0216] In some embodiments, an RGN having the amino acid sequence shown in SEQ ID NO: 566 or 1165-1179, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 5' end of the PAM sequence shown in NNGG. In some embodiments, when bound to a guide RNA comprising a crRNA repeat sequence or an active variant or fragment thereof of SEQ ID NO: 573 or 1180 and a tracrRNA sequence or an active variant or fragment thereof of SEQ ID NO: 574 or 1181, an RGN having the amino acid sequence shown in SEQ ID NO: 566 or 1165-1179, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to and at its 5' end of the PAM sequence shown in NNGG.

[0217] As is well known in this technique, the PAM sequence specificity of a given nuclease is affected by the enzyme concentration (see, for example, Karvelis et al. (2015), *Genome Biology*). Genome Biol (16:253) It can be modified by changing the amount of ribonucleoprotein complex used to express RGN or delivered to target cells, organelles or embryos.

[0218] After identifying its corresponding PAM sequence, an active RGN can cleave one or both strands of the target sequence at specific cleavage sites. As used herein, cleavage sites contain specific nucleotides within the target sequence at which the target strand, non-target strand, or both strands of the target sequence are cleaved by the RGN. Cleavage sites may contain nucleotides 1 and 2, 2 and 3, 3 and 4, 4 and 5, 5 and 6, 7 and 8, or 8 and 9 from the PAM in the 5' or 3' direction. In some embodiments, cleavage sites may contain more than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides from the PAM in the 5' or 3' direction. Because the RGN cleaves the target sequence, producing staggered ends, in some embodiments, the cleavage sites are defined based on the distance between two nucleotides from the PAM on the non-target strand of the target sequence and the distance between two nucleotides from the complementary PAM sequence on the target strand.

[0219] 1. PEgRNA

[0220] In embodiments involving polymerase editors (PE), the PE system utilizes a polymerase to edit a guide RNA (“PEgRNA”). PEgRNA is a guide RNA that specifies a target sequence and provides a template for polymerizing a replacement strand containing the desired edit by means of an engineered extension to the RGN guide RNA or a portion thereof (referred to herein as an extension arm). PEgRNA can be a single guide RNA, wherein the extension arm is located at the 5' or 3' end or within the guide RNA, or it can be multiple polynucleotides (e.g., dual guide RNA). In embodiments where PEgRNA is a dual guide RNA, the extension arm is located at the 5' or 3' end or within a crRNA or tracrRNA molecule. The template polymerized within the extension arm is referred to herein as a DNA synthesis template. In embodiments where the polymerase of the PE is a reverse transcriptase, the DNA synthesis template may be referred to as a reverse transcriptase template (RTT). RGN is guided to the target sequence via PEgRNA, and in embodiments where RGN is a nickase having an inactive HNH domain and an active RuvC domain, the RGN nickase cleaves the non-target strand upstream of the sequence to be edited and upstream of PAM, thereby generating a 3' overhang on the non-target strand. PEgRNA includes a primer binding site (PBS) complementary to the 3' overhang of the non-target strand. The PBS length can be at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides. In some embodiments, the pegRNA comprises PBS of at least 5 nucleotides in length (e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides). In some embodiments, the pegRNA may comprise PBS of 9, 11, 12, 13, or 15 nucleotides in length. Hybridization of PBS with the 3' overhang of the non-target strand allows for the polymerization of the edited replacement strand using a DNA synthesis template during PEgRNA extension. The DNA synthesis template can be at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more nucleotides.In some embodiments, the PEgRNA comprises a DNA synthesis template of at least 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, the PEgRNA comprises a DNA synthesis template of 19, 22, 23, 24, 25, 26, 29, 30, 32, 34, 37, 38, 39, 40, 42, or 46 nucleotides in length. DNA synthesis templates contain the edits that need to be made, which can be the substitution, deletion, or addition of one or more nucleotides.

[0221] The extended arm of PEgRNA can be formed from either RNA or DNA. In the case of RNA extension, the polymerase in the polymerase editor can be an RNA-dependent DNA polymerase (such as reverse transcriptase). In the case of DNA extension, the polymerase in the polymerase editor can be a DNA-dependent DNA polymerase.

[0222] The replacement strand containing the desired edit (e.g., substitution, deletion, or addition) shares the same sequence as the non-target strand of the target sequence to be edited (except that it contains the desired edit). Through DNA repair and / or replication mechanisms, the non-target strand of the target sequence is replaced with a new synthetic replacement strand containing the desired edit. In some cases, polymerase editing can be viewed as a "search and replace" genome editing technique because the polymerase editor not only searches for and locates the desired target sequence to be edited but also simultaneously encodes a replacement strand containing the desired edit, which is installed in place of the corresponding non-target strand of the target sequence. Therefore, in some embodiments, the guide RNA of this disclosure comprises an extension containing an editing template for polymerase editing.

[0223] 2. Nicking guide RNA

[0224] To reduce the likelihood of edits introduced by polymerase editors being removed due to mismatch repair on the edited strand, nick guide RNAs can be used. A nick guide RNA is a guide RNA that targets a sequence on the unedited strand near and opposite the original nick, guiding the RGN nicking enzyme of the PE system to this unedited strand to introduce a single-strand nick. The nick guide RNA can be designed to match the edited sequence introduced by PEgRNA, but not the original unedited sequence, to ensure that nicking occurs after an editing event on the non-target strand.

[0225] III. APG07433.1 RGN variant

[0226] This article provides variants of RGN APG07433.1 that identify alternative PAMs (e.g., NNNNCY, NNNNC, NNNNCT, and NNNNCM, compared to APG07433.1's identification of NNNNCC). This type of RGN may have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and include: a) S at position corresponding to amino acid position 1020 in SEQ ID NO: 1; b) G at position corresponding to amino acid position 1021 in SEQ ID NO: 1; c) N at position corresponding to amino acid position 1022 in SEQ ID NO: 1; d) K at position corresponding to amino acid position 1023 in SEQ ID NO: 1; and e) E at position corresponding to amino acid position 1025 in SEQ ID NO: 1.

[0227] This type of RGN may have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and include: a) S at position corresponding to amino acid position 1020 in SEQ ID NO: 1; b) G at position corresponding to amino acid position 1021 in SEQ ID NO: 1; c) N at position corresponding to amino acid position 1022 in SEQ ID NO: 1; d) K at position corresponding to amino acid position 1023 in SEQ ID NO: 1; or e) E at position corresponding to amino acid position 1025 in SEQ ID NO: 1.

[0228] In some embodiments, the APG07433.1 variant has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the amino acid sequence shown in SEQ ID NO: 579 and includes Q at position corresponding to amino acid position 1023 in SEQ ID NO: 579.

[0229] In some embodiments, the APG07433.1 variant has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the amino acid sequence shown in SEQ ID NO: 584 and includes a T at position corresponding to amino acid position 1021 in SEQ ID NO: 584 and an N at position corresponding to amino acid position 1023 in SEQ ID NO: 584.

[0230] In some embodiments, the APG07433.1 variant has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the amino acid sequence shown in SEQ ID NO: 583 and includes N at position 1021 corresponding to amino acid position 1023 in SEQ ID NO: 583.

[0231] In some embodiments, the APG07433.1 variant has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the amino acid sequence shown in SEQ ID NO: 591 and includes: a) Q at position corresponding to amino acid position 975 in SEQ ID NO: 591; b) A at position corresponding to amino acid position 1020 in SEQ ID NO: 591; c) G at position corresponding to amino acid position 1022 in SEQ ID NO: 591; and d) Q at position corresponding to amino acid position 1023 in SEQ ID NO: 591.

[0232] In some embodiments, the RGN disclosed herein, or its active variants or fragments, recognize the protospacer adjacent motif (PAM) (where N is A, C, T / U, or G) of a shared nucleotide sequence with NNNNC at the 3' end of the target sequence on a non-target strand. In some embodiments, the PE or PE system comprises an RGN (APG07433.1) having the amino acid sequence shown in SEQ ID NO: 7, which recognizes the PAM of a shared nucleotide sequence with NNNNC at the 3' end of the target sequence on a non-target strand.

[0233] In some embodiments, the RGN disclosed herein, or its active variants or fragments, recognize the protospacer adjacent motif (PAM) of a shared nucleotide sequence with NNNNNCY at the 3' end of the target sequence on a non-target strand (where N is A, C, T / U, or G and Y is C or T / U). In some embodiments, the PE or PE system comprises an RGN (LPG10221) having the amino acid sequence shown in SEQ ID NO: 1, which recognizes the PAM of a shared nucleotide sequence with NNNNNCY at the 3' end of the target sequence on a non-target strand.

[0234] In some embodiments, the RGN disclosed herein, or its active variants or fragments, recognize the protospacer adjacent motif (PAM) (where N is A, C, T / U, or G) of a shared nucleotide sequence with NNNNC at the 3' end of the target sequence on a non-target strand. In some embodiments, the PE or PE system comprises an RGN (LPG10209) having the amino acid sequence shown in SEQ ID NO: 579, which recognizes the PAM of a shared nucleotide sequence with NNNNC at the 3' end of the target sequence on a non-target strand. In some embodiments, the PE or PE system comprises an RGN (LPG10212) having the amino acid sequence shown in SEQ ID NO: 583, which recognizes the PAM of a shared nucleotide sequence with NNNNC at the 3' end of the target sequence on a non-target strand.

[0235] In some embodiments, the RGN disclosed herein, or its active variants or fragments, recognize the protospacer adjacent motif (PAM) of a shared nucleotide sequence with NNNNCM at the 3' end of the target sequence on a non-target strand (where N is A, C, T / U, or G and M is C or A). In some embodiments, the PE or PE system comprises an RGN (LPG10210) having the amino acid sequence shown in SEQ ID NO: 584, which recognizes the PAM of a shared nucleotide sequence with NNNNCM at the 3' end of the target sequence on a non-target strand.

[0236] In some embodiments, the RGN disclosed herein, or its active variants or fragments, recognize the protospacer adjacent motif (PAM) (where N is A, C, T / U, or G) of a shared nucleotide sequence with NNNNCT at the 3' end of the target sequence on a non-target strand. In some embodiments, the PE or PE system comprises an RGN (LPG10213) having the amino acid sequence shown in SEQ ID NO: 591, which recognizes the PAM of a shared nucleotide sequence with NNNNCT at the 3' end of the target sequence on a non-target strand.

[0237] In some embodiments, the RGN disclosed herein, or its active variants or fragments, recognize the original spacer adjacent motif (PAM) of a shared nucleotide sequence with NNGRR at the 3' end of the target sequence on a non-target strand (where N is A, C, T / U, or G and R is A or G). In some embodiments, the PE or PE system comprises an RGN (APG01604) having the amino acid sequence shown in SEQ ID NO: 565, which recognizes the PAM of a shared nucleotide sequence with NNGRR at the 3' end of the target sequence on a non-target strand.

[0238] In some embodiments, the RGN disclosed herein, or its active variants or fragments, recognize the protospacer adjacent motif (PAM) (where N is A, C, T / U, or G) of a shared nucleotide sequence with NNGG at the 3' end of the target sequence on a non-target strand. In some embodiments, the PE or PE system comprises an RGN (LPG10145) having the amino acid sequence shown in SEQ ID NO: 566, which recognizes the PAM of a shared nucleotide sequence with NNGG at the 3' end of the target sequence on a non-target strand.

[0239] The RGN or its active variants or fragments disclosed in this invention may have a PAM interaction (PI) domain, which facilitates the recognition of PAM sites in target polynucleotides. The PI domain may contain 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 or more amino acid residues. In some embodiments, the PI domain of the disclosed RGN or its active variants or fragments is located in the carboxyl (C) terminal region of the RGN. The C-terminal region of the PI domain comprising the RGN or its active variants or fragments disclosed herein may include 151, 150, 140, 135, 132, 130, 125, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 amino acid residues of the RGN. In some embodiments, the PI domain of the RGN or its active variants or fragments disclosed herein is located within or includes amino acid residues 939-1071 of the RGN. In some embodiments, the PI domain of the RGN having the amino acid sequence shown in SEQ ID NO: 1, 7, 579, 583, 584, or 591, or an active variant or fragment thereof, is within or includes amino acid residues 939-1071 of the RGN. In some embodiments, the RGN variants described herein contain a PI domain having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with amino acid residues 939-1071 of SEQ ID NO: 1, 7, 579, 583, 584, or 591.In other embodiments, the RGN variant described herein comprises a PI domain having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with amino acid residues 939-1071 of SEQ ID NO: 1, and recognizes a PAM with a shared nucleotide sequence of NNNNCY at the 3' of the target sequence on a non-target strand.

[0240] In other embodiments, the RGN variants described herein comprise a PI domain having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with amino acid residues 939-1071 of SEQ ID NO: 579 or 583, and recognizes a PAM with a shared nucleotide sequence of NNNNC at the 3' of the target sequence on a non-target strand.

[0241] In other embodiments, the RGN variant described herein comprises a PI domain having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with amino acid residues 939-1071 of SEQ ID NO: 584, and recognizes a PAM with a common nucleotide sequence of NNNNCM at the 3' of the target sequence on a non-target strand.

[0242] In other embodiments, the RGN variants described herein comprise a PI domain having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with amino acid residues 939-1071 of SEQ ID NO: 591, and recognizes a PAM with a shared nucleotide sequence of NNNNCT at the 3' end of the target sequence on a non-target strand. The RGN disclosed herein may comprise at least one nuclease domain (e.g., a DNase or RNase domain) and at least one RNA recognition and / or RNA binding domain to interact with guide RNA. In some embodiments, the RGN comprises only one active nuclease domain and thus acts as a nicking enzyme. In some embodiments, the active RGN nuclease domain is a RuvC domain. In some embodiments, the RGN includes an inactive HNH nuclease domain. Other domains that may be found in the RGN include (but are not limited to): a DNA-binding domain, a helicase domain, a protein-protein interaction domain, and a dimerizing domain. In some embodiments, the RGN may contain at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with one or more of the DNA-binding domain, helicase domain, protein-protein interaction domain, and dimerizing domain.

[0243] In various embodiments, the target sequence is bound by the RGN disclosed in this invention. In those cases where the target sequence is double-stranded (e.g., double-stranded DNA), the target strand of the target sequence hybridizes with the guide RNA associated with the RGN. If the polypeptide has nuclease activity, the target strand and / or non-target strand of the target sequence (e.g., the target DNA sequence) can subsequently be cleaved by the RGN. The RGN disclosed in this invention can cleave nucleotides within the polynucleotide, acting as an endonuclease, or can be an exonuclease, removing consecutive nucleotides from the ends (5' and / or 3' ends) of the polynucleotide. In some embodiments, the RGN disclosed in this invention can cleave nucleotides of the target polynucleotide at any position within the polynucleotide and thus acts as both an endonuclease and an exonuclease. Cleavage of the target polynucleotide by the RGN disclosed in this invention can cause staggered breaks or blunt ends.

[0244] The RGN disclosed in this invention may be a wild-type sequence derived from bacterial or archaeal species. Alternatively, the RGN may be a variant or fragment of a wild-type polypeptide. For example, wild-type RGN may be modified to alter nuclease activity or PAM specificity. In some embodiments, the RGN is not naturally occurring.

[0245] In some embodiments, the RGNs disclosed in this invention can act as cleaving enzymes, cleaving only the single strand of a double-stranded target sequence (e.g., a target DNA sequence). These RGNs possess a single-function nuclease domain. In specific embodiments, the cleaving enzyme is capable of cleaving either the target strand (with an active HNH domain) or the non-target strand (with an active RuvC domain) of a double-stranded target sequence (e.g., a target DNA sequence). To achieve cleavage of the single strand of a double-stranded target polynucleotide (e.g., target DNA), at least one inactivating mutation (also referred to herein as a nuclease-reducing mutation) can be introduced into the nuclease domain of the RGN. For example, to deactivate the HNH domain of LPG10221, the following catalytic amino acid residues can be mutated to, for example, alanine or another non-conserved amino acid residue: D603, H604, N618, N627. More than one nuclease-reducing mutation can be introduced into the nuclease domain of the RGN to further reduce its activity. Therefore, in some embodiments, the HNH nuclease domain of the RGN disclosed in this invention comprises alanine (or another non-conserved amino acid residue) at position 604 corresponding to SEQ ID NO: 1 and alanine (or another non-conserved amino acid residue) at position 627 corresponding to SEQ ID NO: 1. In some embodiments, the RGN nickase comprises the sequence shown in SEQ ID NO: 8, or an active fragment or variant thereof, such as a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with SEQ ID NO: 8. RGN nickases having an inactive HNH domain can be used for polymerase editing and can be fused with polymerases as described herein.

[0246] Alternatively, RGN nickases with an inactive RuvC domain are commonly used for base editing and fused with base-editing peptides (such as deaminases that directly chemically modify (e.g., deamination) nucleobases) to convert one nucleobase into another. The base-editing peptide can be fused to the RGN at its N-terminus or C-terminus. Alternatively, the base-editing peptide can be fused to the RGN via a peptide linker. A non-limiting example of a deaminase peptide suitable for such compositions and methods includes cytosine deaminases or adenine deaminases (such as the adenine deaminase base editor described below: Gaudelli et al. (2017) *Nature*). Nature(551:464-471, U.S. Publications 2017 / 0121693 and 2018 / 0073012 and International Publication WO2018 / 027078, or any of the following deaminases disclosed: International Publication WO 2020 / 139783, International Publication WO 2022 / 056254, International Publication WO 2022 / 204093 and PCT International Application PCT / IB2023 / 061192 (filed November 6, 2023), each of which is incorporated herein by reference in its entirety).

[0247] In implementations using nickases and requiring double-strand cutting, two nickases can be used, each nicking a single strand within the double-stranded target sequence. Alternatively, the same RGN nickase can be used, along with two guide RNAs targeting one strand of each double-stranded target sequence.

[0248] In other embodiments, the RGN generally lacks nuclease activity and is referred to herein as nuclease-inactive or nuclease-free. In some embodiments, the nuclease-inactive or nuclease-free form of the enzyme is used to modify nucleic acids, such as for base editing as described herein. Any methods known in this art for introducing mutations into amino acid sequences, such as PCR-mediated mutation induction and site-directed mutagenesis induction, can be used to produce nickase or nuclease-inactive RGNs. See, for example, U.S. Publication No. 2014 / 0068797 and U.S. Patent No. 9,790,490; each of which is incorporated herein by reference in its entirety.

[0249] RGNs, including those containing nuclease activity or lacking nuclease activity, can be used to deliver fusion peptides, polynucleotides, or small molecule payloads to specific genomic locations. In some of these embodiments, the RGN peptide or guide RNA may be fused with a detectable marker or purification tag to allow detection of a specific sequence. As a non-limiting example, a nuclease-inactivated RGN may be fused with a detectable marker (e.g., fluorescent protein) and target a disease-related specific sequence to allow detection of a disease-related sequence. As another non-limiting example, a nuclease-active RGN may be fused with a detectable marker (e.g., fluorescent protein) and target a disease-related specific sequence to allow detection of a disease-related sequence. The detectable marker or purification tag may be located directly or indirectly via a linker peptide at the N-terminus, C-terminus, or internal location of the RGN. In some embodiments, the RGN component of the fusion protein is a nuclease-inactivated RGN. In some embodiments, the RGN component of the fusion protein is an RGN with nickase activity.

[0250] The detectable marker is a molecule that can be visually or otherwise observed. The detectable marker may be fused with RGN to form a fusion protein (e.g., a fluorescent protein) or may be a small molecule conjugated to an RGN polypeptide, which can be detected visually or otherwise. Detectable markers that can be fused with the RGN disclosed in this invention to form a fusion protein include any detectable protein domain, including (but not limited to) fluorescent proteins or protein domains detectable by specific antibodies. Non-limiting examples of fluorescent proteins include green fluorescent proteins (e.g., GFP, EGFP, ZsGreen1) and yellow fluorescent proteins (e.g., YFP, EYFP, ZsYellow1). Non-limiting examples of small molecule detectable markers include radioactive markers, such as… 3 H and 35 S.

[0251] RGN peptides may also contain purification tags, which are any molecules that can be used to isolate proteins or fusion proteins from mixtures (e.g., biological samples, culture media). Non-limiting examples of purification tags include biotin, myc, maltose-binding protein (MBP), glutathione S-transferase (GST), and 3X FLAG tags.

[0252] Alternatively, nuclease-inactivated RGNs can target specific genomic locations to alter the expression of desired genes (i.e., target genes). In some embodiments, the binding of a nuclease-inactivated RGN to a target sequence causes a reduction in the expression of the gene at the target sequence by interfering with the binding of RNA polymerases or transcription factors within the targeted genomic region. In some embodiments, the RGN (e.g., a nuclease-inactivated RGN) or its complexed guide RNA further comprises an expression regulator that, upon binding to the target sequence within the target gene, inhibits or activates the expression of the target gene.

[0253] In some embodiments, the expression regulator of the fusion protein includes a transcriptional repressor domain that interacts with transcriptional control components and / or transcriptional regulatory proteins, such as RNA polymerases and transcription factors, to reduce or terminate transcription of at least one gene. The transcriptional repressor domain is known in this art and includes (but is not limited to) Sp1-like repressors, IκB, and Krüppel-associated box (KRAB) domains.

[0254] In some embodiments, the expression regulator of the fusion protein includes a transcription activation domain that interacts with transcriptional control components and / or transcriptional regulatory proteins, such as RNA polymerases and transcription factors, to increase or activate transcription of at least one gene. Transcription activation domains are known in this art and include (but are not limited to) the herpes simplex virus VP16 activation domain and the NFAT activation domain.

[0255] In some embodiments, expression regulators regulate the expression of target sequences or regulated genes through epigenetic mechanisms. In some embodiments, epigenetic regulators covalently modify DNA or histones to alter histone structure and / or chromosome structure without altering the DNA sequence, resulting in changes in gene expression (e.g., upregulation or downregulation). Non-limiting examples of epigenetic modifications include acetylation or methylation of lysine residues, arginine methylation, serine and threonine phosphorylation, and lysine ubiquitination and ubiquitin-like methylation of histones, and methylation and hydroxymethylation of cytosine residues in DNA. Non-limiting examples of epigenetic regulators include histone acetyltransferases, histone deacetylases, histone methyltransferases, histone demethylases, DNA methyltransferases, and DNA demethylases.

[0256] In some embodiments, nuclease-inactivated RGNs or RGNs with nicking enzyme activity can target specific genomic locations to modify the sequence of a target polynucleotide by fusing with a base-editing polypeptide (e.g., a deaminase polypeptide or an active variant or fragment thereof), which directly chemically modifies (e.g., deaminases) the nucleobases, causing one nucleobase to be converted to another. The base-editing polypeptide can fuse with the RGN at its amino-terminus (N-terminus) or carboxyl-terminus (C-terminus). The base-editing polypeptide can also be operatively fused to the RGN at an internal location. Insertion of a polypeptide (e.g., a base-editing polypeptide or DNA polymerase) at an internal location of the RGN polypeptide is described elsewhere in this document. Alternatively, the base-editing polypeptide can fuse with the RGN via a peptide linker. A “base editor” is a fusion protein comprising a DNA-targeting polypeptide (such as an RGN) and a base-editing polypeptide (such as a deaminase). Non-limiting examples of deaminases or base editors that can be used in such compositions and methods include cytosine deaminases, adenine deaminases, or their corresponding cytosine base editors or adenine base editors (such as the adenine deaminase or base editor described below: Gaudelli et al. (2017) Nature ( Nature(This refers to SEQ ID NO: 551:464-471, U.S. Publication No. 2017 / 0121693 and 2018 / 0073012, and International Publication No. WO 2018 / 027078, or any of the following deaminases or base editors: International Publication No. WO 2020 / 139783, International Publication No. WO 2022 / 056254, International Publication No. WO 2022 / 204093, PCT / IB2023 / 061192 (filed November 6, 2023), each of which is incorporated herein by reference in its entirety). In some embodiments, the deaminase polypeptide that can be used in such compositions and methods is a cytosine deaminase or adenine deaminase comprising an amino acid sequence selected from any one of SEQ ID NO: 484-555. In one embodiment, the deaminase polypeptide suitable for use in such compositions and methods is a cytosine deaminase or adenine deaminase having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with any of the amino acid sequences shown in SEQ ID NO: 484-555. In some embodiments, the deaminase suitable for use in such compositions and methods of the present invention is a deaminase disclosed in Table 17 of International Publication No. WO 2020 / 139783 (which is incorporated herein by reference in its entirety). Furthermore, certain fusion proteins known in this technology between RGN and base editing enzymes (such as cytosine deaminase) may also contain at least one uracil-stabilizing polypeptide that increases the mutation rate of the polynucleotide deaminase in mutating cytidine, deoxycytidine, or cytosine to thymidine, deoxythymidine, or thymine. Non-limiting examples of uracil-stabilizing polypeptides include those disclosed in PCT Publication WO 2021 / 217002 and PCT Publication WO 2022 / 015969 (each of which is incorporated herein by reference in its entirety). The disclosed uracil-stabilizing polypeptides include USP2 (SEQ ID NO: 556) and a uracil glycosidase inhibitor (UGI) domain (SEQ ID NO: 557), which can increase base editing efficiency. Therefore, a base editor may contain the RGN or variants thereof described herein, a deaminase, and, where appropriate, at least one uracil-stabilizing polypeptide, such as UGI or USP2.

[0257] In some embodiments, the RGN fusion protein includes a cleavage domain, which is any domain capable of cleaving polynucleotides (i.e., RNA, DNA, or RNA / DNA hybrids), and includes (but is not limited to) restriction endonucleases and homing endonucleases, such as IIS-type endonucleases (e.g., 2-3 nucleotides). FokI (See, for example, Belfort et al. (1997) Nucleic Acid Research) Nucleic Acids Res. )》25:3379-3388; Linn et al. (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993.

[0258] RGNs fused to peptides or domains can be linked to linkers as described elsewhere herein. The RGNs disclosed herein may include at least one nuclear localization signal (NLS) to enhance protein transport to the cell nucleus, as described elsewhere herein. Other localization signal sequences known in this art for targeting peptides to specific subcellular locations can also be used to target RGNs, including (but not limited to) plasmid localization sequences, mitochondrial localization sequences, and dual-targeting signal sequences targeting both plasmids and mitochondria.

[0259] The RGN disclosed in this invention may include at least one cell-penetrating domain that facilitates cellular uptake of the RGN, as described elsewhere herein.

[0260] Nuclear localization signals, plastid localization signals, mitochondrial localization signals, dual-targeting localization signals, and / or cell-penetrating domains may be located at the N-terminus (N-terminus), C-terminus (C-terminus), and / or internal location of the RGN.

[0261] IV. Polynucleotides encoding polymerase editors, polymerase editor systems, RGNs, and guide RNAs

[0262] This disclosure provides polynucleotides comprising or encoding the guide RNA or PEgRNA disclosed herein and polynucleotides comprising nucleotide sequences encoding RNA-guided nucleases (RGN), PE, or polymerases disclosed herein. The polynucleotides disclosed herein comprise polynucleotides encoding RGN having the amino acid sequence shown in SEQ ID NO: 1 and its active fragments or variants, the active fragments or variants retaining the ability to bind to a target sequence in an RNA-guided sequence-specific manner and comprising: a) S at position corresponding to amino acid position 1020 in SEQ ID NO: 1; b) G at position corresponding to amino acid position 1021 in SEQ ID NO: 1; c) N at position corresponding to amino acid position 1022 in SEQ ID NO: 1; d) K at position corresponding to amino acid position 1023 in SEQ ID NO: 1; and e) E at position corresponding to amino acid position 1025 in SEQ ID NO: 1.

[0263] The polynucleotides disclosed in this invention also include polynucleotides encoding RGN having the amino acid sequence shown in SEQ ID NO: 579 and their active fragments or variants, the active fragments or variants retaining the ability to bind to the target sequence in an RNA-guided sequence-specific manner and containing a Q at position corresponding to amino acid position 1023 in SEQ ID NO: 579.

[0264] The polynucleotides disclosed in this invention also include polynucleotides encoding RGN having the amino acid sequence shown in SEQ ID NO: 584 and their active fragments or variants, the active fragments or variants retaining the ability to bind to a target sequence in an RNA-guided sequence-specific manner and containing a T at position corresponding to amino acid position 1021 in SEQ ID NO: 584 and an N at position corresponding to amino acid position 1023 in SEQ ID NO: 584.

[0265] The polynucleotides disclosed in this invention also include polynucleotides encoding RGN having the amino acid sequence shown in SEQ ID NO: 583 and their active fragments or variants, the active fragments or variants retaining the ability to bind to a target sequence in an RNA-guided sequence-specific manner and containing N at position 1021 corresponding to amino acid position 1021 in SEQ ID NO: 583 and N at position 1023 corresponding to amino acid position 1023 in SEQ ID NO: 583.

[0266] The polynucleotides disclosed in this invention also include polynucleotides encoding an RGN having the amino acid sequence shown in SEQ ID NO: 591 and its active fragments or variants, the active fragments or variants retaining the ability to bind to a target sequence in an RNA-guided sequence-specific manner and comprising: a) Q at position 975 corresponding to amino acid position 975 in SEQ ID NO: 591; b) A at position 1020 corresponding to amino acid position 1020 in SEQ ID NO: 591; c) G at position 1022 corresponding to amino acid position 1022 in SEQ ID NO: 591; and d) Q at position 1023 corresponding to amino acid position 1023 in SEQ ID NO: 591.

[0267] This document also provides polynucleotides encoding a polymerase comprising a polymerase operatively linked to an RGN, wherein the RGN is LG10221, LPG10209, LPG10210, LPG10212, LPG10213, APG07433.1, APG05586, APG01604, or LPG10145, or an active variant or fragment thereof. The compositions and methods disclosed in this invention may also comprise polynucleotides encoding polymerases (e.g., reverse transcriptases), wherein they are provided in a trans configuration with the polynucleotide encoding the RGN polypeptide.

[0268] The use of the terms "polynucleotide" or "nucleic acid molecule" is not intended to limit this disclosure to polynucleotides containing DNA. Those skilled in the art will recognize that polynucleotides can comprise ribonucleotides (RNA) and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogs. These include peptide nucleic acids (PNA), PNA-DNA chimeras, locked nucleic acids (LNA), and phosphothioate linker sequences. The polynucleotides disclosed herein also encompass all forms of sequences, including (but not limited to) single-stranded, double-stranded, DNA-RNA hybrids, triple-helix structures, stem-loop structures, circular RNA (circRNA), and similar forms.

[0269] In some embodiments of the compositions and methods disclosed in this invention that comprise a polynucleotide encoding an RGN, polymerase, or PE, the polynucleotide is an RNA polynucleotide (e.g., an mRNA (messenger RNA) molecule). mRNA refers to any polynucleotide that encodes a polypeptide of interest and is capable of being translated to produce the encoded polypeptide of interest in vitro, in vivo, in situ, or ex vivo. In some embodiments, the basic components of the mRNA molecule include at least a coding region, a 5' untranslated region (UTR), a 3' UTR, a 5' cap, and a poly-A tail. The 5' UTR, located at the 5' of the coding sequence and transcribed as part of the mRNA, may contain various regulatory components, including, for example, a 5' cap structure, a G-quadruplex structure (G4), a stem-loop structure, and an internal ribosome entry site (IRES), which control the initiation of mRNA translation. The 3' UTR, located at the 3' of the coding sequence and transcribed as part of the mRNA, may be involved in numerous regulatory processes, including transcript cleavage, stability and polyadenylation, translation, and mRNA localization. The 3' UTR can serve as a binding site for many regulatory proteins and small non-coding RNAs (such as microRNAs). 5' UTRs and / or 3' UTRs that are heterologous to mRNA originate from an organism or species different from the one from which the mRNA originated, or, if they originate from the same organism or species as the mRNA, are substantially modified from their native form at the constituent and / or genomic loci through intentional artificial intervention.

[0270] In some embodiments, including a heterologous 5' UTR and / or 3' UTR to the mRNA encoding the disclosed RGN peptide, polymerase, or PE can improve polypeptide synthesis from mRNA in tissues (e.g., liver or in vitro cells such as stem cells, hepatocytes, or lymphocytes). The heterologous 5' UTR and / or 3' UTR can increase protein synthesis, for example, by increasing the retention time of mRNA in translational polyribosomes (information stability) and / or the rate of ribosome-initiated mRNA translation (information translation efficiency). Therefore, including a heterologous 5' UTR and / or 3' UTR to the mRNA encoding the disclosed RGN peptide, polymerase, or PE can prolong and / or increase polypeptide synthesis, enabling improved cleavage or modification of the target polynucleotide by the RGN peptide or PE or PE system. In some embodiments, the enhanced polypeptide synthesis from mRNA is performed in a tissue-specific manner. Heterologous UTR sequences are described, for example, in US 2023 / 0050143 and US 2017 / 0252461.

[0271] In some embodiments, the mRNA encoding the RGN, polymerase, or PE suitable for use in the methods and compositions disclosed in this invention may include one or more structural and / or chemical modifications or alterations that impart suitable properties to the polynucleotide. For example, suitable properties of the mRNA include a lack of substantial induction of an innate immune response in the cell to which the mRNA is introduced. A “structural” feature or modification is a feature or modification in which two or more linking nucleotides are inserted, deleted, duplicated, inverted, or randomized in the mRNA without significant chemical modification to the nucleotides themselves. Because structural modifications necessarily require the breaking and reforming of chemical bonds, structural modifications are chemical in nature and are therefore chemical modifications. However, structural modifications will result in different nucleotide sequences. Chemical modifications to mRNA may involve 5-methylcytosine, N1-methyl-pseuuridine, pseudouridine, 2-thiouridine, 4-thiouridine, 5-methoxyuridine, 2'-fluoroguanidine, 2'-fluorouridine, 5-bromouridine, 5-(2-methoxycarbonylvinyl)uridine, 5-[3(1-E-propenylamino)]uridine, α-thiocytidine, N6-methyladenosine, 5-methylcytidine, N4-acetylcytidine, 5-formylcytidine, or combinations thereof.

[0272] Polynucleotides encoding RGN, polymerase, PE, and / or guide RNA can be codon-optimized for expression in organisms of interest. "Codon-optimized" sequences are polynucleotide sequences whose codon usage frequencies are designed to mimic the frequencies of optimal codon usage or transcriptional conditions in a specific host cell. Expression in a specific host cell or organism is enhanced by one or more codon changes at the nucleic acid level, without altering the translated amino acid sequence. Polynucleotides can be codon-optimized, either fully or partially. This technique provides codon tables and other references that offer information on preferences for various organisms (for discussions of plant-preferred codon usage, see, for example, Campbell and Gowri (1990), *American Journal of Plant Physiology*). Plant Physiol. (92:1-11). This technique provides a method for synthesizing plant-preferred genes or codon-optimized coding sequences of mammals (e.g., humans). See, for example, U.S. Patent Nos. 5,380,831 and 5,436,391, and Murray et al. (1989), Nucleic Acid Research (Nucleic Acids Research). Nucleic Acids Res. (17:477-498) is incorporated herein by reference.

[0273] Polynucleotides encoding the RGN, polymerase, PE, and / or gRNA provided herein may be provided in an expression cassette for expression in vitro or in cells, organelles, embryos, or organisms of interest. The cassette will include 5' and 3' regulatory sequences operatively linked to the polynucleotide encoding the RGN, polymerase, PE, and / or gRNA provided herein, allowing expression of the polynucleotide. The cassette may additionally contain at least one additional gene or genetic component to be co-transformed into an organism. Where additional genes or components are included, these components are operatively linked. The term "operatively linked" is intended to refer to a functional link between two or more components. For example, an operative link between a promoter and a coding region of interest (e.g., a region encoding RGN and / or gRNA) is a functional link that allows expression of the coding region of interest. Operablely linked components may be connected or unconnected. When used to refer to the conjugation (by fusion or insertion) of two protein-coding regions, "operatively linked" or "operatively fused" means that these coding regions are in the same reading frame, even if one coding region is inserted into another. In some embodiments, a "operably fused" or "operably linked" polypeptide means that the structure and / or biological activity of the individual peptides are also present in the fusion. Alternatively, additional genes or components may be provided on multiple expression cassettes. For example, the nucleotide sequence encoding the RGN, polymerase, or PE disclosed in this invention may be present on one expression cassette, while the nucleotide sequence encoding the guide RNA may be on another expression cassette. Such expression cassettes have multiple restriction sites and / or recombination sites to allow the insertion of polynucleotides under transcriptional regulation in the regulatory region. The expression cassette may additionally contain optional marker genes.

[0274] The expression cassette will include, in the 5'-3' direction of transcription, a transcription (and in some embodiments, translation) initiation region (i.e., promoter), a polynucleotide encoding the RGN, polymerase, PE, and / or gRNA of the present invention, and a transcription (and in some embodiments, translation) termination region (i.e., termination region) functional in the cell or organism of interest. The promoter of this disclosure is capable of directing or driving the expression of the coding sequence in a host cell. Regulatory regions (e.g., promoters, transcription regulatory regions, and translation termination regions) may be endogenous or heterologous to the host cell or to each other. As used herein, “heterogeneous” when referring to a sequence means a sequence derived from a foreign species, or, if derived from the same species, substantially modified from its native form at the constituent and / or genomic loci through intentional artificial intervention. As used herein, chimeric genes comprise coding sequences operatively linked to a transcription initiation region heterologous to the coding sequence.

[0275] Appropriate termination regions include those derived from simian virus (SV40), human growth hormone (hGH), bovine growth hormone (BGH), and rabbit β-globulin (rbGlob). See also Proudfoot (1991), *Cell*. Cell )》 64:671-674; Munroe et al. (1990) "Genes ( Gene )》91:151-158; Schek et al. (1992) Molecular and Cell Biology ( Molecular and Cellular Biology )》12(12):5386-5393; Gil and Proudfoot (1987) "Cell ( Cell )》49(3):399-406; Goodwin and Rottman (1992) Journal of Biochemistry ( The Journal of Biological Chemistry )》267(23):16330-16334; and Lanoix and Acheson (1988) Journal of the European Society for Molecular Biology ( EMBO J. )》7(8):2515-2522. Suitable termination areas can be obtained from Agrobacterium tumefaciens ( A.tumefaciens Ti-plasts, such as the termination regions of octopine synthase and carmine synthase, are also mentioned. See also Guerineau et al. (1991), Molecular Genetics and Genomics (Molecular Genetics and Genomics). Mol.Gen. Genet. )》262:141-144; Proudfoot (1991) "Cell ( Cell )》64:671-674; Sanfacon et al. (1991) "Genes and Development" Genes Dev. )》5:141-149; Mogen et al. (1990) "Plant Cells ( Plant Cell )》2:1261-1272; Munroe et al. (1990) "Genes ( Gene )》91:151-158; Ballas et al. (1989) "Nucleic Acid Research" Nucleic Acids Res. )》17:7891-7903; and Joshi et al. (1987) "Nucleic Acid Research" Nucleic Acids Res. )》15:9627-9639.

[0276] Additional regulatory signals include (but are not limited to) transcription initiation sites, operons, activators, enhancers, other regulatory components, ribosome binding sites, start codons, termination signals, and analogues. See, for example, U.S. Patents 5,039,523 and 4,853,331; EPO 0480762A2; Sambrook et al. (1992), *Molecular Cloning: A Laboratory Manual*, edited by Maniatis et al. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), hereinafter “Sambrook 11”; Davis et al. (1980), *Advanced Bacterial Genetics* (Cold Spring Harbor Laboratory Press), Cold Spring Harbor, NY, and the references cited therein.

[0277] During expression cassette preparation, various DNA fragments can be manipulated to provide the DNA sequence in the appropriate reading frame at the appropriate orientation and time. For this purpose, adaptors or linkers can be used to attach the DNA fragments, or other manipulations may be involved to provide facilitated restriction sites, remove redundant DNA, remove restriction sites, or the like. For this purpose, in vitro mutation induction, primer repair, restriction, annealing, and substitution, such as transitions and transversions, may be involved.

[0278] Multiple promoters can be used in practice with this disclosure. Promoters can be selected based on desired results. Nucleic acids can be combined with constitutive, inducible, growth phase-specific, cell type-specific, tissue-biased, tissue-specific activators, or other promoters for expression in organisms of interest. See, for example, the promoters described below: WO 99 / 43838 and U.S. Patent Nos. 8,575,425; 7,790,846; 8,147,856; 8,586,832; 7,772,369; 7,534,939; 6,072,050; 5,659,026; 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611, which are incorporated herein by reference.

[0279] Exemplary constitutive promoters for expression in cells according to this disclosure include: the SV40 early promoter; the mouse mammary tumor virus long terminal repeat (LTR) promoter; the adenovirus major late promoter (Ad MLP); the herpes simplex virus (HSV) promoter; cytomegalovirus (CMV) promoters, such as the CMV immediate early promoter region (CMVIE); Raoult's sarcoma virus (RSV) promoter; the human ubiquitin C promoter (UBC); the human U6 small nucleus promoter (U6); the enhanced U6 promoter; the human H1 promoter (H1) derived from RNA polymerase III; the human elongation factor 1 α promoter (EF1A); the human β-actin promoter (ACTB); the human or mouse phosphoglycerate kinase 1 promoter (PGK); the chicken β-actin promoter (CAGG) in conjunction with the CMV early enhancer; the yeast transcription elongation factor promoter (TEF1); and analogues thereof. See, for example, Miyagishi et al. (2002), Nature Biotechnology (… Nature Biotechnology )》20:497-500; Xia et al. (2003) Nucleic Acids Res. 31(17):e100-e100; Pasleau et al. (1985) Gene ( Gene )》38:227-232; Martin-Gallardo et al. (1988) "Genes ( Gene )》70: 51-56; Oellig and Seliger (1990) Journal of Neuroscience Research ( J Neurosci Res )》26:390-396; Manthorpe et al. (1993) "Human Gene Therapy ( Hum Gene Ther )》4:419-431; Yew et al. (1997) Human Gene Therapy ( Hum Gene Ther )》8:575-584; Xu et al. (2001) "Genes ( Gene )》272:149-156; Nguyen et al. (2008) Journal of Surgical Research ( J Surg Res )》148:60-66; Costa et al. (2005) "Natural Methods ( Nat Meth. )》2:259-260; Lam and Truong (2020)《ACS Synthetic Biology》2:259-260; ACS Synth.Biol. )》9(10):2625-2631.

[0280] For expression in plants, constitutive promoters also include the CaMV 35S promoter (Odell et al. (1985), Nature). Nature)》313:810-812); Rice actin (McElroy et al. (1990)《Plant Cell ...) Plant Cell )》2:163-171); ubiquitin (Christensen et al. (1989) "Plant Molecular Biology" Plant Mol.Biol. )》12:619-632 and Christensen et al. (1992) Plant Molecular Biology ( Plant Mol.Biol. )》18:675-689), pEMU (Last et al. (1991) "Theoretical and Applied Genetics ( Theor.Appl.Genet. )》81:581-588); and MAS (Velten et al. (1984) "Proceedings of the European Society for Molecular Biology" ( EMBO J. )》3:2723-2730).

[0281] Examples of inducible promoters include stress-regulated promoters such as the Adh1 promoter, Hsp70 promoter, and Hsp90 promoter (Wurm et al. (1986), Proceedings of the National Academy of Sciences of the United States of America). Proc. Natl. Acad. Sci. USA. ()》83:5414-5418; Nover L. "Heat Shock Response ( Heat Shock Response )》. CRC Press; BocaRaton, FL, USA: 1991); photoinducible promoters, such as the PPDK promoter and the phosphoenolpyruvate carboxylase promoter; metal-regulated promoters (Mayo et al. (1982) "Cell"). Cell )》. 29:99-108; Searle et al. (1985) Molecular and Cellular Biology ( Mol. Cell. Biol. )》 5:1480-1489); Hormone-responsive promoters, including glucocorticoid-responsive promoters (Hynes et al. (1981) Proceedings of the National Academy of Sciences of the United States of America (PNAS) Proc. Natl. Acad. Sci. USA. )》78:2038-2042; Klock et al. (1987) Nature ( Nature )》. 329:734-736). Chemically regulated promoters include: the safener-induced In2-2 promoter (US Patent No. 5,364,780); the auxin-induced, tapetum-specific, and callus-active Axig1 activator (PCT US01 / 22169); steroid-responsive promoters (see, for example, the estrogen-induced ERE promoter and glucocorticoid-induced promoters below: Schena et al. (1991), Proceedings of the National Academy of Sciences of the United States of America (PNAS) Proc.Natl.Acad.Sci.USA )》88:10421-10425 and McNellis et al. (1998) "Plant Journal" ( Plant J.)》14(2):247-257); Tetracycline-inducible and tetracycline-repressive promoters (see, for example, Gatz et al. (1991) Molecular Genetics and Genomics ( Mol.Gen. Genet. )》227:229-237; Gossen et al. (1993) "Trends in Biochemical Sciences" Trends Biochem Sci. )》18:471-475; Gossen and Bujard (1992) Proceedings of the National Academy of Sciences of the United States of America (PNAS) Proc. Natl Acad. Sci. USA )》89:5547-5551; Zhou et al. (2006) "Gene Therapy ( Gene Ther. (13:1382-1390; and US Patent Nos. 5,814,618 and 5,789,156); promoters regulated by isopropyl-β-D-thiopiperanol galactoside (IPTG); and promoters regulated by lactose. Inducible expression can be achieved using operon systems including AlcR / acetaldehyde, ArgR / L-arginine, BirA / biotinyl-AMP, CymR / cumate, EthR / 2-phenylethyl butyrate, HdnoR / 6-hydroxynicotinic acid, HucR / uric acid, MphR(A) / macrolide, PIP / streptocin, Rex / NADH, RheA / thermolysin, ScbR / SCB1, TraR / 3-side-oxy-C8-HSL, and TtgR / phlorizin; see, for example, U.S. Patent No. 8,728,759B2; U.S. Patent No. 7,745,592B2; Weber and Fussenegger (2004), Methods in Molecular Biology (… Methods Mol. Biol. )》267:451-466; Hartenbach et al. (2007) Nucleic Acid Research ( Nucleic Acids Res. )》35:e136; Weber et al. (2009) Metabolic Engineering ( Metab. Eng. )》11:117-124; Weber et al. (2008) Proceedings of the National Academy of Sciences of the United States of America (PNAS) Proc. Natl. Acad. Sci. USA. Nucleic Acid Research (Nucleic Acids Research) 105:9994-9998; Malphettes et al. (2005) Nucleic Acids Res. )》33:e107; Kemmer et al. (2010) Nature Biotechnology ( Nat. Biotechnol. )》28:355-360; Weber et al. (2002) Nature Biotechnology ( Nat. Biotechnol. )》20:901-907; Fussenegger et al. (2000) Nature Biotechnology ( Nat. Biotechnol. )》18:1203-1208; Weber et al. (2006) Metabolic Engineering ( Metab. Eng. )》8:273-280; Weber et al. (2003) "Nucleic Acid Research" Nucleic Acids Res. )》31:e69; Weber et al. (2003) "Nucleic Acid Research" Nucleic Acids Res. )》31:e71; Neddermann et al. (2003) Report of the European Society for Molecular Biology ( EMBO Rep. ) 4:159-165; and Gitzinger et al. (2009) Proceedings of the National Academy of Sciences of the United States of America (PNAS) Proc. Natl. Acad. Sci. USA. )》106:10638-10643. Inducible expression can be obtained using protein-protein interaction systems, including: rapamycin-induced interaction between FKBP12 (FK506-binding protein 12) and mTOR (Rivera et al. (1996) Nature Medicine (106:10638-10643). Nat. Med. )》2:1028-1032; Belshaw et al. (1996) Proceedings of the National Academy of Sciences of the United States of America (PNAS) Proc. Natl. Acad. Sci. USA. )》93:4604-46077); The interaction between PYL1 (abscisic acid receptor) and ABI1 (protein phosphatase 2C56) regulated by abscisic acid (ABA) (Liang et al. (2011) Science Signals ( Sci. Signal. )》4 (164): rs2-rs2); and photoinduced protein-protein interaction systems (Wang et al. (2012) Nature Methods ( Nat. Methods. )》9:266-269; Yamada et al. (2018) Cell Reports ( Cell. Rep. )》25:487-500).

[0282] Tissue-specific or tissue-preferred promoters can be used to target the expression of constructs within specific tissues. In some embodiments, tissue-specific or tissue-preferred activators are active in mammalian tissues. Examples of tissue-specific or tissue-preferred promoters include promoters that preferentially initiate transcription in certain tissues, such as leukocytes (e.g., CD4 T cells), heart, kidney, liver, CNS, eye, pancreas, skeletal muscle, and testis. In some embodiments, tissue-specific or tissue-preferred activators are active in plant tissues. Examples of promoters under developmental control in plants include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, fruits, seeds, or flowers. A “tissue-specific” activator is an activator that initiates transcription only in certain tissues. Unlike constitutive gene expression, tissue-specific expression is the result of several levels of interaction in gene regulation. Therefore, promoters from homologous or closely related species are preferred for achieving efficient and reliable expression of transgenes in specific tissues. In some embodiments, expression includes tissue-preferred promoters. "Tissue-preferred" activators are activators that preferentially initiate transcription, but not necessarily completely or only in certain tissues.

[0283] In some implementations, the polynucleotide encoding RGN, polymerase PE, and / or gRNA contains a cell-type-specific promoter. A “cell-type-specific” promoter is a promoter that primarily drives the expression of certain cell types in one or more organs. Examples of cell-type-specific promoters that primarily target active cells include, for example, primary cells, neurons, glial cells, adipocytes, cardiomyocytes, smooth muscle cells, photoreceptor cells, and retinal ganglion cells. Functional cell-type-specific promoters in plants may primarily target active plant cells, including, for example, BETL cells, vascular cells in roots, leaves, and stem cells, and stem cells. The polynucleotide may also include cell-type-preferred promoters. A “cell-type-preferred” promoter is a promoter that primarily, but not necessarily completely or only drives expression in certain cell types in one or more organs. Examples of cell-type-preferred promoters that preferentially target active cells include, for example, primary cells, neurons, adipocytes, cardiomyocytes, smooth muscle cells, and photoreceptor cells. In plants, some examples of functional cell type-preferred promoters that preferentially activate plant cells include, for example, vascular cells in BETL cells, roots, leaves, stem cells, and stem cells.

[0284] Nucleic acid sequences encoding RGN, polymerase, PE, and / or gRNA are operatively linked to a promoter sequence recognized by a bacteriophage RNA polymerase, for example, for in vitro mRNA synthesis. In such embodiments, the in vitro transcribed RNA can be purified for use in the methods described herein. For example, the promoter sequence can be a T7, T3, or SP6 promoter sequence or a variant of the T7, T3, or SP6 promoter sequence. In such embodiments, the expressed protein and / or RNA can be purified for use in the genome modification methods described herein.

[0285] In some embodiments, the polynucleotide encoding RGN, polymerase, PE, and / or gRNA may be linked to a polyadenylation signal (e.g., SV40 polyA signal and other signals functional in plants) and / or at least one transcription termination sequence. Alternatively, the sequence encoding RGN, polymerase, or PE may be linked to a sequence encoding at least one nuclear localization signal, at least one cell-penetrating domain, and / or at least one signal peptide capable of transporting the protein to a specific subcellular location, as described elsewhere herein.

[0286] Polynucleotides encoding RGN, polymerase, PE, and / or gRNA may be present in one or more vectors. A “vector” refers to a polynucleotide composition used to transfer, deliver, or introduce nucleic acids into a host cell. Suitable vectors include plasmid vectors, phage particles, mucinous plasmids, artificial / miniature chromosomes, transposons, and viral vectors (e.g., lentiviral vectors, adeno-associated virus vectors, baculovirus vectors). Vectors may contain additional expression control sequences (e.g., enhancer sequences, Kozak sequences, polyadenylation sequences, transcription termination sequences), optional marker sequences (e.g., antibiotic resistance genes), origin of replication, and similar sequences. Additional information can be found in *Current Protocols in Molecular Biology*, Ausubel et al., John Wiley & Sons, New York, 2003, or *Molecular Cloning: A Laboratory Manual*, Sambrook and Russell, Cold Spring Harbor Press, Cold Spring Harbor, NY, 3rd edition, 2001.

[0287] The vector may also contain optional marker genes for selecting transformed cells. Optional marker genes are used to select transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), and genes conferring resistance to herbicides such as glufosinate ammonium, bromoxynil, imidazolinone, and 2,4-dichlorophenoxyacetate (2,4-D). Marker genes may include genes that allow selection of growth on specific nutrients or substances, such as dihydrofolate reductase (DHFR); Simonsen and Levinson (1983), Proceedings of the National Academy of Sciences (PNAS). Proc.Natl.Acad.Sci.U.S.A. )》 80:2495-2499), histamine dehydrogenase (hisD; Hartman and Mulligan (1988) Proceedings of the National Academy of Sciences of the United States of America (PNAS) Proc.Natl.Acad.Sci.U.S.A. )》 85:8047-8051), puromycin-N-acetyltransferase (PAC or puro; de la Luna et al. (1988) "Gene ( Gene )》62:121-126), Thymidine kinase (TK; Littlefield (1964) Science ( Science )》145:709-710) and xanthine-guanine phosphoribosyltransferase (XGPRT or gpt; Mulligan and Berg (1981) Proceedings of the National Academy of Sciences of the United States of America (PNAS) Proc.Natl.Acad.Sci.U.S.A. )》78:2072-2076).

[0288] Expression cassettes or vectors containing polynucleotides encoding RGN polypeptides, polymerases, and / or PEs may further contain polynucleotides encoding gRNAs. The polynucleotide sequence encoding the gRNA may be operatively linked to at least one transcriptional control sequence for expressing the gRNA in an organism of interest or host cell. For example, the polynucleotide encoding the gRNA may be operatively linked to a promoter sequence recognized by RNA polymerase III (Pol III). Examples of suitable Pol III promoters include, but are not limited to, mammalian U6, U3, H1, and 7SL RNA promoters and rice U6 and U3 promoters, such as the promoter shown in SEQ ID NO:609, and the promoter disclosed in International Application Publication No. WO 2022 / 261394, which is incorporated herein by reference in its entirety.

[0289] As indicated, expression constructs comprising nucleotide sequences encoding RGN, polymerase, PE, and / or gRNA can be used to transform organisms of interest. Transformation methods involve introducing the nucleotide construct into the organism of interest. "Introduction" is defined as introducing the nucleotide construct into a host cell in a manner that allows the construct to enter the host cell. The methods of the present invention do not require a specific method for introducing the nucleotide construct into the host organism, only that the nucleotide construct can enter the interior of at least one cell of the host organism. The host cell can be a eukaryotic or prokaryotic cell. In some embodiments, the eukaryotic host cell is a plant cell, mammalian cell, avian cell, or insect cell. In some embodiments, the eukaryotic cell comprising or expressing the RGN, polymerase, PE, and / or gRNA disclosed herein is a human cell. In some embodiments, the eukaryotic cell comprising or expressing the RGN, polymerase, PE, and / or gRNA disclosed herein is a primary cell. The term "primary cell" refers to a cell directly isolated from a multicellular organism. Primary cells typically undergo minimal population multiplication and are therefore more representative of the major functional components of their derived tissues compared to continuous (tumor or artificially immortalized) cell lines. In some cases, primary cells are isolated cells that are then used immediately. In other cases, primary cells cannot divide indefinitely and therefore cannot be cultured in vitro for extended periods. In some embodiments, the primary cells are primary T cells. In some embodiments, eukaryotic cells containing or expressing the RGN, polymerase, PE, and / or gRNA disclosed herein are hematopoietic cells, such as immune cells (i.e., cells of the innate or adaptive immune system), including (but not limited to) B cells, T cells, natural killer (NK) cells, chimeric antigen receptor T (CAR-T) cells, monocytes, macrophages, and dendritic cells. In some embodiments, the eukaryotic cells are pluripotent stem cells or induced pluripotent stem cells. In some embodiments, the eukaryotic cells containing or expressing the RGN, polymerase, PE and / or gRNA disclosed herein are eye cells, muscle cells (e.g., skeletal muscle cells), epithelial cells (e.g., lung epithelial cells) or diseased cells (e.g., tumor cells).

[0290] Methods for introducing nucleotide constructs into plant and host cells are known in this technology, including (but not limited to) stable transformation methods, transient transformation methods, and virus-mediated methods.

[0291] These methods produce transformed organisms, such as plants, including whole plants and plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, propagules, embryos, and their offspring. Plant cells may be differentiated or undifferentiated (e.g., callus, suspension culture cells, protoplasts, leaf cells, root cells, phloem cells, pollen). In some embodiments, the methods disclosed in this invention can produce transformed organisms or cell lines derived from these transformed cells.

[0292] "Transgenic organism," "transformed organism," or "stable transformed" organism, cell, or tissue refers to an organism incorporating or integrating polynucleotides encoding the RGN, polymerase, PE, and / or gRNA disclosed herein. It should be recognized that other exogenous or endogenous nucleic acid sequences or DNA fragments may also be incorporated into host cells. Agrobacterium ( Agrobacterium Transformation mediated by gene gun and other methods remains the two main approaches used for plant cell transformation. However, host cell transformation can be achieved through infection, transfection, microinjection, electroporation, microprojection, gene gun or particle bombardment, electroporation, silica / carbon fiber, ultrasound-mediated transformation, PEG-mediated transformation, calcium phosphate coprecipitation, polycationic DMSO technology, DEAE polydextrose program, and virus-mediated, liposome-mediated transformation and similar methods. Virus-mediated introduction of polynucleotides encoding RGN, polymerase, PE, and / or gRNA includes introduction and expression mediated by retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses, as well as the use of caulimoviruses (e.g., cauliflower mosaic virus), geminiviruses (e.g., soybean golden mosaic virus or maize stripe virus), and RNA plant viruses (e.g., tobacco mosaic virus).

[0293] Transformation protocols and protocols for introducing polypeptide or polynucleotide sequences into plants vary depending on the type of host cell targeted for transformation (e.g., monocotyledonous or dicotyledonous cells). Transformation methods are known in this art and include those described in the following U.S. patents: No. 8,575,425; No. 7,692,068; No. 8,802,934; No. 7,541,517; each of which is incorporated herein by reference. See also Rakoczy-Trojanowska, M. (2002) Cell & Molecular Biology Letters (… Cell Mol Biol Lett. )》7:849-858; Jones et al. (2005) "Plant Methods ( Plant Methods )》1:5; Rivera et al. (2012) Biophysical Review ( Physics of Life Reviews )》9:308-345; Bartlett et al. (2008) "Plant Methods ( Plant Methods )》4:1-12; Bates, GW (1999) "Methods of Molecular Biology" Methods in Molecular Biology )》 111:359-366; Binns and Thomashow (1988) "Annals of Microbiology" Annual Reviews in Microbiology )》 42:575-606; Christou, P. (1992) "Plant Journal" ( The Plant Journal )》2:275-281; ​​Christou, P. (1995) "Green Algae ( Euphytica )》 85:13-27; Tzfira et al. (2004) "Trends in Genetics" TRENDS in Genetics )》20:375-383; Yao et al. (2006) Journal of Experimental Botany ( Journal of Experimental Botany )》57:3737-3746; Zupan and Zambryski (1995) American Journal of Plant Physiology ( Plant Physiology )》107:1041-1047; Jones et al. (2005) "Plant Methods ( Plant Methods )》1:5.

[0294] Transformation can result in either stable or transient integration of nucleic acids into the cell. "Stable transformation" refers to the integration of the introduced nucleotide construct into the host cell's genome and its heritability by its progeny. "Temporary transformation" refers to the introduction of a polynucleotide into the host cell without integration into its genome.

[0295] Methods for chloroplast transformation are known in this technique. See, for example, Svab et al. (1990), Proceedings of the National Academy of Sciences (PNAS). Proc.Nail.Acad.Sci.USA )》87:8526-8530; Svab and Maliga (1993) Proceedings of the National Academy of Sciences of the United States of America (PNAS) Proc.Natl.Acad.Sci.USA )》90:913-917; Svab and Maliga (1993) Journal of the European Society for Molecular Biology ( EMBO J. This method relies on particle gun delivery of DNA containing optional markers and targeting of the DNA to the plastid genome via homologous recombination. Additionally, plastid transformation can be achieved by tissue-preferred expression of nuclear-encoded and plastid-directed RNA polymerases, leading to transactivation of silent plastid-borne transgenes. Such systems have been reported in McBride et al. (1994) in the *Proceedings of the National Academy of Sciences* (PNAS). Proc.Natl.Acad.Sci.USA )》91:7301-7305.

[0296] Transformed cells can be grown into transgenic organisms such as plants in a well-known manner. See, for example, McCormick et al. (1986), Plant Cell Reports (…). Plant Cell Reports (See 5:81-84). These plants can then be grown and pollinated with the same or different transformed varieties, and hybrids with constitutive expression of the desired phenotypic characteristics can be identified. Two or more generations can be grown to ensure the stable maintenance and inheritance of the polynucleotide encoding RGN, polymerase, PE, and / or gRNA, followed by seed collection to ensure the presence of the polynucleotide encoding RGN, polymerase, PE, and / or gRNA. In this way, this disclosure provides a transformed plant or plant part into which the nucleotide constructs of this disclosure, such as the expression cassette of this disclosure, are stably incorporated into the genome. Seeds into which the expression cassette of this disclosure is stably incorporated into the genome may be called "transgenic seeds".

[0297] Alternatively, the transformed cells can be introduced into an organism. These cells can be derived from an organism in which the cells were transformed in vitro. These cells can be autologous (derived from and returned to the same subject) or allogeneic (the donor and recipient are of the same species).

[0298] The sequences provided herein can be used for transformation of any plant species, including (but not limited to) monocots and dicots. Examples of plants of interest include (but are not limited to) corn, sorghum, wheat, sunflower, tomato, cruciferous plants, pepper, potato, cotton, rice, soybean, sugar beet, sugarcane, tobacco, barley and rapeseed, Brassica sp., alfalfa, rye, chestnut, safflower, peanut, sweet potato, cassava, coffee, coconut, pineapple, citrus, cocoa, tea, banana, avocado, fig, guava, mango, olive, papaya, cashew, macadamia nut, almond, oat, vegetables, ornamental plants, and conifers.

[0299] Vegetables include (but are not limited to) tomatoes, lettuce, mung beans, lima beans, peas, and members of the genus *Cucumis*, such as cucumbers, cantaloupes, and honeydew melons. Ornamental plants include (but are not limited to) azaleas, hydrangeas, hibiscus, roses, tulips, daffodils, petunias, carnations, poinsettias, and chrysanthemums. In specific embodiments, the plants of this invention are crop plants (e.g., corn, sorghum, wheat, sunflowers, tomatoes, cruciferous plants, peppers, potatoes, cotton, rice, soybeans, sugar beets, sugarcane, tobacco, barley, rapeseed, etc.).

[0300] As used herein, the term plant includes plant cells, plant protoplasts, plant cell tissue cultures from which plants can regenerate, plant callus, plant clusters, and intact plant cells in a plant or plant part, such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, kernels, spikes, rachis, bracts, pedicels, roots, root tips, pollen sacs, and the like. For purposes other than growth or regeneration of species, grain is intended to refer to mature seeds produced by commercial growers. Subsequent generations, variants, and mutants of regenerated plants are also included within the scope of this invention, limited by the fact that these parts contain the introduced polynucleotides. Processed plant products or byproducts retaining the sequences disclosed herein are also provided, including, for example, soybean flour.

[0301] Polynucleotides encoding RGN, polymerase, PE, and / or gRNA, or containing gRNA, can be used to transform any eukaryotic species, including (but not limited to) animals (e.g., mammals, humans, insects, fish, birds, and reptiles), plants, fungi, amoebas, algae, and yeast. In some embodiments, polynucleotides encoding RGN, polymerase, PE, and / or gRNA, or containing gRNA, can be used to transform any prokaryotic species, including (but not limited to) archaea and bacteria (e.g., Bacillus). Bacillus sp.), Klebsiella spp. Klebsiella sp.), Streptomyces ( Streptomyces sp.), Rhizobium (spe.), Rhizobium genus ( Rhizobium sp.), Escherichia coli ( Escherichia sp.), Pseudomonas spp. Pseudomonas sp.), Salmonella (spe.), Salmonella spp. Salmonella sp.), Shigella spp. Shigella sp.), Vibrio genus ( Vibrio sp.), Yersinia spp. Yersinia sp.), Mycoplasma genus ( Mycoplasma sp.), Agrobacterium, Lactobacillus (sp ... Lactobacillus sp.)).

[0302] Well-known viral and nonviral gene transfer methods can be used to introduce nucleic acids into mammalian, insect, or avian cells or target tissues. These methods can be used to administer nucleic acids encoding components of the RGN system to cells in culture or in a host organism. Nonviral vector delivery systems include DNA plasmids, RNA (such as transcripts of vectors described herein), naked nucleic acids, and nucleic acids complexed with a delivery medium such as liposomes. Viral vector delivery systems include DNA and RNA viruses that have a free or integrated genome after delivery to cells. Non-limiting examples include vectors utilizing cauliflower mosaic viruses (e.g., cauliflower mosaic virus), geminiviruses (e.g., bean golden mosaic virus or maize stripe virus), and RNA plant viruses (e.g., tobacco mosaic virus). For reviews of gene therapy procedures, see Anderson, Science 256:808-813 (1992); Nabel and Feigner, Trends in Biotechnology 11:211-217 (1993); Mitani and Caskey, Trends in Biotechnology 11:162-166 (1993); Dillon, Trends in Biotechnology 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10):1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer and Perricaudet, British Medical Bulletin 51(1):31-44 (1995); Haddadada et al., Current Topics in Microbiology and Immunology, Doerfler and Bohm (eds.) (1995); and Yu et al., Gene Therapy 1:13-26 (1994).

[0303] Non-viral methods for delivering nucleic acids include liposome transfection, nuclear transfection, microinjection, gene gun, virions, liposomes, immunoliposomes, polycationic or lipid:nucleic acid conjugates, naked DNA, artificial viral particles, and drug-enhanced DNA uptake. Liposome transfection is described, for example, in U.S. Patents 5,049,386, 4,946,787, and 4,897,355, and the liposome transfection reagents are commercially available (e.g., Transfectam). TM and Lipofectin TM Cationic and neutral lipids suitable for receptor-recognition liposome transfection with polynucleotides include those listed in Feigner, WO 91 / 17424; WO 91 / 16024. They can be delivered to cells (e.g., in vitro or ex vivo administration) or to target tissues (e.g., in vivo administration). The preparation of lipid:nucleic acid complexes (including targeted liposomes, such as immunolipid complexes) is well known to those skilled in the art (see, for example, Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Therapy 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Research... Res. 52:4817-4820 (1992); U.S. Patent Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028 and 4,946,787.

[0304] The delivery of nucleic acids using RNA or DNA virus-based systems leverages highly evolved processes to target viruses to specific cells in vivo and to deliver viral payloads to the cell nucleus. Viral vectors can be administered directly to patients (in vivo) or used in cells for in vitro therapy, and modified cells can be administered to patients as appropriate (ex vivo). Well-known virus-based systems include retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, and herpes simplex virus vectors for gene transfer. Using retroviruses, lentiviruses, and adeno-associated viruses for gene transfer, integration into the host genome is possible, typically resulting in long-term expression of the inserted transgene. Furthermore, high transduction efficiency has been observed in many different cell types and target tissues.

[0305] Retroviral tropism can be altered by incorporating foreign envelope proteins and amplifying potential target populations of cells. Lentiviral vectors are retroviral vectors capable of transducing or infecting non-dividing cells and typically produce high viral potency. Therefore, the choice of retroviral gene transfer system depends on the target tissue. Retroviral vectors contain ipsilateral long terminal repeats (LTRs) capable of encapsulating foreign sequences of up to 6-10 kb. Minimal ipsilateral LTRs are sufficient for vector replication and encapsulation, subsequently used to integrate therapeutic genes into target cells to provide permanent transgenic expression. Widely used retroviral vectors include those based on murine leukemia virus (MuLV), gibberish leukemia virus (GaLV), simmon immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, for example, Buchscher et al., Journal of Virology 66:2731-2739 (1992); Johann et al., Journal of Virology 66:1635-1640 (1992); Sommnerfelt et al., Journal of Virology 176:58-59 (1990); Wilson et al., Journal of Virology 63:2374-2378 (1989); Miller et al., Journal of Virology 65:2220-2224 (1991); PCT / US94 / 05700).

[0306] In applications where transient expression is preferred, adenovirus-based systems can be used. Adenovirus-based vectors exhibit extremely high transduction efficiency in many cell types and do not require cell division. High titers and high expression levels have been achieved with these vectors. These vectors can be mass-produced in relatively simple systems. Adeno-associated virus (“AAV”) vectors can also be used, for example, in the in vitro production of nucleic acids and peptides and for in vivo and in vitro gene therapy procedures, to transduce cells with target nucleic acids (see, for example, West et al., Virology 160:38-47 (1987); U.S. Patent No. 4,797,368; WO 93 / 24641; Katin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994).

[0307] As used herein, the term "adeno-associated virus" or "AAV" refers to a member of the viral class associated with this name and belonging to the genus *dependoparvovirus* within the family Parvoviridae. Multiple serotypes of this virus are known to be suitable for gene delivery; all known serotypes can infect cells from a wide variety of tissue types. At least 11 have been described, numbered sequentially. Non-limiting exemplary serotypes that can be used in the compositions and methods disclosed herein include any of the 11 serotypes (e.g., AAV2, AAV5, AAV6, AAV8), or variant serotypes such as AAV-DJ. AAV is superior to other viral vectors used for in vivo gene delivery (e.g., encoding gene-editing components) due to its low virulence and low probability of inducing insertional mutations, as it typically does not integrate into the host genome. AAV has a packaging limitation of approximately 4.5 to 4.75 kb.

[0308] The construction of recombinant AAV vectors is described in numerous publications, including U.S. Patent No. 5,173,414; Tratschin et al., *Molecular Biology* 5:3251-3260 (1985); Tratschin et al., *Molecular Biology* 4:2072-2081 (1984); Hermonat and Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., *Journal of Virology*. 1.Viral. 63:03822-3828 (1989). Packaging cells are commonly used to form viral particles capable of infecting host cells. These cells include 293 cells that encapsulate adenoviruses and ψJ2 or PA317 cells that encapsulate retroviruses.

[0309] Viral vectors used in gene therapy are typically produced by generating cell lines that encapsulate nucleic acid vectors into viral particles. The vectors typically contain the minimum viral sequence required for encapsulation and subsequent integration into the host, with other viral sequences replaced by expression cassettes for the polynucleotides to be expressed. Deprived viral function is typically supplied trans-form by the packaging cell line. For example, AAV vectors used in gene therapy typically contain only the ITR sequence from the AAV genome, which is required for encapsulation and integration into the host genome. Viral DNA is encapsulated in a cell line containing helper plasmids encoding other AAV genes (i.e., rep and cap) but lacking the ITR sequence. Cell lines can also be infected using adenovirus as a helper virus. The helper virus promotes AAV vector replication and AAV gene expression via the helper plasmid. Due to the lack of the ITR sequence, the helper plasmid is not encapsulated in large quantities. Adenovirus contamination can be reduced, for example, by heat treatment, as adenovirus is more sensitive to heat treatment than AAV. Additional methods for delivering nucleic acids into cells are known to those skilled in the art. See, for example, US20030087817, which is incorporated herein by reference.

[0310] In some embodiments, host cells are transiently or non-transiently transfected with one or more of the vectors described herein. In some embodiments, cells are transfected while they are naturally present in the subject. In some embodiments, the transfected cells are obtained from the subject.

[0311] In some embodiments, the transfected cells are eukaryotic cells. In some embodiments, the eukaryotic cells are animal cells (e.g., mammalian, human, insect, fish, bird, and reptile). In some embodiments, the transfected cells are human cells. In some embodiments, the transfected cells are hematopoietic cells, such as immune cells (i.e., cells of the innate or adaptive immune system), including (but not limited to) B cells, T cells, natural killer (NK) cells, pluripotent stem cells, induced pluripotent stem cells, chimeric antigen receptor T (CAR-T) cells, monocytes, macrophages, and dendritic cells.

[0312] In some embodiments, the cells are derived from cells obtained from a subject, such as cell lines. In some embodiments, the cells or cell lines are prokaryotic. In some embodiments, the cells or cell lines are eukaryotic. In other embodiments, the cells or cell lines are derived from insect, bird, plant, or fungal species. In some embodiments, the cells or cell lines may be mammals, such as humans, monkeys, mice, cattle, pigs, goats, hamsters, rats, cats, or dogs. Various cell lines for tissue culture are known in this art. Examples of cell lines include (but are not limited to) C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLaS3, Huhl, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panel, PC-3, TFl, CTLL-2, CIR, Rat6, CVI, RPTE, AlO, T24, 182, A375, ARH-77, Calul, SW480, SW620, SKOV3, SK-UT, CaCo2, P388Dl, SEM-K2, WEHI-231, HB56, TIB55, lurkat, 145.01LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A, BS-C-1 monkey kidney epithelial cells, BALB / 3T3 mouse embryonic fibroblasts, 3T3 Swiss, 3T3-Ll, 132-d5 human fetal fibroblasts; 10.1 mouse fibroblasts, 293-T, 3T3, 721, 9L, A2780, A2780ADR, A2780cis, A172, A20, A253, A431, A-549, ALC, B16, B35, BCP-I cells, BEAS-2B, bEnd.3, BHK-21, BR 293. BxPC3, C3H-10Tl / 2, C6 / 36, Cal-27, CHO, CHO-7, CHO-IR, CHO-Kl, CHO-K2, CHO-T, CHO Dhfr- / -, COR-L23, COR-L23 / CPR, COR-L235010, CORL23 / R23, COS-7, COV-434, CML Tl, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3, EMT6 / AR1, EMT6 / AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa, Hepalclc7, HL-60, HMEC, HT-29, lurkat, lYCells, K562 cells, Ku812, KCL22, KGl, KYOl, LNCap, Ma-Mel 1-48, MC-38, MCF-7, MCF-10A, MDA-MB-231, MDA-MB-468, MDA-MB-435, MDCKII, MDCKII, MOR / 0.2R, MONO-MAC 6, MTD-1A, MyEnd, NCI-H69 / CPR, NCI-H69 / LX10, NCI-H69 / LX20, NCI-H69 / LX4, NIH-3T3, NALM-1, NW-145, OPCN / OPCT cell lines, Peer, PNT-1A / PNT 2. RenCa, RIN-5F, RMA / RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell lines, U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1, YAR, and their transgenic strains. Cell lines can be obtained from a variety of sources known to those skilled in the art (see, for example, the American Type Culture Collection (ATCC) (Manassas, Va.)).

[0313] In some embodiments, novel cell lines containing sequences derived from one or more vectors are established using cells transfected with one or more polynucleotides or vectors described herein. In some embodiments, novel cell lines containing cells containing modified sequences but lacking any other exogenous sequences are established using cells transiently transfected with components of an RGN or PE system as described herein (e.g., transient transfection with one or more vectors or with RNA) and modified by the activity of the RGN or PE system. In some embodiments, cells transiently or non-transiently transfected with one or more vectors described herein, or cell lines derived from such cells, are used to evaluate one or more test compounds.

[0314] In some embodiments, non-human transgenic animals or transgenic plants are produced using one or more vectors described herein. In some embodiments, the transgenic animal is an insect. In other embodiments, the insect is a pest, such as a mosquito or tick. In some embodiments, the insect is a plant pest, such as a corn rootworm or fall armyworm. In some embodiments, the transgenic animal is a bird, such as a chicken, turkey, goose, or duck. In some embodiments, the transgenic animal is a mammal, such as a human, mouse, rat, hamster, monkey, ape, rabbit, pig, cow, horse, goat, sheep, cat, or dog.

[0315] V. Variants and fragments of polypeptides and polynucleotides

[0316] This disclosure provides an RNA-guided nuclease (RGN), polymerase, or an active variant or fragment thereof containing a PE. The RGN may have the amino acid sequence shown in SEQ ID NO: 1, 2, 7, 565, 566, 579, 583, 584, 591 or 1165-1179, and the corresponding gRNA, or an active variant or fragment thereof. In some embodiments, this disclosure provides: active variants and fragments of RGN having the amino acid sequences shown in SEQ ID NO: 1, 2, 7, 565, 566, 579, 583, 584, 591 or 1165-1179; active variants and fragments of naturally occurring CRISPR repeat sequences including the nucleotide sequences shown in SEQ ID NO: 3, 5, 470, 471, 475, 476, 571, 573 and 1180; active variants and fragments of tracrRNA including the sequences shown in SEQ ID NO: 4, 6, 472, 473, 474, 477, 478, 479, 480, 572, 574 and 1181; having SEQ ID NO: Active variants and fragments of PE containing any one of the sequences 9-11, 111-130, 137-173, 364, 774-787, 790-814, 817-901, 926, 1143-1155, 1164, 1182-1185, 1189-2085, and 2121-2125; having SEQ ID NO: Active variants and fragments of PE having any one of the sequences of SEQ ID NO: 174, 1208, 1209, 1210, 1221, 1224, 1225, 1226, 1337, 1361, 1371, 1425, 1432, 1443, 1444, 1445, 1447, 1448, 1516, 1577, 1592, 1622, 1640, 1658, 1666, 1682, 1683, 1692, 1694, 1696 and 1718; active variants and fragments of polymerase having any one of the sequences shown in SEQ ID NO: 174, 175 and 2127-2131; and polynucleotides encoding them.

[0317] Although the activity of variants or fragments may vary relative to the polynucleotide or peptide of interest, variants and fragments should retain the functionality of the polynucleotide or peptide of interest. For example, variants or fragments may have increased activity, decreased activity, a different range of activity, or any other variation in activity compared to the polynucleotide or peptide of interest.

[0318] Fragments and variants of RGN polypeptides (or PEs containing thereof) having any of the amino acid sequences shown in SEQ ID NO: 1, 2, 7, 565, 566, 579, 583, 584, 591 or 1165-1179, such as those disclosed herein, will retain sequence-specific RNA-guided DNA-binding activity. In some embodiments, fragments and variants of the RGN polypeptides disclosed herein will retain nuclease (e.g., nickase) activity.

[0319] When used as part of guide RNA, the CRISPR RNA (crRNA) repeat sequences or fragments and variants of tracrRNA disclosed herein will retain the ability to bind RGN (or PE containing it) in a sequence-specific manner and guide it to the target sequence (e.g., the target DNA sequence).

[0320] When used as part of a PE, the fragments and variants of the polymerase disclosed herein will retain the ability to catalyze the addition of nucleotides to the polynucleotide chain at the nick using a template.

[0321] The PE fragments and variants disclosed herein retain the ability to edit double-stranded polynucleotides by replacing the target sequence with the template sequence of the PEgRNA when associated with PEgRNA.

[0322] The term "fragment" refers to a portion of the polynucleotide or polypeptide sequence disclosed herein. A "fragment" or "bioactive portion" includes a polynucleotide containing a sufficient number of linked nucleotides to retain biological activity (i.e., to bind RGN in a sequence-specific manner and direct RGN to the target nucleotide sequence when included within guide RNA). A "fragment" or "bioactive portion" includes a polypeptide containing a sufficient number of linked amino acid residues to retain biological activity (i.e., to bind to the target sequence in a sequence-specific manner when complexed with guide RNA). Fragments of RGN proteins (or PEs containing them) include fragments shorter than the full-length sequence due to the use of alternative downstream initiation sites. The bioactive portion of an RGN protein may be a polypeptide comprising 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 1000 or more linked amino acid residues, such as any one of SEQ ID NO: 1, 2, 7, 565, 566, 579, 583, 584, 591 or 1165-1179. The bioactive portion of PE may be a polypeptide comprising, for example, any one of SEQ ID NO: 9-11, 111-130, 137-173, 364, 774-787, 790-814, 817-901, 926, 1143-1155, 1164, 1182-1185, 1189-2085, and 2121-2125, containing 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 1000 or more linked amino acid residues. In some embodiments, the bioactive portion of PE may be a polypeptide comprising, for example, SEQ ID NO: 364, 1208, 1209, 1210, 1221, 1224, 1225, 1226, 1337, 1361, 1371, 1425, 1432, 1443, 1444, 1445, 1447, 1448, 1516, 1577, 1592, 1622, 1640, 1658, 1666, 1682 A polypeptide of 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 1000 or more linked amino acid residues from any of the following: 1683, 1692, 1694, 1696 and 1718.The bioactive portion of the polymerase may be a polypeptide comprising, for example, any one of SEQ ID NOs: 174, 175, and 2127-2131, 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 linked amino acid residues. The bioactive fragment of the crRNA repeat sequence may comprise at least eight linked amino acids of SEQ ID NOs: 3, 5, 470, 471, 475, 476, 571, 573, or 1180. The biologically active portion of a CRISPR repeat sequence may be a polynucleotide comprising, for example, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 linked nucleotides of SEQ ID NO: 3, 5, 470, 471, 475, 476, 571, 573, or 1180. The biologically active fragment of a tracrRNA sequence may comprise at least 10 linked amino acids of SEQ ID NO: 4, 6, 472, 473, 474, 477, 478, 479, 480, 572, 574, or 1181. The bioactive portion of a tracrRNA sequence may be a polynucleotide comprising 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or 105 linked nucleotides, such as SEQ ID NO: 4, 6, 472, 473, 474, 477, 478, 479, 480, 572, 574, or 1181. Such bioactive portions can be prepared and their activity evaluated using recombinant techniques.

[0323] Generally, "variant" is intended to refer to substantially similar sequences. For polynucleotides, a variant comprises the deletion and / or addition of one or more nucleotides at one or more internal sites within the native polynucleotide, and / or the substitution of one or more nucleotides at one or more sites within the native polynucleotide. As used herein, "native" or "wild-type" polynucleotides or peptides comprise naturally occurring nucleotide or amino acid sequences, respectively. For polynucleotides, conserved variants include those sequences that encode the native amino acid sequence of the gene of interest due to genetic code degeneracy. Naturally occurring allelic variants, such as these allelic variants, can be identified using well-known molecular biology techniques, such as polymerase chain reaction (PCR) and hybridization techniques as outlined below. Variant polynucleotides also include synthetically derived polynucleotides, such as those induced by, for example, site-directed mutagenesis, but which still encode peptides or polynucleotides of interest. Generally, as determined by sequence alignment procedures and parameters described elsewhere in this document, variants of the specific polynucleotides disclosed herein will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the specific polynucleotides.

[0324] Variants of the specific polynucleotides disclosed herein (i.e., reference polynucleotides) can also be assessed by comparing the percentage of sequence identity between the polypeptide encoded by the variant polynucleotide and the polypeptide encoded by the reference polynucleotide. The percentage of sequence identity between any two polypeptides can be calculated using the sequence alignment procedures and parameters described elsewhere herein. When any given pair of polynucleotides disclosed herein is assessed by comparing the percentage of sequence identity shared by the two polypeptides encoded by the polynucleotides, the percentage of sequence identity between the two encoded polypeptides is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher.

[0325] The present invention discloses a polynucleotide encoding polymerase, an RGN polypeptide (or a PE containing the same). In some embodiments, the polynucleotide encoding amino acid sequence disclosed herein has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with any one of the amino acid sequences shown in SEQ ID NO: 1, 2, 7, 565, 566, 579, 584, 591 or 1165-1179. In some embodiments, the polynucleotide encoding amino acid sequence disclosed in this invention has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with the amino acid sequence shown in any one of SEQ ID NO: 174, 175 and 2127-2131. In some embodiments, the polynucleotide encoding amino acid sequences disclosed in this invention have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with any one of the amino acid sequences shown in SEQ ID NO: 9-11, 111-130, 137-173, 364, 774-787, 790-814, 817-901, 926, 1143-1155, 1164, 1182-1185, 1189-2085 and 2121-2125.In some embodiments, the polynucleotide encoding amino acid sequences disclosed in this invention are identical to SEQ ID NO: 364, 1208, 1209, 1210, 1221, 1224, 1225, 1226, 1337, 1361, 1371, 1425, 1432, 1443, 1444, 1445, 1447, 1448, 1516, 1577, 1592, 1622, 1640, 1658, 1666, 1682, 1683, 1692, 1694, The PEs of any one of the amino acid sequences shown in 1696 and 1718 have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity.

[0326] Bioactive variants of the RGN polypeptide disclosed herein may differ by as few as about 1-15 amino acid residues, as few as about 1-10, such as about 6-10, as few as 5, as few as 4, as few as 3, as few as 2, or as few as 1 amino acid residue. In some embodiments, the polypeptide may include an N-terminal or C-terminal truncation, which may have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, or more amino acids deleted from the N-terminus or C-terminus of the polypeptide.

[0327] In some embodiments, the polynucleotides disclosed in this invention comprise or encode crRNA repeat sequences having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the nucleotide sequences shown in SEQ ID NO: 3, 5, 470, 471, 475, 476, 571, 573 or 1180.

[0328] In some embodiments, the polynucleotides disclosed in this invention comprise or encode tracrRNAs with nucleotide sequences having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the nucleotide sequences shown in SEQ ID NO: 4, 6, 472, 473, 474, 477, 478, 479, 480, 572, 574 or 1181.

[0329] The guide RNA variants disclosed herein include guide RNAs with modified nucleotide, sugar, phosphate backbone and / or nucleobases. Variant guide RNAs may include modifications such as: 2'-O-methyl (2'-O-Me) modification; 2'-fluorine (2'-F) modification; 2'F-4'C α-OMe modification; 2',4'-di-C α-OMe modification; 2'-O-methyl 3'-phosphothioate (MS) modification; 2'-O-methyl 3'-thiophosphonoacetate (MSP; 2'-O-methyl 3'-thioPACE) modification; 2'-O-methyl 3'-phosphonoacetate (MP) modification; phosphothioate (PS) modification; and bridged nucleic acid (BNA) modification (e.g., 2',4' BNA, locked nucleic acid (LNA), N-methyl-substituted bridged nucleic acid BNA). NC [N-Me], 2'-O,4'-C-extrinsic ethyl-bridged nucleic acids (2',4'-ENA) and S-constrained ethyl (cEt)); or combinations thereof. Chemical modifications of spacer regions, crRNA repeat sequences, crRNA, tracrRNA and guide RNA are described in International Application No. PCT / IB2023 / 058418, filed August 25, 2023 (which is incorporated herein by reference in its entirety).

[0330] The bioactive variants of the guide RNA disclosed herein may differ by as few as about 1-15 nucleotides, as few as about 1-10, such as about 6-10, as few as 5, as few as 4, as few as 3, as few as 2, or as few as 1 nucleotide. In some embodiments, the polynucleotide may contain a 5' or 3' truncation that may have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more nucleotides deleted from the 5' or 3' end of the polynucleotide.

[0331] It should be understood that the polymerases, RGN peptides (or PEs containing them), crRNA repeat sequences, or tracrRNAs provided herein can be modified to produce variant proteins and polynucleotides. Artificially designed variations can be introduced using site-directed mutagenesis techniques. Alternatively, native, unknown, or unidentified polynucleotides and / or peptides structurally and / or functionally related to the sequences disclosed herein can be identified and fall within the scope of this invention. Conserved amino acid substitutions can be performed in non-conserved regions without altering the function of the polymerase, RGN protein, or PE. Alternatively, modifications can be made to improve the activity of RGN or PE.

[0332] Variant polynucleotides and proteins also encompass sequences and proteins derived from mutation-induced and recombination procedures, such as DNA shuffling. Using such procedures, one or more different polymerases, RGN proteins, or PEs disclosed herein are manipulated to produce novel polymerases, RGN proteins, or PEs with desired properties. In this manner, recombinant polynucleotide libraries are generated from a population of related sequence polynucleotides containing sequence regions with substantial sequence identity and capable of homologous recombination in vitro or in vivo. For example, using this method, sequence motifs encoding domains of interest can be shuffled between the polymerase, RGN, or PE sequences provided herein and other known polymerase, RGN, or PE genes to obtain sequences encoding modified properties of interest (such as increased K in the case of the enzyme). m This involves creating new genes for proteins. Such DNA shuffling strategies are known in this technique. See, for example, Stemmer (1994), *Proceedings of the National Academy of Sciences*. Proc.Natl.Acad.Sci.USA )》91:10747-10751; Stemmer (1994) Nature ( Nature )》370:389-391; Crameri et al. (1997) Nature Biotechnology ( Nature Biotech. )》15:436-438; Moore et al. (1997) Journal of Molecular Biology ( J. Mol. Biol. )》272:336-347; Zhang et al. (1997) Proceedings of the National Academy of Sciences of the United States of America (PNAS) Proc.Natl.Acad.Sci.USA )》94:4504-4509; Crameri et al. (1998) Nature ( Nature (See 391:288-291; and U.S. Patent Nos. 5,605,793 and 5,837,458). A “recombined” nucleic acid is a nucleic acid produced by a recombining procedure (such as any recombining procedure described herein). Recombined nucleic acids are produced by, for example, manually and, as appropriate, recursively recombinating (physical or virtual) two or more nucleic acids (or strings). Generally, one or more screening steps are used during the recombining process to identify the nucleic acid of interest; this screening step may be performed before or after any recombination step. In some (but not all) recombining implementations, multiple rounds of recombination are required before selection to increase the diversity of the pool to be screened. The entire process of recombination and selection is repeated recursively, as appropriate. Depending on the context, recombining may refer to the entire process of recombination and selection, or alternatively, only the recombination portion of the overall process.

[0333] As used herein, “sequence identity” or “identity” in the context of two polynucleotide or polypeptide sequences refers to identical residues in two sequences when aligned to maximum correspondence within a specified comparison window. It is recognized that inconsistent residue positions are often distinguished by conserved amino acid substitutions, in which an amino acid residue substitutes for another amino acid residue with similar chemical properties (e.g., charge or hydrophobicity) and therefore does not alter the functional properties of the molecule. Protein sequences that differ due to such conserved substitutions are thus referred to as having “sequence similarity” or “identity.” Methods for measuring sequence similarity are well known to those skilled in the art. Typically, this involves scoring conserved substitutions as partial mismatches rather than complete mismatches. Thus, for example, where a consistent amino acid is assigned a score of 1 and a non-conservative substitution is assigned a score of 0, the score for a conserved substitution is between 0 and 1. The scoring of conserved substitutions is calculated, for example, in the program PC / GENE (Intelligenetics, MountainView, California).

[0334] As used herein, "sequence identity percentage" refers to a value determined by comparing two best-aligned sequences within a comparison window, where the polynucleotide sequence portion of the comparison window may contain additions or deletions (i.e., vacancies) compared to a reference sequence (which does not contain additions or deletions) to best align the two sequences. The percentage is calculated as follows: determine the number of positions in the two sequences where there are identical nucleic acid bases or amino acid residues, obtain the number of matching positions, divide this number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the sequence identity percentage.

[0335] Unless otherwise stated, the sequence identity / similarity values ​​provided herein refer to values ​​obtained using GAP version 10 with the following parameters: nucleotide sequence identity % and similarity % using a vacancy weight of 50 and a length weight of 3, and an nwsgapdna.cmp scoring matrix; amino acid sequence identity % and similarity % using a vacancy weight of 8 and a length weight of 2, and a BLOSUM62 scoring matrix; or any equivalent procedure thereof. “Equivalent procedure” means any sequence comparison procedure that, for any two sequences in question, generates an alignment that has consistent nucleotide or amino acid residue matching and consistent sequence identity percentages compared to the corresponding alignment generated by GAP version 10.

[0336] When two sequences are aligned to achieve the highest possible score for the pair using a defined amino acid substitution matrix (e.g., BLOSUM62), a vacancy presence penalty, and a vacancy expansion penalty, these sequences are considered “optimally aligned.” The amino acid substitution matrix and its use in quantifying the similarity between two sequences are well-known in this technique and described, for example, in Dayhoff et al. (1978), “A model of evolutionary change in proteins,” *Atlas of Protein Sequence and Structure*, Vol. 5, Supplement 3 (edited by MO Dayhoff), pp. 345-352, *National Biomedical Research Foundation*, Washington, DC, and Henikoff et al. (1992), *Proceedings of the National Academy of Sciences*, 89:10915-10919. The BLOSUM62 matrix is ​​commonly used as a pre-defined scoring substitution matrix in sequence alignment schemes. A penalty is applied for the introduction of a single amino acid vacancy into one of the aligned sequences, and a penalty is applied for vacancy expansion at each additional empty amino acid position inserted into an already opened vacancy. Alignment is defined by the start and end amino acid positions of each sequence and, depending on the case, by inserting one or more vacancy positions in one or two sequences to achieve the highest possible score. Although optimal alignment and scoring can be performed manually, this process is facilitated by alignment algorithms implemented using a calculator, such as those described in Altschul et al. (1997) in *Nucleic Acid Research*. Nucleic Acids Res. The vacancy BLAST 2.0 described in 25:3389-3402 of the National Center for Biotechnology Information (NCBI, NLM, NIH.gov) is publicly available. Best alignments, including multiple alignments, can be made using, for example, PSI-BLAST as described in Altschul et al. (1997), Nucleic Acid Research (Nucleic Acids Research). Nucleic Acids Res. )》25:3389-3402.

[0337] Regarding the amino acid sequence for optimal alignment with the reference sequence, the amino acid residues "correspond" to the positions in the reference sequence where they pair with each other in the alignment. These "positions" are indicated by the numbering of each amino acid in the reference sequence, identified sequentially relative to its N-terminus. Because deletions, insertions, truncations, and fusions must be considered when determining optimal alignment, the amino acid residue numbers in the test sequence determined by counting only from the N-terminus will generally not be the same as their corresponding positions in the reference sequence. For example, in the case of a deletion in the aligned test sequence, there will be no amino acid at the position corresponding to the deletion site in the reference sequence. In the case of an insertion in the aligned reference sequence, the insertion will not correspond to any amino acid position in the reference sequence. In the case of truncation or fusion, there may be amino acid extensions in the reference or aligned sequence that do not correspond to any amino acid in the corresponding sequence.

[0338] VI. Antibodies

[0339] This also covers antibodies against the polymerases, RGN peptides, PEs, or ribonucleoprotein complexes comprising polymerases, RGN peptides, or PEs of this disclosure, including those RGN peptides (or PEs comprising them) having an amino acid sequence having any one of the amino acid sequences shown in SEQ ID NO: 1, 2, 7, 579, 583, 584, or 591, or their active variants or fragments. Methods for generating antibodies are well known in the art (see, for example, Harlow and Lane (1988), *Antibodies: A Laboratory Manual*, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; and U.S. Patent No. 4,196,265). These antibodies can be used in kits for the detection and separation of polymerases, RGN peptides, PEs, or ribonucleoprotein complexes. Therefore, this disclosure provides kits containing antibodies that specifically bind to the polypeptides or ribonucleoprotein complexes described herein, including, for example, RGN polypeptides (or PEs containing the thereof) having an amino acid sequence shown in any one of SEQ ID NO: 1, 2, 7, 579, 583, 584 or 591.

[0340] VII. RGN or PE systems and ribonucleoprotein complexes for binding and modifying target sequences, and methods for their preparation.

[0341] This disclosure provides an RNA-guided nuclease (RGN) system for binding to a target sequence of interest (e.g., a target DNA sequence), wherein the RGN system comprises at least one RGN polypeptide (or a polynucleotide sequence encoding it) and one or more guide RNAs (or one or more polynucleotide sequences encoding it) capable of forming a complex (ribonucleoprotein complex) with the RGN polypeptide. The guide RNA hybridizes to the target strand of the target sequence of interest and also forms a complex with the RGN polypeptide, thereby directing the RGN polypeptide to bind to the target sequence. This document also provides a PE system for binding to and modifying a target sequence of interest, wherein the PE system comprises a polymerase (or a polynucleotide encoding it) and an RGN (or a polynucleotide encoding it), along with one or more PEgRNAs (or one or more polynucleotide sequences encoding it). The PE system may comprise a PE, wherein the polymerase is operatively linked to the RGN or the RGN may be two separate polypeptides.

[0342] In some embodiments, the RGN (in the RGN system or the PE system comprising it) has the amino acid sequence shown in SEQ ID NO: 1, or an active variant or fragment thereof, and binds to a target nucleotide sequence adjacent to and at its 5' end of the PAM sequence shown in NNNNCY. In some embodiments, the RGN (in the RGN system or the PE system comprising it) has the amino acid sequence shown in SEQ ID NO: 1, or an active variant or fragment thereof, and binds to a guide RNA comprising a crRNA repeat sequence or an active variant or fragment thereof of any one of SEQ ID NO: 3, 470, and 471 and a tracrRNA sequence or an active variant or fragment thereof of any one of SEQ ID NO: 4, 472, 473, and 474, and binds to a target nucleotide sequence adjacent to and at its 5' end of the PAM sequence shown in NNNNCY.

[0343] In some embodiments, the RGN (in the RGN system or the PE system comprising it) has the amino acid sequence shown in SEQ ID NO: 2, or an active variant or fragment thereof, and binds to a target nucleotide sequence adjacent to and at its 5' end of the PAM sequence shown in NNRYA. In some embodiments, the RGN (in the RGN system or the PE system comprising it) has the amino acid sequence shown in SEQ ID NO: 2, or an active variant or fragment thereof, and binds to a guide RNA comprising a crRNA repeat sequence or an active variant or fragment thereof of any one of SEQ ID NO: 5, 475, and 476 and a tracrRNA sequence or an active variant or fragment thereof of any one of SEQ ID NO: 6, 477, 478, 479, and 480, and binds to a target nucleotide sequence adjacent to and at its 5' end of the PAM sequence shown in NNRYA.

[0344] In some embodiments, the RGN (in the RGN system or the PE system comprising it) has the amino acid sequence shown in SEQ ID NO: 7, or an active variant or fragment thereof, and binds to a target nucleotide sequence adjacent to and at its 5' end of the PAM sequence shown in NNNNCC. In some embodiments, the PE or PE system comprises an RGN having the amino acid sequence shown in SEQ ID NO: 7, or an active variant or fragment thereof, and binds to a guide RNA comprising a crRNA repeat sequence or an active variant or fragment thereof from any of SEQ ID NO: 3, 470, and 471 and a tracrRNA sequence or an active variant or fragment thereof from any of SEQ ID NO: 4, 472, 473, and 474, when bound to a guide RNA, and also binds to a target nucleotide sequence adjacent to and at its 5' end of the PAM sequence shown in NNNNCC. In some embodiments, the RGN system comprises an RGN polypeptide heterologous to the guide RNA, wherein the RGN polypeptide and the guide RNA are not found to complex with each other in nature (i.e., bind to each other).

[0345] In some embodiments, the RGN (in the RGN system or the PE system comprising it) has the amino acid sequence shown in SEQ ID NO: 579, or an active variant or fragment thereof, and binds to a target nucleotide sequence adjacent to and at its 5' end of the PAM sequence shown in NNNNC. In some embodiments, the PE or PE system comprises an RGN having the amino acid sequence shown in SEQ ID NO: 579, or an active variant or fragment thereof, and binds to a guide RNA comprising a crRNA repeat sequence or an active variant or fragment thereof from any of SEQ ID NO: 3, 470, and 471 and a tracrRNA sequence or an active variant or fragment thereof from any of SEQ ID NO: 4, 472, 473, and 474, when bound to a guide RNA, and also binds to a target nucleotide sequence adjacent to and at its 5' end of the PAM sequence shown in NNNNC. In some embodiments, the RGN system comprises an RGN polypeptide heterologous to the guide RNA, wherein the RGN polypeptide and the guide RNA are not found to complex with each other in nature (i.e., bind to each other).

[0346] In some embodiments, the RGN (in the RGN system or the PE system comprising it) has the amino acid sequence shown in SEQ ID NO: 583, or an active variant or fragment thereof, and binds to a target nucleotide sequence adjacent to and at its 5' end of the PAM sequence shown in NNNNC. In some embodiments, the PE or PE system comprises an RGN having the amino acid sequence shown in SEQ ID NO: 583, or an active variant or fragment thereof, and binds to a guide RNA comprising a crRNA repeat sequence or an active variant or fragment thereof from any of SEQ ID NO: 3, 470, and 471 and a tracrRNA sequence or an active variant or fragment thereof from any of SEQ ID NO: 4, 472, 473, and 474, when bound to a guide RNA, and also binds to a target nucleotide sequence adjacent to and at its 5' end of the PAM sequence shown in NNNNC. In some embodiments, the RGN system comprises an RGN polypeptide heterologous to the guide RNA, wherein the RGN polypeptide and the guide RNA are not found to complex with each other in nature (i.e., bind to each other).

[0347] In some embodiments, the RGN (in the RGN system or the PE system comprising it) has the amino acid sequence shown in SEQ ID NO: 584, or an active variant or fragment thereof, and binds to a target nucleotide sequence adjacent to and at its 5' of the PAM sequence shown in NNNNCM. In some embodiments, the PE or PE system comprises an RGN having the amino acid sequence shown in SEQ ID NO: 584, or an active variant or fragment thereof, and binds to a guide RNA comprising a crRNA repeat sequence or an active variant or fragment thereof from any of SEQ ID NO: 3, 470, and 471 and a tracrRNA sequence or an active variant or fragment thereof from any of SEQ ID NO: 4, 472, 473, and 474, when bound to a guide RNA, and also binds to a target nucleotide sequence adjacent to and at its 5' of the PAM sequence shown in NNNNCM. In some embodiments, the RGN system comprises an RGN polypeptide heterologous to the guide RNA, wherein the RGN polypeptide and the guide RNA are not found to complex with each other in nature (i.e., bind to each other).

[0348] In some embodiments, the RGN (in the RGN system or the PE system comprising it) has the amino acid sequence shown in SEQ ID NO: 591, or an active variant or fragment thereof, and binds to a target nucleotide sequence adjacent to and at its 5' end of the PAM sequence shown in NNNNCT. In some embodiments, the PE or PE system comprises an RGN having the amino acid sequence shown in SEQ ID NO: 591, or an active variant or fragment thereof, and binds to a guide RNA comprising a crRNA repeat sequence or an active variant or fragment thereof from any of SEQ ID NO: 3, 470, and 471 and a tracrRNA sequence or an active variant or fragment thereof from any of SEQ ID NO: 4, 472, 473, and 474, when bound to a guide RNA, and also binds to a target nucleotide sequence adjacent to and at its 5' end of the PAM sequence shown in NNNNCT. In some embodiments, the RGN system comprises an RGN polypeptide heterologous to the guide RNA, wherein the RGN polypeptide and the guide RNA are not found to complex with each other in nature (i.e., bind to each other).

[0349] In some embodiments, the RGN (in the RGN system or the PE system comprising it) has the amino acid sequence shown in SEQ ID NO: 565, or an active variant or fragment thereof, and binds to a target nucleotide sequence adjacent to and at its 5' of the PAM sequence shown in NNGRR. In some embodiments, the PE or PE system comprises an RGN having the amino acid sequence shown in SEQ ID NO: 565, or an active variant or fragment thereof, and binds to a target nucleotide sequence adjacent to and at its 5' of the PAM sequence shown in NNGRR when bound to a guide RNA comprising a crRNA repeat sequence of SEQ ID NO: 571 or an active variant or fragment thereof and a tracrRNA sequence of SEQ ID NO: 572 or an active variant or fragment thereof. In some embodiments, the RGN system comprises an RGN polypeptide heterologous to the guide RNA, wherein the RGN polypeptide and the guide RNA are not found to complex with each other in nature (i.e., bind to each other).

[0350] In some embodiments, the RGN (in the RGN system or the PE system comprising it) has the amino acid sequence shown in SEQ ID NO: 566, or an active variant or fragment thereof, and binds to a target nucleotide sequence adjacent to and at its 5' end of the PAM sequence shown in NNGG. In some embodiments, the PE or PE system comprises an RGN having the amino acid sequence shown in SEQ ID NO: 566, or an active variant or fragment thereof, and binds to a target nucleotide sequence adjacent to and at its 5' end of the PAM sequence shown in NNGG when bound to a guide RNA comprising a crRNA repeat sequence or an active variant or fragment thereof of SEQ ID NO: 573 or 1180 and a tracrRNA sequence or an active variant or fragment thereof of SEQ ID NO: 574 or 1181. In some embodiments, the RGN system comprises an RGN polypeptide heterologous to the guide RNA, wherein the RGN polypeptide and the guide RNA are not found to complex with each other in nature (i.e., bind to each other).

[0351] In some embodiments, a dual PE (DPE) system for dual polymerase editing is provided, wherein the system comprises: a first PE system comprising a first PE (or a polynucleotide encoding thereof) and a first PEgRNA (or a polynucleotide encoding thereof); and a second PE system comprising a second PE (or a polynucleotide encoding thereof) and a second PEgRNA (or a polynucleotide encoding thereof). The first PE and the second PE may be the same or different and may be any of the PEs described herein. The DPE system may also comprise a PE (or a polynucleotide encoding thereof) and a first PEgRNA (or a polynucleotide encoding thereof) and a second PEgRNA (or a polynucleotide encoding thereof). The first PEgRNA and the second PEgRNA bind to opposite strands of the target DNA such that the intended repair event is mounted on both strands of the target DNA. The DNA synthesis templates for the first PEgRNA and the second PEgRNA each encode a single-stranded DNA sequence that is complementary to each other (completely or partially) to replace the DNA region between the two nick sites.

[0352] For large insertions, the DPE system comprises PEgRNA, wherein the DNA synthesis template is designed such that the replacement sequence (containing a complementary single-stranded DNA sequence encoded by a first DNA synthesis template and a second DNA synthesis template) contains a first recombinase site; donor DNA containing a second recombinase site; and site-specific recombinases that recognize the first and second recombinase sites. Recombination of the replacement sequence with the donor DNA results in the insertion of exogenous DNA.

[0353] As used herein, the term "recombinase" refers to a site-specific enzyme that catalyzes DNA recombination between recombinase sites, causing excision, integration, inversion, or exchange of DNA fragments between these sites. Non-restrictive examples of recombinases include serine recombinases (e.g., Hin, Gin, Tn3, β-six, CinH, ParA, γδ, Bxb1, ...). C31, TP901, TG1 BT1, R4 RV1 FC1, MR11, A118, U153 and gp29) and tyrosine recombinases (e.g. Cre, FLP, R, λ, HK101, HK022 and pSAM2). See, for example, Brown et al., "Serine recombinases as tools for genome engineering," *Methods*, 2011;53(4):372-9; Hirano et al., "Site-specific recombinases as tools for heterologous gene integration," *Applied Microbiol. Biotechnol.*, 2011;92(2):227-39; Chavez and Calos, "Therapeutic applications of the ΦC31 integrase system," *Curr. Gene Ther.*, 2011;11(5):375-81; Turan and Bode, "Site-specific recombinases: from tag- and target-based genome modification to tag- and exchange-based genome modification." Tag-and-target to tag-and-exchange-based genomic modifications” Journal of the Federation of American Societies for Experimental Biology (FASEB J) 2011;25(12):4088-107; Venken and Bellen, Genome-wide manipulations of Drosophila melanogaster with transposons, Flprecombinase, and ΦC31 integrase” Methods in Molecular Biology 2012;859:203-28; Murphy, Phage recombinases and their applications” Adv. Virus Research)》2012;83:367-414; Zhang et al., "Conditional gene manipulation: Creating a new biological era" J. Zhejiang Univ. Sci. B. 2012;13(7):511-24; Karpenshif and Bernstein, "From yeast to mammals: recent advances in genetic control of homologous recombination" DNA Repair (Amst). 2012;1;11(10):781-8; Each of these is incorporated herein by reference in full. The names of serine and tyrosine recombinases are derived from conserved nucleophilic amino acid residues that attack DNA and are covalently linked to DNA during strand exchange.

[0354] As used herein, the term "recombinase site" refers to a target nucleotide sequence recognized by a recombinase that undergoes strand exchange with another nucleotide sequence having a similar recombinase site. Non-limiting examples of recombinase sites include those derived from HK022 and... The attB / attP site recognized by the C31 recombinase.

[0355] The RGN or PE systems provided herein for binding to target sequences of interest may include ribonucleoprotein (RNP) complexes, which are at least one RNA molecule that binds to at least one protein. The RNP complexes provided herein comprise at least one guide RNA as an RNA component and an RGN polypeptide or PE as a protein component. The guide RNA of the RNP complexes disclosed herein may include a spacer region that hybridizes to a eukaryotic target sequence. In some embodiments, the eukaryotic target sequence is a mammalian target sequence. Such RNP complexes can be purified from cells or organisms that naturally express RGN polypeptides and are engineered to express specific guide RNAs that are specific to the target sequence of interest. In some embodiments, the RNP complexes can be purified from cells or organisms that have been transformed with one or more polynucleotides encoding an RGN polypeptide (e.g., mRNA encoding an RGN polypeptide) or a PE containing an RGN polypeptide and a guide RNA (or a polynucleotide containing a guide RNA) and cultured under conditions that allow the expression of the RGN polypeptide (or PE) and the guide RNA.

[0356] Methods for preparing polymerase, RGN peptide, PE, or RNP complexes are provided. These methods involve culturing cells under conditions expressing the polymerase, RGN peptide, or PE (and in some embodiments, guide RNA), the cells containing a polynucleotide sequence encoding the polymerase, RGN peptide, or PE, and in some embodiments, a polynucleotide sequence encoding or containing guide RNA. The polymerase, RGN peptide, PE, or RNP complex can then be purified from the lysate of the cultured cells. In some embodiments, the polynucleotide sequence encoding the polymerase, RGN peptide, or PE comprises mRNA (messenger RNA). In some embodiments, methods for assembling RNP complexes involve combining one or more of the guide RNAs disclosed herein with one or more of the polymerases, RGN peptides, or PEs disclosed herein under conditions suitable for forming RNP complexes.

[0357] Methods for purifying polymerases, RGN peptides, PE or RNP complexes from lysates of biological samples are known in this technique (e.g., size exclusion and / or affinity chromatography, 2D-PAGE, HPLC, reverse phase chromatography, immunoprecipitation). In certain methods, polymerases, RGN peptides, or PEs are generated recombinantly and include purification tags to aid in their purification. These purification tags include, but are not limited to, glutathione S-transferase (GST), chitin-binding protein (CBP), maltose-binding protein, thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tags, myc, AcV5, AU1, AU5, E, ECS, E2, FLAG (e.g., 3X FLAG tags), HA, nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV-G, 6xHis, 10xHis, biotinylate carboxyl carrier protein (BCCP), and calmodulin. Generally, immobilized metal affinity chromatography is used to purify labeled polymerases, RGN peptides, PEs, or RNP complexes. It should be understood that other similar methods known in this technique, including other forms of chromatography or, for example, immunoprecipitation, can be used alone or in combination.

[0358] "Isolated" or "purified" polypeptides or their bioactive portions are substantially or substantially free of components typically found in their natural environment that accompany or interact with the polypeptide. Therefore, when produced by recombinant technology, isolated or purified polypeptides are substantially free of other cellular material or culture media, or when chemically synthesized, substantially free of chemical precursors or other chemicals. Proteins substantially free of cellular material include protein formulations having less than about 30%, 20%, 10%, 5%, or 1% (dry weight) of contaminating proteins. When the proteins of the present invention or their bioactive portions are produced recombinantly, most preferably, the culture medium represents less than about 30%, 20%, 10%, 5%, or 1% (dry weight) of chemical precursors or non-protein chemicals of interest. Similarly, "isolated" polynucleotides or nucleic acid molecules are removed from their natural environment. When chemically synthesized or removed from genomic loci by breaking phosphodiester bonds, isolated polynucleotides are substantially free of chemical precursors or other chemicals. Isolated polynucleotides may be part of a carrier, a composition of substances, or may be contained within cells, provided that the cells are not the original environment of the polynucleotide.

[0359] The specific methods provided herein for binding and / or cleaving target polynucleotides containing the target sequence of interest involve the use of in vitro assembled RNP complexes. In vitro assembly of the RNP complex can be performed using any method known in this art, wherein the RGN peptide or PE is contacted with the guide RNA under conditions that allow binding to the guide RNA. As used herein, “contact,” “contacting,” and “contacted” refer to placing the components of the desired reaction together under conditions suitable for carrying out the desired reaction. The RGN peptide or PE may be purified from a biological sample, cell lysate, or culture medium, generated via in vitro translation, or chemically synthesized. The guide RNA may be purified from a biological sample, cell lysate, or culture medium, transcribed in vitro, or chemically synthesized. The RGN peptide or PE and the guide RNA may be contacted in solution (e.g., buffered saline solution) to allow for the in vitro assembly of the RNP complex.

[0360] Some categories of this disclosure provide kits that contain one or more components of the RGN or PE system described herein, including: guide RNA, polymerase, RGN polypeptide or PE or polynucleotide encoding guide RNA, polymerase, RGN polypeptide or PE; cells; and the complete RGN or PE system. In some embodiments, the kit includes instructions suitable for reagents, buffers and / or use of one or more components of the RGN or PE system, such as instructions for use in vitro or in vivo nucleic acid editing. Reagents are available in any suitable container, such as vials, bottles or tubes. Reagents can be used in methods utilizing one or more of the components of the RGN system. For example, a restriction enzyme may be included for selecting polynucleotides encoding polymerase, RGN or PE into a vector. In some embodiments, the kit includes instructions for designing and using a suitable guide RNA for targeted editing of a target sequence. Reagents may be provided in a form suitable for use in a particular assay or in a form requiring the addition of one or more other components prior to use (e.g., in a concentrate or lyophilized form). The buffer may be any buffer, including (but not limited to) sodium carbonate buffer, sodium bicarbonate buffer, borate buffer, Tris buffer, MOPS buffer, HEPES buffer, and combinations thereof. In some embodiments, the buffer is alkaline. In some embodiments, the buffer has a pH of about 7 to about 10.

[0361] Kits comprising one or more components of the RGN or PE system of this disclosure can be used for a wide range of applications, including modification (e.g., deletion, insertion of donor polynucleotides, translocation, inactivation, activation, base editing, polymerase editing) of target sequences in a large number of cell types. Therefore, kits comprising one or more components of the RGN or PE system of this disclosure are suitable for applications such as gene therapy, drug screening, disease diagnosis, and prognosis.

[0362] In some embodiments, the kits disclosed herein include pharmaceutical kits comprising the pharmaceutical compositions described herein. In some embodiments, the pharmaceutical kit may include: (a) a container containing a lyophilized form of the compositions disclosed herein and (b) a second container containing a pharmaceutically acceptable diluent for injection (e.g., sterile water). The pharmaceutically acceptable diluent may be used to reconstitute or dilute the lyophilized compounds of this disclosure. As appropriate, the precautions associated with such containers may be in the form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceutical or biological products, reflecting approval for human administration by such agency.

[0363] VIII. Methods for binding, cleaving, or modifying target polynucleotides

[0364] This disclosure provides methods for binding, cleaving, and / or modifying a target polynucleotide of interest (e.g., target DNA) containing a target sequence. These methods include delivering an RGN or PE system containing at least one guide RNA or a polynucleotide encoding thereof and at least one RGN polypeptide (or PE, or a trans-provided polymerase and RGN polypeptide) or a polynucleotide encoding thereof) to the target sequence or to a cell, organelle, or embryo containing the target sequence. The methods for binding, cleaving, and / or modifying the target polynucleotide of interest (e.g., target DNA) containing the target sequence can be performed in vitro or ex vivo. In some embodiments, the methods for binding, cleaving, and / or modifying the target polynucleotide of interest (e.g., target DNA) are not methods for therapeutically treating human or animal bodies or methods for modifying human germline genetic identity.

[0365] In a particular embodiment, the polymerase, RGN, PE, and / or guide RNA are heterologous to the cell, organelle, or embryo in which the polymerase, RGN, PE, and / or guide RNA (or a polynucleotide encoding at least one of the polymerase, RGN, PE, and guide RNA) is introduced.

[0366] Polynucleotides encoding guide RNA, polymerase, PE, and / or RGN peptides can be delivered to cells or embryos in vitro, in vivo, or ex vivo. Cells or embryos can be cultured under conditions expressing guide RNA, polymerase, PE, and / or RGN peptides. In some embodiments, the method includes contacting the target polynucleotide with an RNP complex. Contact can be performed in vitro, in vivo, or ex vivo. In some embodiments, the method includes introducing the RNP complex into cells, organelles, or embryos containing the target polynucleotide. The RNP complex can be a complex that has been purified from a biological sample, generated in a recombinant manner and subsequently purified, or assembled in vitro as described herein. In those embodiments where the RGN or PE ribonucleoprotein complex in contact with the target polynucleotide, cell, organelle, or embryo has been assembled in vitro, the method may further include assembling the complex in vitro prior to contact with the target polynucleotide, cell, organelle, or embryo. Purified or in vitro assembled RGN or PE ribonucleoprotein complexes can be introduced into cells, organelles, or embryos using any method known in this art (e.g., electroporation). In some embodiments, the delivery of the polynucleotide encoding guide RNA, polymerase, PE, and / or RGN polypeptide to cells or embryos is not a method for therapeutically treating the human or animal body or a method for modifying human germline genetic identity. In some embodiments, the method of contacting the target polynucleotide with the RNP complex is not a method for therapeutically treating the human or animal body or a method for modifying human germline genetic identity. In some embodiments, the embryo is a non-human embryo.

[0367] Following delivery to or contact with the target polynucleotide or cells, organelles, or embryos containing the target polynucleotide, the guide RNA directs the RGN peptide or PE to bind to the target sequence within the target polynucleotide in a sequence-specific manner. In embodiments where the RGN has nuclease activity, the RGN peptide cleaves the target sequence of interest upon binding. The target sequence (e.g., the target DNA sequence) can then be modified via endogenous repair mechanisms, such as non-homologous end joining or homology-directed repair using the provided donor polynucleotide. In embodiments where the PE contains an RGN cleavage enzyme, a single strand of the double-stranded DNA target sequence is cleaved, and a DNA synthesis template within the extended arm of the associated PEgRNA serves as a template to introduce the desired edit into the target sequence.

[0368] Methods for measuring the binding of RGN peptides (or PEs containing them) to target sequences are known in this art and include chromatin immunoprecipitation assays, gel migration variation assays, DNA pull-down assays, reporter assays, microplate capture assays, and detection assays. Similarly, methods for measuring the cleavage or modification of target polynucleotides containing target sequences are known in this art and include in vitro or in vivo cleavage assays where cleavage is confirmed using PCR, sequencing, or gel electrophoresis, with or without appropriate labeling (e.g., radioisotopes, fluorescent substances) attached to the target sequence to facilitate the detection of degradation products. Alternatively, nicking-triggered exponential amplification reaction (NTEXPAR) assays can be used (see, for example, Zhang et al. (2016) Chemical Sciences (…)). Chem.Sci. (7:4951-4957). Surveyor analysis can be used to assess in vivo dissection (Guschin et al. (2010) Molecular Biology Methods ( Methods Mol Biol (649:247-256)

[0369] In some implementations, these methods involve the use of a single type of RGN or PE complexed with more than one guide RNA. More than one guide RNA can target different regions of a single gene or can target multiple genes.

[0370] In some embodiments, the method comprises dual polymerase editing, wherein a single PE (or polynucleotide encoding it) and two PEgRNAs (or polynucleotides encoding it) associated with the PE are contacted with a target DNA molecule to programmatically replace or excise DNA sequences. Alternatively, a first PE (or polynucleotide encoding it) and its associated first PEgRNA (or polynucleotide encoding it) and a second PE (or polynucleotide encoding it) and its associated second PEgRNA (or polynucleotide encoding it) are contacted with the target DNA molecule, wherein the first PE and the second PE are different from each other. In both cases, the two PEgRNAs bind to opposite strands of the target DNA such that the intended repair event is installed on both strands of the target DNA. The DNA synthesis templates for the first PEgRNA and the second PEgRNA each encode a single-stranded DNA sequence that is complementary (completely or partially) to each other to replace the DNA region between the two nick sites.

[0371] For large insertions, the DPE DNA synthesis template is designed such that the replacement sequence contains a first recombinase site and introduces donor DNA containing a second recombinase site, along with a site-specific recombinase that recognizes both the first and second recombinase sites. This allows for site-specific integration of the foreign DNA into the target DNA molecule. In embodiments where a donor polynucleotide is not provided, double-strand breaks introduced by the RGN peptide can be repaired via a non-homologous end joining (NHEJ) repair process. Due to the error-prone nature of NHEJ, the repair of double-strand breaks can lead to modifications of the target sequence. As used herein, “modification” of a polynucleotide refers to a change in the nucleotide sequence of the polynucleotide, which may be the deletion, insertion, or substitution of one or more nucleotides, or a combination thereof. Modification of the target polynucleotide containing the target sequence can lead to altered expression of the protein product or inactivation of the coding sequence.

[0372] In embodiments containing donor polynucleotides, the donor sequence in the donor polynucleotide can be integrated into or exchanged with the target nucleotide sequence during the repair of introduced double-strand breaks, thereby introducing an exogenous donor sequence. Therefore, the donor polynucleotide contains a donor sequence that needs to be introduced into the target sequence of interest. In some embodiments, the donor sequence alters the original target nucleotide sequence so that the newly integrated donor sequence will not be recognized and cleaved by the RGN. In some embodiments, the donor polynucleotide is single-stranded DNA. Integration of the donor sequence can be enhanced by including side-joint sequences within the donor polynucleotide, referred herein as “homologous arms,” which have substantial sequence identity with the sequence of the side-joint target nucleotide sequence, thereby allowing for homology-directed repair processes. In some embodiments, the homology arm has a length of at least 30 base pairs, at least 35 base pairs, at least 40 base pairs, at least 45 base pairs, at least 50 base pairs, at least 55 base pairs, at least 60 base pairs, at least 65 base pairs, at least 70 base pairs, at least 75 base pairs, at least 80 base pairs, at least 85 base pairs, at least 90 base pairs, at least 95 base pairs, at least 100 base pairs, and up to 2000 base pairs or more, and has at least 90%, at least 95%, or higher sequence homology with its corresponding sequence within the target nucleotide sequence.

[0373] In embodiments where the RGN peptide introduces double-strand cross-linking breaks, the donor polynucleotide may comprise a donor sequence with a compatible dangling appendage, thereby allowing the donor sequence to be directly linked to the cleaved target nucleotide sequence, which contains the dangling appendage generated during double-strand break repair via a non-homologous repair process.

[0374] In embodiments where the method involves the use of an RGN that is a cleavage enzyme (i.e., capable of cleaving only the single strand of a double-stranded polynucleotide), the method may include introducing two RGN cleavage enzymes that target identical or overlapping target sequences and cleave different strands of the polynucleotide. For example, an RGN cleavage enzyme that cleaves only the positive (+) strand of the double-stranded polynucleotide may be introduced together with a second RGN cleavage enzyme that cleaves only the negative (-) strand of the double-stranded polynucleotide.

[0375] In some embodiments, a method is provided for binding to and detecting a target nucleotide sequence, wherein the method comprises introducing at least one guide RNA or a polynucleotide encoding thereof and at least one RGN polypeptide or a polynucleotide encoding thereof into a cell, organelle, or embryo; expressing the guide RNA and / or the RGN polypeptide (if the coding sequence is introduced), wherein the RGN polypeptide is a nuclease-inactivated RGN and further comprises a detectable marker; and the method further comprises detecting the detectable marker. The detectable marker may be fused to the RGN into a fusion protein (e.g., a fluorescent protein) or may be a small molecule conjugated to or incorporated into the RGN polypeptide, which can be detected visually or otherwise.

[0376] This document also provides methods for regulating the expression of a target gene of interest containing a target sequence or a gene regulated by a target sequence. The methods involve introducing at least one guide RNA or a polynucleotide encoding it and at least one RGN polypeptide or a polynucleotide encoding it into a cell, organelle, or embryo, expressing the guide RNA and / or the RGN polypeptide (if the coding sequence is introduced), wherein the RGN polypeptide is a nuclease-inactivated RGN. In some of these embodiments, the nuclease-inactivated RGN is a fusion protein containing an expression regulator domain (i.e., an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repression domain) as described herein.

[0377] This disclosure also provides methods for binding and / or modifying a target polynucleotide of interest containing a target sequence. These methods include delivering a system comprising a polynucleotide containing at least one guide RNA or encoding therethe and at least one fusion polypeptide or polynucleotide encoding a fusion polypeptide containing the RGN of this disclosure and a base-editing polypeptide (e.g., cytosine deaminase or adenine deaminase) to a target sequence or a cell, organelle, or embryo containing the target sequence.

[0378] In some embodiments utilizing a fusion polypeptide comprising an RGN and a base-editing polypeptide, the binding of the fusion polypeptide to the target sequence causes modification of the nucleotides adjacent to the target sequence. The nucleotides adjacent to the target sequence modified by a deaminase can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 base pairs from the 5' or 3' end of the target sequence.

[0379] In some embodiments utilizing the PE or PE system disclosed herein, by using a DNA synthesis template of varying lengths such that the editing window of a polymerase editor can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 base pairs from the 5' or 3' end of the target sequence, binding of the PE or PE system causes modification of nucleotides within or adjacent to the target sequence.

[0380] To minimize the removal of DNA mismatch repair mechanisms due to editing by a polymerase editor, one or more components of the mismatch repair system can be deactivated, for example, in a dominant-negative form. A non-limiting example of a component of the mismatch repair system that can be deactivated or have its activity reduced is MLH1. Dominant-negative MLH1 or the polynucleotide encoding it can be introduced into the cell along with, before, or after the introduction of the PE system. A non-limiting example of dominant-negative MLH1 is the sequence shown in SEQ ID NO: 334. In some embodiments, the DNA construct encoding PE contains a dominant-negative MLH1 coding sequence at its N-terminus or C-terminus, and the dominant-negative MLH1 coding sequence is linked to the PE coding sequence via a peptide linker and / or NLS. Non-limiting examples of DNA constructs encoding PE containing APG07433.1 nickase and dominant-negative MLH1 are SEQ ID NO: 1146-1153.

[0381] Those skilled in the art will understand that any of the methods disclosed in this invention can be used to target a single target sequence or multiple target sequences. Therefore, the methods include using a single RGN polypeptide or PE and various different guide RNAs, which can target multiple different sequences within a single gene and / or multiple genes. This document also covers methods incorporating multiple different guide RNAs and multiple different RGN polypeptides or PEs. These guide RNAs and guide RNA / RGN or guide RNA / PE systems can target multiple different sequences within a single gene and / or multiple genes.

[0382] IX. Target polynucleotides

[0383] This disclosure provides methods for expressing modified target polynucleotides containing a target sequence or modified target polynucleotides in eukaryotic cells, which can be performed in vivo, in vitro, or ex vivo. In some embodiments, the method includes sampling cells or cell populations from human or non-human animals or plants (including microalgae) and modifying one or more of the cells. Culture can occur in vitro at any stage. One or more cells can even be reintroduced into non-human animals or plants (including microalgae).

[0384] Utilizing natural variability, plant breeders combine the most useful genes to achieve desired qualities such as yield, quality, uniformity, hardiness, and resistance to pests. These desired qualities also include growth, day length preference, temperature requirements, onset date of flowering or reproductive development, fatty acid content, insect resistance, disease resistance, nematode resistance, fungal resistance, herbicide resistance, and tolerance to various environmental factors, including drought, heat, humidity, cold, wind, and unfavorable soil conditions including high salinity. Sources of these useful genes include natural or exotic varieties, native species, close relatives of wild plants, and induced mutations, such as treatment of plant material with mutation inducers. Using this invention, plant breeders are equipped with new tools for inducing mutations. Therefore, those skilled in the art can analyze genomes targeting the sources of useful genes and employ this invention in varieties with desired characteristics or traits to induce the generation of useful genes more precisely than previous mutation inducers, thus promoting and improving plant breeding procedures.

[0385] The target polynucleotide of the RGN or PE system can be any polynucleotide, whether endogenous or exogenous in eukaryotic cells. For example, the target polynucleotide can be a polynucleotide present in the nucleus of a eukaryotic cell. The target polynucleotide can be a sequence encoding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or junk DNA).

[0386] The target polynucleotides of the RGN or PE system disclosed herein may include a variety of disease-related genes and polynucleotides, as well as genes and polynucleotides related to signal transduction biochemical pathways. Examples of target polynucleotides include sequences related to signal transduction biochemical pathways, such as genes or polynucleotides related to signal transduction biochemical pathways. Examples of target polynucleotides include disease-related genes or polynucleotides. A “disease-related” gene or polynucleotide refers to any gene or polynucleotide that produces a transcription or translation product in abnormal amounts or in abnormal forms in cells derived from disease-affected tissues or cells compared to non-disease control tissues or cells. It may be a gene whose expression becomes abnormally high; it may be a gene whose expression becomes abnormally low, wherein the altered expression is associated with the onset and / or progression of the disease. A disease-related gene also refers to a gene having a mutation or genetic variation (e.g., a mutation) that directly causes the cause of the disease or is in linkage disequilibrium with a gene that causes the cause of the disease. The transcribed or translated product may be known or unknown, and may further be in normal or abnormal amounts. In some embodiments, the disease is an animal disease. In some embodiments, the disease may be a avian disease. In some embodiments, the disease may be a mammalian disease. In some implementations, the disease may be a human disease. Non-limiting examples of disease-related genes and polynucleotides in humans are available from the McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and the National Center for Biotechnology Information (Bethesda, Md.) of the National Library of Medicine, and are available on the Global Information Network.

[0387] The method involves contacting a target polynucleotide containing a target sequence with an RGN, PE, or PE system of the present disclosure, wherein the target polynucleotide is contacted with the RGN, PE, or PE system in an effective amount and under conditions suitable for editing the target sequence (e.g., cleavage, insertion of donor polynucleotide, modified expression, base editing, polymerase editing). In some embodiments, the target sequence comprises a sequence associated with a disease or symptom, and editing of the target sequence produces a sequence unrelated to the disease or symptom. In some embodiments, the target sequence is present in the alleles of a crop plant, wherein a specific allele is associated with traits that cause lower agronomic value in the plant. Editing of the target sequence produces alleles associated with traits that increase agronomic value in the plant.

[0388] In some embodiments, the target DNA sequence contains a T→C or A→G point mutation associated with a disease or condition, and deamination of the mutated C or G base produces a sequence unrelated to the disease or condition. In some embodiments, deamination corrects the point mutation in the disease or condition-related sequence.

[0389] In some embodiments, the disease- or condition-associated sequence encodes a protein, and editing of the target sequence introduces a stop codon into the disease- or condition-associated sequence, resulting in a truncation of the encoded protein. In some embodiments, exposure is performed in vivo in subjects susceptible to or diagnosed with the disease or condition. In some embodiments, the disease or condition is associated with a point mutation or a single-base mutation in the genome. In some embodiments, the disease is a genetic disorder, cancer, metabolic disorder, or cytosolic storage disease.

[0390] X. Cells containing polynucleotide gene modifications

[0391] This document provides cells and organisms containing a targeted polynucleotide that has been modified using a method mediated by an RGN or PE system, as described herein. The modified cells can be eukaryotic (e.g., mammalian, plant, insect, avian cells) or prokaryotic. Prokaryotic cells can originate from species including (but not limited to) archaea and bacteria (e.g., Bacillus spp.). Bacillus sp.), Klebsiella spp. Klebsiella sp.), Streptomyces ( Streptomyces sp.), Rhizobium (spe.), Rhizobium genus ( Rhizobium sp.), Escherichia coli ( Escherichia sp.), Pseudomonas spp. Pseudomonas sp.), Salmonella (spe.), Salmonella spp. Salmonella sp.), Shigella spp. Shigella sp.), Vibrio genus ( Vibrio sp.), Yersinia spp. Yersinia sp.), Mycoplasma genus ( Mycoplasma sp.), Agrobacterium, Lactobacillus (sp ... Lactobacillus sp.)).

[0392] Eukaryotic cells may include cells derived from animals (e.g., mammals, humans, insects, fish, birds, and reptiles), plants, fungi, amoebas, algae, and yeast. In some embodiments, cells modified by the methods disclosed in this invention include hematopoietic cells, such as immune system cells, including (but not limited to) B cells, T cells, natural killer (NK) cells, chimeric antigen receptor T (CAR-T) cells, monocytes, macrophages, and dendritic cells. In some embodiments, the cells are pluripotent stem cells or induced pluripotent stem cells. In some embodiments, cells modified by the methods disclosed in this invention include primary cells. In some embodiments, the primary cells are primary T cells.

[0393] The methods described herein can be used for the modification of any plant species, including (but not limited to) monocots and dicots. Examples of plants of interest include (but are not limited to) corn, sorghum, wheat, sunflower, tomato, cruciferous plants, pepper, potato, cotton, rice, soybean, sugar beet, sugarcane, tobacco, barley and rapeseed, brassica, alfalfa, rye, chestnut, safflower, peanut, sweet potato, cassava, coffee, coconut, pineapple, citrus, cocoa, tea, banana, avocado, fig, guava, mango, olive, papaya, cashew, macadamia nut, almond, oats, vegetables, ornamental plants, and conifers.

[0394] Vegetables include (but are not limited to) tomatoes, lettuce, mung beans, lima beans, peas, and members of the Cucumber genus, such as cucumbers, cantaloupes, and honeydew melons. Ornamental plants include (but are not limited to) azaleas, hydrangeas, hibiscus, roses, tulips, daffodils, petunias, carnations, poinsettias, and chrysanthemums. In specific embodiments, the plants of the present invention are crop plants (e.g., corn, sorghum, wheat, sunflowers, tomatoes, cruciferous plants, peppers, potatoes, cotton, rice, soybeans, sugar beets, sugarcane, tobacco, barley, rapeseed, etc.).

[0395] Organelles and embryos are also provided, which contain at least one target sequence modified by means of an RGN or PE system as described herein. The genetically modified cells, organisms, organelles and embryos may be hetero-conjugated or homo-conjugated with respect to the modified target sequence.

[0396] Chromosomal modifications to cells, organisms, organelles, or embryos can cause altered (upregulated or downregulated) expression of protein products or the expression of inactivated or altered sequences. In embodiments where chromosomal modifications result in gene inactivation or the expression of nonfunctional protein products, the genetically modified cell, organism, organelle, or embryo is referred to as a “gene knockout.” Gene knockout phenotypes can result from deletion mutations (i.e., the removal of at least one nucleotide), insertion mutations (i.e., the insertion of at least one nucleotide), or nonsense mutations (i.e., substitution of at least one nucleotide, resulting in the introduction of a stop codon). In some embodiments, chromosomal modifications cause upregulation of the expression of protein products that are absent or reduced due to mutations in the target sequence.

[0397] Alternatively, chromosomal modifications of cells, organisms, organelles, or embryos can produce “gene embeddings,” which arise from the integration of nucleotide sequences encoding proteins into the chromosome. In some of these embodiments, the coding sequence is integrated into the chromosome such that the chromosomal sequence encoding the wild-type protein is inactive, but the exogenously introduced protein is expressed.

[0398] In some embodiments, mutations introduced using the RGN or PE systems disclosed in this invention result in the production of variant protein products. The expressed variant protein products may have at least one amino acid substitution and / or at least one amino acid addition or deletion. When compared to wild-type proteins, variant protein products encoded by altered chromosomal sequences may exhibit improved properties or activities, including (but not limited to) altered enzyme activity or receptor specificity. In some embodiments, chromosomal modifications may cause changes in the protein's expression pattern. As a non-limiting example, chromosomal alterations in the regulatory regions controlling protein product expression may cause overexpression or downregulation of the protein product or changes in its tissue or temporal expression pattern. In some embodiments, gene expression is reduced or eliminated due to mutations introduced by these RGN or PE systems.

[0399] Modified cells can be introduced into organisms. In the case of autologous cell transplantation, these cells can be derived from the same organism (e.g., a human), where the cells are modified in vitro. Alternatively, in the case of allogeneic cell transplantation, the cells are derived from another organism within the same species (e.g., another human). Modified cells can be grown into organisms, such as plants, in a well-known manner. See, for example, McCormick et al. (1986), Plant Cell Reports (…). Plant Cell Reports (See 5:81-84). These plants can then grow and be pollinated with the same or different modified varieties, and the resulting hybrids have genetic modifications. This disclosure provides genetically modified seeds. Progeny, variants, and mutants of regenerated plants are also included within the scope of this disclosure, limited by the fact that these portions contain genetic modifications. A processed plant product or byproduct retaining genetic modifications is also provided, including, for example, soybean flour.

[0400] XI. Pharmaceutical Composition

[0401] Pharmaceutical compositions disclosed herein are provided that may comprise: an RGN polypeptide (or a PE containing thereof) or an active variant and fragment thereof, and a polynucleotide encoding thereof; a gRNA or an active variant and fragment thereof, or a polynucleotide encoding thereof, as described herein; an RGN system comprising an RGN polypeptide and / or gRNA as described herein; a polymerase; a PE system (including a DPE system); or a cell as described herein comprising an RGN polypeptide (or a PE containing thereof) or a polynucleotide encoding a polymerase, an RGN or PE, a gRNA or a polynucleotide encoding a gRNA, or a polymerase or an RGN or PE system (including a DPE system); and a pharmaceutically acceptable carrier.

[0402] The pharmaceutical composition is a composition for preventing, reducing the intensity of, curing or otherwise treating a target symptom or disease, comprising an active ingredient (i.e., an RGN polypeptide, a polynucleotide encoding RGN, a polymerase, a polynucleotide encoding a polymerase, a PE, a polynucleotide encoding a PE, a gRNA, a polynucleotide encoding a gRNA, an RGN system, a PE system (including a DPE system), or a cell comprising any of these) and a pharmaceutically acceptable carrier.

[0403] As used herein, a "pharmaceutically acceptable carrier" is a substance that does not significantly irritate an organism and does not eliminate the activity and properties of the active ingredient (i.e., RGN polypeptides, polynucleotides encoding RGN, polymerases, polynucleotides encoding polymerases, PE, polynucleotides encoding PE, gRNA, polynucleotides encoding gRNA, RGN systems, PE systems (including DPE systems), or cells containing any of these). The carrier must be of sufficiently high purity and sufficiently low toxicity to be suitable for administration to the treated subject. The carrier may be inert or may have a pharmaceutical benefit. In some embodiments, a pharmaceutically acceptable carrier comprises one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to humans or other vertebrates. In some embodiments, the pharmaceutically acceptable carrier is not naturally occurring. In some embodiments, the pharmaceutically acceptable carrier and active ingredient are not found together in nature.

[0404] The pharmaceutical compositions used in the methods disclosed in this invention can be formulated with suitable carriers, excipients, and other agents that provide suitable transfer, delivery, tolerability, and similar properties. A variety of suitable formulations are known to those skilled in the art. See, for example, Remington, *The Science and Practice of Pharmacy* (21st edition, 2005). Suitable formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipid-containing vesicles (such as LIPOFECTIN vesicles), lipid nanoparticles, DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbowax (polyethylene glycol of various molecular weights), semi-solid gels containing carbowax, and semi-solid mixtures. Pharmaceutical compositions for oral or non-enteral use can be prepared in unit dose dosage forms appropriate to the dosage of the active ingredient. Such unit dose dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc.

[0405] This disclosure provides pharmaceutical compositions comprising lipid-based formulations, which include an active ingredient (i.e., an RGN polypeptide, a polynucleotide encoding an RGN, a polymerase, a polynucleotide encoding a polymerase, a PE, a polynucleotide encoding a PE, gRNA, a polynucleotide encoding gRNA, an RGN system, a PE system (including a DPE system), or a cell comprising any of these). Lipid-based formulations may include liposomes. Lipid-based formulations may include lipid nanoparticles (LNPs). In some embodiments, the active ingredient is encapsulated in liposomes and / or disposed on the surface of liposomes. In some embodiments, the active ingredient is covalently attached to liposomes. In some embodiments, the active ingredient is non-covalently associated with liposomes. Covalent attachment includes sharing electrons in a chemical bond. Non-covalent interactions include dispersed electromagnetic interactions such as hydrogen bonds, ionic bonds, van der Waals interactions, and hydrophobic bonds.

[0406] In some embodiments, the active ingredient is encapsulated in liposomes. The term "encapsulation" means to close, surround, or cover. Encapsulation can be substantial, complete, or partial, as it relates to the formulation of the compounds of this disclosure. The term "substantially encapsulated" or "substantially encapsulated" means that more than 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9% or more of the pharmaceutical composition or active ingredient of this disclosure is closed, surrounded, or covered within a delivery agent (e.g., liposomes or LNPs). The term "partially encapsulated" or "partial encapsulation" means that less than 50%, 40%, 30%, 20%, 10%, or less of the pharmaceutical composition or active ingredient of this disclosure is closed, surrounded, or covered within a delivery agent. Encapsulation can be determined by measuring the escape or activity of the pharmaceutical composition or active ingredient of this disclosure using fluorescence and / or electron microscopy. For example, at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9% or more of the pharmaceutical composition or active ingredient of this disclosure are encapsulated in a delivery agent (e.g., liposomes or LNPs).

[0407] Liposomes are spherical vesicle structures consisting of a single or multiple lipid bilayers surrounding an internal aqueous compartment and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes have attracted considerable attention as drug delivery carriers because they are biocompatible, non-toxic, capable of delivering both hydrophilic and lipophilic drug molecules, protecting their load from degradation by plasma enzymes, and transporting their load across biological membranes and the blood-brain barrier (BBB) ​​(see, for example, Spuch and Navarro (2011), Journal of Drug Delivery, 2011).

[0408] Liposomes can be made from several different types of lipids (e.g., ionizable lipids, structural lipids, accessory lipids, and PEGylated lipids); however, phospholipids are most commonly used to generate liposomes as drug carriers. Although liposomes spontaneously form when lipid membranes are mixed with aqueous solutions, liposome formation can be accelerated by applying force in an oscillatory manner using homogenizers, sonic processors, or extrusion devices (see, for example, Spuch and Navarro (2011), Journal of Drug Delivery, 2011).

[0409] Liposome preparations are well known to mainly contain natural phospholipids and substances such as 1,2-distearate- sn Phospholipids of glycero-3-phosphatidylcholine (DSPC), sphingomyelin, lecithin, and monosialotetrahexosylganglioside. In some embodiments, 1,2-dioleoyl- sn 3-glycerol-3-phosphate ethanolamine (DOPE) increases the stability of liposomes.

[0410] Additives can be added to liposomes to improve their structure and properties. In some embodiments, cholesterol and / or sphingomyelin can be added to the liposome mixture to help stabilize the liposome structure and prevent leakage of the internal carriers. In some embodiments, adding cholesterol to a well-known liposome formulation can reduce the rapid release of encapsulated active ingredients (i.e., RGN peptides, polynucleotides encoding RGN, polymerases, polynucleotides encoding polymerases, PE, polynucleotides encoding PE, gRNA, polynucleotides encoding gRNA, RGN systems, PE systems (including DPE systems), or cells containing any of these) into the plasma. In some embodiments, the liposomes are prepared from hydrogenated lecithin choline or lecithin choline, cholesterol, and dicetyl phosphate. In some embodiments, the average liposome vesicle size is adjusted to approximately 50 or 100 nm. In some embodiments, Trojan horse liposomes (also known as molecular Trojans or PEGylated immunoliposomes) can be used in pharmaceutical compositions to deliver the active ingredient across the blood-brain barrier (BBB) ​​(described on the Global Information Network at cshprotocols.cshlp.org / content / 2010 / 4 / pdb.prot5407.long). Without being bound by any theory, it is believed that neutral liposomes with specific antibodies conjugated to their surface allow for crossing the BBB via endocytosis. In some embodiments, pharmaceutical compositions containing Trojan horse liposomes can be used to deliver the active ingredient (i.e., RGN peptides, polynucleotides encoding RGN, polymerases, polynucleotides encoding polymerases, PE, polynucleotides encoding PE, gRNA, polynucleotides encoding gRNA, the RGN system, the PE system (including the DPE system), or cells containing any of these) to the brain via intravascular injection.

[0411] In some embodiments, the liposomes comprise stable nucleic acid-liposomes (SNALP) (see, for example, Morrissey et al. (2005), Nature Biotechnology 23(8):1002-1007; Zimmerman et al. (2006), Nature 441:111-114). SNALPs comprise a mixture of cationic lipids and fusion lipids, coated with polyethylene glycol (PEG) to allow for cellular uptake and endosome release of the active ingredient carrier. In some embodiments, SNALPs are a class of LNPs and include ionizable lipids that are cationic at low pH (e.g., DLinDMA, COATSOME® SS-OC), neutral accessory lipids (e.g., DSPC), cholesterol, and diffusible polyethylene glycol (PEG)-lipids (e.g., BrijS100). In some embodiments, the SNALP formulation comprises the following lipids: 3- N-(-Methoxypoly(ethylene glycol)2000)carbamoyl-1,2-bis(tetradecanoyloxy)-propylamine (PEG-cDMA); 1,2-Dilinoleyloxy- N , N -Dimethyl-3-aminopropane (DLinDMA); 1,2-distearate- sn - Glyceryl-3-phosphate choline (DSPC); and cholesterol. In some embodiments, SNALP comprises synthetic cholesterol, dipalmitoylphosphatidylcholine (DOPC), PEG-cDMA, and DLinDMA (see, for example, Geisbert et al. (2010), *The Lancet* 375:1896-1905). In some embodiments, SNALP comprises synthetic cholesterol, DSPC, PEG-cDMA, and DLinDMA (see, for example, Judge et al. (2009), *J. Clin. Invest.* 119:661-673). In some embodiments, SNALP formulations comprise COATSOME® SS-OC, DSPC, Brij S100, and cholesterol. In some embodiments, SNALP formulations comprise ionizable lipids, DSPC, cholesterol, and PEG lipids. In some embodiments, the SNALP formulation includes ionizable lipids and / or PEG lipids disclosed in WO2022173531 or WO2022173531, each of which is incorporated herein by reference in its entirety.

[0412] In some implementations, the size of SNALP liposomes is approximately 80–100 nm. SNALP has been used as an efficient delivery molecule for delivery to highly vascularized HepG2-derived liver tumors (see, for example, Li et al. (2012) Gene Therapy 19:775–780).

[0413] Unbound by any single theory, during SNALP formulation, ionizable lipids are used to condense lipids with the active ingredient (e.g., polynucleotides) during particle formation. When positively charged under increased acidity in endosome conditions, ionizable lipids mediate the fusion of SNALP with the endosome membrane, allowing the active ingredient to be released into the cytoplasm. PEG-lipids stabilize the particles and reduce aggregation during formulation, and subsequently provide a neutral hydrophilic exterior that improves pharmacokinetic properties. In some embodiments, SNALP liposomes are prepared by formulating DLinDMA and PEG-cDMA with DSPC, cholesterol, and the active ingredient in the following ratios: 25:1 lipid:active ingredient ratio and 48:40:10:2 molar ratio of cholesterol:DLinDMA:DSPC:PEG-cDMA.

[0414] In some embodiments, the pharmaceutical compositions of this disclosure include LNPs. In some embodiments, lipids may be formulated with the active ingredient of this disclosure to form LNPs. LNPs comprise multiple lipid molecules physically associated with each other by intermolecular forces. In some embodiments, LNPs comprise liposomes. In some embodiments, LNPs differ from liposomes in that they do not have a continuous lipid bilayer. In some embodiments, LNPs comprise solid particles having a mixture of solid and liquid lipids. In some embodiments, LNPs comprise dendritic polymeric lipid nanoparticles (DLNPs), SNALPs, and lipid-like nanoparticles (LLNPs). Generally, "nanoparticles" refers to any particle with a diameter less than 1000 nanometers (nm). In some embodiments, the diameter of the nanoparticles is 500 nm or less. In some embodiments, the diameter of the nanoparticles is in the range of 25 nm to 200 nm or 100 nm or less. In some embodiments, the diameter of the nanoparticles is in the range of 35 nm to 60 nm. In some embodiments, LNPs comprise liposomes with a size between about 1 nm and about 100 nm.

[0415] LNPs comprise four components: ionizable cationic lipids, fused zwitterionic phospholipids, cholesterol, and PEGylated lipids. In some embodiments, the ionizable cationic lipid component complexes with negatively charged polynucleotides and enhances endosome escape. In some embodiments, the phospholipid component functions in modifying the lipid bilayer structure. In some embodiments, the cholesterol component helps stabilize LNPs. In some embodiments, the PEGylated lipid component reduces LNP aggregation and nonspecific uptake.

[0416] In some embodiments, LNPs include ionizable lipids that are cationic at low pH (e.g., DLinDMA, COATSOME® SS-OC), neutral accessory lipids (e.g., DSPC), cholesterol, and diffusible polyethylene glycol (PEG)-lipids (e.g., Brij S100). In some embodiments, LNPs include the following lipids: 3- N -(-Methoxypoly(ethylene glycol)2000)carbamoyl-1,2-bis(tetradecanoyloxy)-propylamine (PEG-cDMA); 1,2-Dilinoleyloxy- N , N -Dimethyl-3-aminopropane (DLinDMA); 1,2-distearate- sn- Glyceryl-3-phosphate choline (DSPC); and cholesterol. In some embodiments, the LNP includes synthetic cholesterol, dipalmitoylphosphatidylcholine (DOPC), PEG-cDMA, and DLinDMA (see, for example, Geisbert et al. (2010) Lancet 375:1896-1905). In some embodiments, the LNP includes synthetic cholesterol, DSPC, PEG-cDMA, and DLinDMA (see, for example, Judge et al. (2009) J. Clin. Invest. 119:661-673).

[0417] Ionizable cationic lipids suitable for use with LNP include: COATSOME® SS-OC, 1,2-dilinoleoyl-3-dimethylammonium-propane (DLinDAP); DLinDMA; 1,2-dilinoleyloxy-keto- N,N -Dimethyl-3-aminopropane (DlinK-DMA); 1,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxane (DlinKC2-DMA); 5A2-SC8 (Zhou et al. (2016) Proc. Natl Acad. Sci. USA 113:520-525); C12-200 (Love et al. (2010) Proc. Natl Acad. Sci. USA 107:1864-1869); 246C10 (Kim et al. (2021) Sci Adv 7 (9):eabf4398); cKK-E12 (Fenton et al. (2016) Advanced Materials Materials) 28(15):2939-2943); 1,2-Distearateloxy- N,N -Dimethyl-3-aminopropane (DSDMA); 1,2-Dioleoyloxy- N , N -Dimethyl-3-aminopropane (DODMA); 1,2-dimethyllinolenic acid- N , N-Dimethyl-3-aminopropane (DLenDMA); and dilinoleylmethyl-4-dimethylaminobutyrate (Dlin-MC3-DMA; Jayaraman et al. (2012) Angew Chem Int Ed Engl. 51(34): 8529-8533). The cationic lipids are further described in International Publications Nos. WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365, and WO201204463. The following are references to U.S. Patent Nos. 8, WO2010080724, WO201021865, WO2022173531, WO2022150485 and WO2008103276, U.S. Patent Nos. 7,893,302 and 7,404,969 and U.S. Patent Publication No. US20100036115, each of which is incorporated herein by reference in its entirety.

[0418] Zwitterionic phospholipids suitable for LNP include DSPC, DOPE, and DOPC. PEG lipids suitable for LNP include: 1,2-dimyristoyl-rac-glycero-3-methoxy polyethylene glycol (PEG-DMG); (3-o-[2''-(methoxy-polyethylene glycol 2000)succinyl]-1,2-dimyristoyl-sn-diol (PEG-S-DMG); R-3-[(ω-methoxy-poly(ethylene glycol) 2000)aminoformyl]-1,2-dimyristoyloxypropyl-3-amine (PEG-C-DOMG); and C16 PEG-ceramide. In some embodiments, LNP comprises DlinKC2-DMA or C12-200:DSPC:cholesterol:PEG-DMG in a 50:10:38.5:1.5 molar ratio (e.g., Basha et al. (2011) Molecular Therapy). Therapy, 19(12):2186-2200. In some embodiments, the LNP comprises 26.5:20:52:1.5 ionizable lipid:DOPE:cholesterol:PEG lipid (e.g., Han et al. (2022) Sci Advances 8(3): eabj6901; Kim et al. (2021) Sci Advances 7(9): eabf4398). PEG lipids are further described in WO2012099755, WO2022173531 and WO2022150485, each of which is incorporated herein by reference in its entirety. In some embodiments, the ratio of PEG in the LNP formulation may be increased or decreased and / or the carbon chain length of the PEG lipid may be modified within C14 to C18 to alter the pharmacokinetics and / or biodistribution of the LNP formulation.

[0419] In some embodiments, the LNP formulation includes COATSOME® SS-OC, DSPC, Brij S100, and cholesterol. In some embodiments, the LNP formulation includes ionizable lipids, DSPC, cholesterol, and PEG lipids. In some embodiments, the LNP formulation includes ionizable lipids and / or PEG lipids disclosed in WO2022173531 or WO2022150485, each of which is incorporated herein by reference in its entirety. In some embodiments, the LNP formulation includes ionizable lipids and / or PEG lipids, DSPC, and cholesterol disclosed in WO2022173531 or WO2022150485 (each of which is incorporated herein by reference in its entirety). In some embodiments, the LNP formulation includes any one of CAT1-CAT35 and any one of CHM-001-CHM-016 disclosed in WO2022173531 or WO2022150485 (each of which is incorporated herein by reference in its entirety). In some embodiments, the LNP formulation includes any one of CAT1-CAT35 and any one of CHM-001-CHM-016, DSPC, and cholesterol as disclosed in WO2022173531 or WO2022150485 (each of which is incorporated herein by reference in its entirety).

[0420] In some embodiments, the LNP of this disclosure comprises 44-60 mol% cationic lipids, 19-25 mol% accessory lipids, 25-33 mol% structural lipids, and 0.2-0.8 mol% PEG-lipids, including endpoints. In some embodiments, the LNP of this disclosure comprises 44-54 mol% cationic lipids, 19-25 mol% accessory lipids, 24-32 mol% structural lipids, and 1.2-1.8 mol% PEG-lipids, including endpoints. In some embodiments, the LNP of this disclosure comprises 44-54 mol% cationic lipids, 8-14 mol% accessory lipids, 35-43 mol% structural lipids, and 1.2-1.8 mol% PEG-lipids, including endpoints. In some embodiments, the LNP of this disclosure comprises 45-55 mol% cationic lipids, 5-9 mol% accessory lipids, 36-44 mol% structural lipids, and 2.5-3.5 mol% PEG-lipids, including endpoints.

[0421] In some embodiments, the LNP of this disclosure comprises 49 mol% cationic lipids, 22 mol% accessory lipids, 28.5% structural lipids, and 0.05 mol% PEG-lipids. In some embodiments, the LNP of this disclosure comprises 49 mol% SS-OC, 22 mol% DSPC, 28.5 mol% cholesterol, and 0.05 mol% Brij S100.

[0422] In some embodiments, the LNP of this disclosure comprises 50 mol% cationic lipids, 7 mol% accessory lipids, 40% structural lipids, and 3 mol% PEG-lipids. In some embodiments, the LNP of this disclosure comprises 50 mol% ionizable lipids, 7 mol% DSPC, 40 mol% cholesterol, and 0.05 mol% PEG-lipids. In some embodiments, the LNP of this disclosure comprises 50 mol% of any one of CAT1-CAT35 disclosed in WO2022173531 or WO2022150485 (each of which is incorporated herein by reference in its entirety), 7 mol% DSPC, 40 mol% cholesterol, and 0.05 mol% of any one of CHM-001-CHM-016 disclosed in WO2022173531 or WO2022150485 (each of which is incorporated herein by reference in its entirety). In some embodiments, the LNP of this disclosure comprises CAT7, 7 mol% DSPC, 40 mol% cholesterol, and CHM-006 disclosed in 50 mol% WO2022173531 or WO2022150485 (each of which is incorporated herein by reference in its entirety).

[0423] In some implementations, the LNP size is approximately 80-100 nm. In some implementations, the LNP size is approximately 80 nm, approximately 81 nm, approximately 82 nm, approximately 83 nm, approximately 84 nm, approximately 85 nm, approximately 86 nm, approximately 87 nm, approximately 88 nm, approximately 89 nm, approximately 90 nm, approximately 91 nm, approximately 92 nm, approximately 93 nm, approximately 94 nm, approximately 95 nm, approximately 96 nm, approximately 97 nm, approximately 98 nm, approximately 99 nm, or approximately 100 nm. The size of individual LNPs within a LNP population can vary by approximately +5 nm.

[0424] In some implementations, the charge of the LNP is taken into consideration. Cationic lipids can be combined with negatively charged lipids to induce non-bilayer structures that facilitate intracellular delivery. Since charged LNPs are rapidly cleared from circulation after intravenous injection, ionizable cationic lipids with pKa values ​​below 7 have been developed (see, for example, Basha et al. (2011), Molecular Therapy 19(12):2186-2200). Negatively charged polymers, such as polynucleotides, can be loaded into LNPs at low pH values ​​(e.g., pH 4) where ionizable lipids exhibit a positive charge. However, at physiological pH values, LNPs exhibit a low surface charge compatible with longer circulation times.

[0425] LNP preparation and encapsulation of active ingredients are described in, for example, Basha et al. (2011) Molecular Therapy 19(12):1286-2200; Han et al. (2022) Sci Advances 8(3):eabj6901; Kim et al. (2021) Sci Advances 7(9):eabf4398; Finn et al. (2018) Cell Reports 22:2227-2235; Wei et al. (2020) Nature Communications 11:3232; WO2011127255; and WO2008103276. The lipids are commercially available (e.g., from Tekmira Pharmaceuticals, Vancouver, Canada; Avanti Polar Lipids, Alabaster, AL) or synthetic (e.g., Kim et al. (2021) Sci Advances 7(9): eabf4398). The synthesis of cationic lipids is described in International Publications Nos. WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365, WO2012044638, WO2010080724 and WO201021865. Cholesterol is available (e.g. from Sigma-Aldrich, St Louis, MO).

[0426] In some embodiments, encapsulation can be performed by dissolving a lipid mixture comprising cationic lipids (e.g., Dlin-DMA):phospholipids (e.g., DSPC, DOPE):cholesterol:PEG-lipids (e.g., 40:10:40:10 molar ratio) in ethanol. The active ingredient (e.g., a polynucleotide containing or encoding the guide RNA or RGN disclosed herein) can be dissolved in an acidic buffer (e.g., citrate, acetate) at pH 3 or 4. In some embodiments, the lipid and active ingredient solutions can be mixed using a microfluidic system (Chen et al. (2012), Journal of the American Chemical Society, 134:6948-6951; e.g., NanoAssemblr from Precision Nanosystems) or by adding the lipid solution dropwise to the active ingredient solution. Ethanol removal and neutralization with the prepared buffer can be performed by dialyzing against phosphate-buffered saline (PBS) for, for example, 16 hours or overnight using a dialysis cartridge (e.g., a 3500 molecular weight cutoff cartridge from Life Technologies). Dynamic light scattering can be used to assess LNP size, polydispersity index (PDI), and zeta potential. Encapsulation efficiency of active ingredients (such as RNA) can be measured using methods such as Quant-it. TM Analytical determination using Ribogreen (Thermo Fisher) assay. In some embodiments where the encapsulated active ingredient is a polynucleotide, the polynucleotide is extracted from self-eluting nanoparticles and quantified at 260 nm. The LNP pKa can be evaluated u...

Claims

1. One or more polynucleotides encoding a polymerase editor (PE) comprising a DNA polymerase and an RNA-guided nuclease (RGN) polypeptide, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, 2, 7, 565, 566, 579, 583, 584, 591 or 1165-1179.

2. The one or more polynucleotides according to claim 1, wherein the RGN polypeptide comprises an amino acid sequence having the following elements: a) Has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and includes: i) S at position corresponding to amino acid position 1020 in SEQ ID NO: 1; ii) G at position 1021 corresponding to amino acid position in SEQ ID NO: 1; iii) N at position 1022 corresponding to amino acid position in SEQ ID NO: 1; iv) K at position corresponding to amino acid position 1023 in SEQ ID NO: 1; and v) E at position corresponding to amino acid position 1025 in SEQ ID NO: 1; b) Having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 579, wherein the RGN polypeptide contains Q at position corresponding to amino acid position 1023 in SEQ ID NO: 579; c) Having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 584, wherein the RGN polypeptide contains a T at position 1021 corresponding to amino acid position 1021 in SEQ ID NO: 584 and an N at position 1023 corresponding to amino acid position 1023 in SEQ ID NO: 584; d) Having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 583, wherein the RGN polypeptide contains N at position 1021 corresponding to amino acid position 1023 in SEQ ID NO: 583; or e) Having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 591, wherein the RGN polypeptide comprises: i) Q at position 975 corresponding to amino acid position in SEQ ID NO: 591; ii) A at position A corresponding to amino acid position 1020 in SEQ ID NO: 591; iii) G at position 1022 corresponding to amino acid position in SEQ ID NO: 591; and iv) Q at position 1023 corresponding to amino acid position in SEQ ID NO:

591.

3. One or more polynucleotides according to claim 1, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequences shown in SEQ ID NO: 1, 2, 7, 565, 566, 579, 583, 584, 591 or 1165-1179.

4. One or more polynucleotides according to any one of claims 1 or 3, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1, 2, 7, 565, 566, 579, 583, 584, 591 or 1165-1179.

5. One or more polynucleotides according to any one of claims 1 to 4, comprising at least a first polynucleotide and a second polynucleotide, wherein the first polynucleotide comprises a nucleotide sequence encoding the DNA polymerase and the second polynucleotide comprises a nucleotide sequence encoding the RGN polypeptide.

6. One or more polynucleotides according to any one of claims 1 to 4, wherein the nucleotide sequence encoding the DNA polymerase and the nucleotide sequence encoding the RGN polypeptide are contained within a single polynucleotide.

7. One or more polynucleotides according to claim 6, wherein a single promoter is operatively linked to the nucleotide sequence encoding the DNA polymerase and the nucleotide sequence encoding the RGN polypeptide.

8. One or more polynucleotides according to claim 7, wherein the single polynucleotide further comprises a nucleotide sequence encoding a self-cleaving peptide, the nucleotide sequence encoding the self-cleaving peptide being between the nucleotide sequence encoding the DNA polymerase and the nucleotide sequence encoding the RGN polypeptide.

9. One or more polynucleotides according to any one of claims 1 to 7, wherein the nucleotide sequence encoding the DNA polymerase and the nucleotide sequence encoding the RGN polypeptide are translated into two independent polypeptides.

10. One or more polynucleotides according to claim 8 or 9, wherein the DNA polymerase, the RGN polypeptide, or both the DNA polymerase and the RGN polypeptide are operatively fused with one or more nuclear localization signals (NLS).

11. One or more polynucleotides according to claim 10, wherein the NLS is selected from the group consisting of SEQ ID NO: 107-109, 2086-2101, 2118 and 2119.

12. One or more polynucleotides according to claim 10, wherein the DNA polymerase, the RGN polypeptide, or both the DNA polymerase and the RGN polypeptide are operatively fused to the NLS at their N-terminus, C-terminus, or both their N-terminus and C-terminus.

13. One or more polynucleotides according to any one of claims 10 to 12, wherein the DNA polymerase, the RGN polypeptide, or both the DNA polymerase and the RGN polypeptide are further operablely fused with one or more adapter sequences, the one or more adapter sequences being intermediate between the one or more NLS and the DNA polymerase, the RGN polypeptide, or both the DNA polymerase and the RGN polypeptide.

14. One or more polynucleotides according to claim 7, wherein the DNA polymerase is operatively fused to the RGN polypeptide.

15. One or more polynucleotides according to claim 14, wherein the PE comprises the DNA polymerase fused at the amino terminus of the RGN polypeptide.

16. One or more polynucleotides according to claim 14, wherein the PE comprises the DNA polymerase fused at the carboxyl terminus of the RGN polypeptide.

17. One or more polynucleotides according to any one of claims 14 to 16, wherein the PE is operably fused with one or more NLS.

18. One or more polynucleotides according to claim 17, wherein the NLS is selected from the group consisting of SEQ ID NO: 107-109, 2086-2101, 2118 and 2119.

19. One or more polynucleotides according to claim 17 or 18, wherein the PE is operably fused to the NLS at its N-terminus, C-terminus, within the PE, or in a combination thereof.

20. One or more polynucleotides according to any one of claims 14 to 19, wherein the PE is further operablely fused with one or more adapter sequences.

21. One or more polynucleotides according to claim 20, wherein at least one of the one or more adapter sequences is between the DNA polymerase and the RGN polypeptide.

22. One or more polynucleotides according to claim 21, wherein the one or more linker sequences have the formula -(SGGS) x -NLS m -(SGGS) y -NLS n -(SGGS) z - where x, y, or z are 0, 1, 2, 3, or 4 respectively; and where m or n are 0 or 1 respectively.

23. The one or more polynucleotides of claim 21, wherein the one or more linker sequences between the DNA polymerase and the RGN polypeptide comprise one or more copies of the amino acid sequence SGGS.

24. One or more polynucleotides according to any one of claims 21 to 23, wherein the one or more linker sequences between the DNA polymerase and the RGN polypeptide have a length of 42 amino acids.

25. One or more polynucleotides according to any one of claims 21 to 24, wherein the one or more adapter sequences between the DNA polymerase and the RGN polypeptide contain at least one NLS.

26. One or more polynucleotides according to claim 25, wherein the one or more linker sequences between the DNA polymerase and the RGN polypeptide comprise two NLS.

27. One or more polynucleotides according to any one of claims 21 to 26, wherein the one or more adapter sequences between the DNA polymerase and the RGN polypeptide comprise the sequence shown in SEQ ID NO: 110, 366, 603, 605, 2120 or 2126.

28. One or more polynucleotides according to claim 14, wherein the PE comprises the DNA polymerase inserted into the RGN polypeptide.

29. One or more polynucleotides according to claim 28, wherein the DNA polymerase is inserted into the adapter domain 2, wedge domain, RuvC domain, HNH domain, Rec-2 domain or PAM interaction domain of the RGN polypeptide, or wherein the DNA polymerase is inserted between the adapter domain 2, wedge domain, RuvC domain, HNH domain, Rec-2 domain or PAM interaction domain of the RGN polypeptide and another domain located at the 5' or 3' of the adapter domain 2, wedge domain, RuvC domain, HNH domain, Rec-2 domain or PAM interaction domain.

30. One or more polynucleotides according to claim 29, wherein the RuvC domain is a RuvCIII domain.

31. One or more polynucleotides according to claim 28, wherein the DNA polymerase is inserted into the adapter domain 2, wedge domain or RuvCIII domain of the RGN polypeptide.

32. One or more polynucleotides according to claim 28, wherein the DNA polymerase is inserted into the RGN polypeptide immediately following an amino acid selected from the group consisting of: a) The amino acid position corresponding to position 678 of SEQ ID NO: 2; b) The amino acid position corresponding to position 736 of SEQ ID NO: 2; c) The amino acid position corresponding to position 922 of SEQ ID NO: 2; d) The amino acid position corresponding to position 642 of SEQ ID NO: 1, 7, 579, 583, 584 or 591; e) The amino acid position 772 corresponding to SEQ ID NO: 1, 7, 579, 583, 584 or 591; f) The amino acid position corresponding to position 739 of SEQ ID NO: 565; g) The amino acid position corresponding to position 744 of SEQ ID NO: 565; h) The amino acid position corresponding to position 680 of SEQ ID NO: 566; i) The amino acid position corresponding to position 785 of SEQ ID NO: 566; and j) The amino acid position corresponding to position 910 of SEQ ID NO:

566.

33. One or more polynucleotides according to any one of claims 28 to 32, wherein the PE is operably fused with one or more NLS.

34. One or more polynucleotides according to claim 33, wherein the NLS is selected from the group consisting of SEQ ID NO: 107-109, 2086-2101, 2118 and 2119.

35. One or more polynucleotides according to claim 33 or 34, wherein the PE is operably fused to the NLS at its N-terminus, C-terminus, within the PE, or in a combination thereof.

36. One or more polynucleotides according to any one of claims 33 to 35, wherein the PE is operably fused to one or more adapter sequences.

37. One or more polynucleotides according to claim 36, wherein the one or more adapter sequences comprise one or more copies of the amino acid sequence SGGS.

38. One or more nucleic acid molecules according to claim 36 or 37, wherein at least one of the one or more adapter sequences is between the DNA polymerase and the fragment of the RGN polypeptide.

39. One or more polynucleotides according to any one of claims 1 to 38, wherein when bound to guide RNA (gRNA), the RGN polypeptide is capable of binding to a target sequence in the target polynucleotide in an RNA-guided sequence-specific manner, wherein the target sequence comprises a target strand and a non-target strand, and wherein the gRNA is capable of hybridizing with the target strand of the target sequence.

40. One or more polynucleotides according to claim 39, wherein the RGN polypeptide recognizes a protospacer adjacent motif (PAM) at the 3' of the target sequence.

41. One or more polynucleotides according to claim 40, wherein: a) The RGN polypeptide containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1 recognizes PAM having a common nucleotide sequence shown in NNNNCY; b) The RGN polypeptide containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 7 recognizes PAM having a common nucleotide sequence shown in NNNNCC; c) The RGN polypeptide containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 2 recognizes PAM having a common nucleotide sequence shown in NNRYA; d) The RGN polypeptide containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 565 recognizes PAM having a common nucleotide sequence shown as NNGRR; e) The RGN polypeptide containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 566 recognizes PAM having a common nucleotide sequence shown in NNGG; f) The RGN polypeptide containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 579 or 583 recognizes PAM having a common nucleotide sequence shown in NNNNC; g) The RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 584 recognizes PAM having a common nucleotide sequence shown in NNNNCM; or h) The RGN polypeptide containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 591 recognizes PAM having a common nucleotide sequence shown in NNNNCT.

42. One or more polynucleotides according to any one of claims 39 to 41, wherein the RGN polypeptide is capable of cleaving the target polynucleotide upon binding.

43. One or more polynucleotides according to claim 42, wherein the RGN polypeptide is capable of producing double-strand breaks.

44. One or more polynucleotides according to claim 42, wherein the RGN polypeptide is capable of generating single-strand breaks.

45. One or more polynucleotides according to claim 44, wherein the RGN polypeptide comprises an RGN nickase, the RGN nickase comprising at least one mutated HNH domain having reduced or eliminated nuclease activity.

46. ​​One or more polynucleotides according to claim 44, wherein the RGN polypeptide does not contain an HNH domain.

47. One or more polynucleotides according to claim 46, wherein the HNH domain of the RGN polypeptide has been replaced by the DNA polymerase.

48. One or more polynucleotides according to claim 47, wherein the DNA polymerase is operatively fused to one or more adapter sequences.

49. One or more polynucleotides according to claim 48, wherein at least one of the one or more adapter sequences is between the DNA polymerase and the RGN polypeptide.

50. One or more polynucleotides according to claim 45, wherein the RGN nickase comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 8, 106, 365, 567 or 568.

51. One or more polynucleotides according to claim 45 or 50, wherein the RGN nickase comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 8, 106, 365, 567 or 568.

52. One or more polynucleotides according to any one of claims 45, 50 and 51, wherein the RGN nickase comprises the amino acid sequence shown in SEQ ID NO: 8, 106, 365, 567 or 568.

53. One or more polynucleotides according to claim 45, wherein the HNH domain comprises at least two mutations that reduce or eliminate its nuclease activity.

54. One or more polynucleotides according to claim 53, wherein the RGN nickase comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 367, 368, 369, 569 or 570.

55. One or more polynucleotides according to claim 53 or 54, wherein the RGN nickase comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 367, 368, 369, 569 or 570.

56. One or more polynucleotides according to any one of claims 53 to 55, wherein the RGN nickase comprises the amino acid sequence shown in SEQ ID NO: 367, 368, 369, 569 or 570.

57. One or more polynucleotides according to any one of claims 39 to 41, wherein the RGN polypeptide is free of nuclease activity.

58. One or more polynucleotides according to any one of claims 1 to 57, wherein one or more nucleotide sequences encoding the PE are codon-optimized for expression in eukaryotic cells.

59. One or more polynucleotides according to claim 58, wherein the eukaryotic cell is a mammalian cell.

60. One or more polynucleotides according to any one of claims 1 to 59, wherein the DNA polymerase is a reverse transcriptase (RT).

61. One or more polynucleotides according to claim 60, wherein the RT is Moloney murine leukemia virus RT (MMLV RT).

62. One or more polynucleotides according to claim 60, wherein the RT has an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NO: 174 and 2127-2131.

63. One or more polynucleotides according to claim 60 or 62, wherein the RT has an amino acid sequence having at least 95% sequence identity with any one of SEQ ID NO:174 and 2127-2131.

64. One or more polynucleotides according to any one of claims 60, 62 and 63, wherein the RT has an amino acid sequence of any one of SEQ ID NO: 174 and 2127-2131.

65. One or more polynucleotides according to claim 60, wherein the RT lacks the RNase H domain.

66. One or more polynucleotides according to claim 65, wherein the RT is truncated after the amino acid at the position corresponding to D497 in SEQ ID NO:

174.

67. One or more polynucleotides according to claim 65 or 66, wherein the RT has an amino acid sequence having at least 90% sequence identity with SEQ ID NO:

175.

68. One or more polynucleotides according to any one of claims 65 to 67, wherein the RT has an amino acid sequence having at least 95% sequence identity with SEQ ID NO:

175.

69. One or more polynucleotides according to any one of claims 65 to 68, wherein the RT has the amino acid sequence shown in SEQ ID NO:

175.

70. One or more polynucleotides according to any one of claims 1 to 69, wherein the PE has an amino acid sequence having at least 90% sequence identity or at least 95% sequence identity with any one of SEQ ID NO: 9-11, 111-130, 137-173, 364, 774-787, 790-814, 817-901, 926, 1143-1155, 1164, 1182-1185, 1189-2085 and 2121-2125.

71. One or more polynucleotides according to any one of claims 1 to 70, wherein the PE has an amino acid sequence having at least 90% sequence identity or at least 95% sequence identity with any one of SEQ ID NO: 364, 1208, 1209, 1210, 1221, 1224, 1225, 1226, 1337, 1361, 1371, 1425, 1432, 1443, 1444, 1445, 1447, 1448, 1516, 1577, 1592, 1622, 1640, 1658, 1666, 1682, 1683, 1692, 1694, 1696 and 1718.

72. One or more polynucleotides according to any one of claims 1 to 71, wherein the PE has SEQ ID NO: 9-11, 111-130, 137-173, 364, 774-787, 790-814, 817-901, 926, 1143-1155, 1164, 1182-1185, 1189-2085, 2121-2125, 1208, 1209, 1210, 1221, 1224, 1225, 12 The amino acid sequence of any one of the following: 26, 1337, 1361, 1371, 1425, 1432, 1443, 1444, 1445, 1447, 1448, 1516, 1577, 1592, 1622, 1640, 1658, 1666, 1682, 1683, 1692, 1694, 1696, and 1718.

73. One or more polynucleotides according to any one of claims 1 to 72, wherein at least one of the one or more polynucleotides is an RNA polynucleotide.

74. One or more polynucleotides according to claim 73, wherein the RNA polynucleotide is mRNA.

75. One or more polynucleotides according to claim 73, wherein the RNA polynucleotide is circRNA.

76. One or more carriers comprising one or more polynucleotides according to any one of claims 1 to 72.

77. The one or more vectors according to claim 76, wherein the one or more vectors further comprises at least one nucleotide sequence encoding polymerase editing guide RNA (PEgRNA), wherein the PEgRNA comprises an extension arm, wherein the extension arm comprises a primer binding site and a DNA synthesis template sequence, and wherein the PEgRNA is capable of binding to the RGN of the PE.

78. One or more vectors according to claim 77, wherein the extended arm is located at the 3' end of the PEgRNA.

79. One or more vectors according to claim 77 or 78, wherein the DNA synthesis template sequence is 19, 22, 23, 24, 25, 26, 29, 30, 32, 34, 37, 38, 39, 40, 42 or 46 nucleotides in length.

80. One or more vectors according to any one of claims 77 to 79, wherein the DNA synthesis template sequence comprises an RT template (RTT) sequence.

81. One or more vectors according to any one of claims 77 to 80, wherein the primer binding site is 9, 11, 12, 13 or 15 nucleotides in length.

82. One or more vectors according to any one of claims 77 to 81, wherein the PEgRNA comprises CRISPR RNA, the CRISPR RNA comprising a nucleotide sequence containing any one of SEQ ID NO: 3, 470 and 471 or a CRISPR RNA (crRNA) repeat sequence differing from any one of SEQ ID NO: 3, 470 and 471 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, 7, 579, 583, 584 or 591.

83. One or more vectors according to any one of claims 77 to 82, wherein the PEgRNA comprises CRISPR RNA, the CRISPR RNA comprises a CRISPR RNA (crRNA) repeat sequence containing a nucleotide sequence of any one of SEQ ID NO: 3, 470 and 471, wherein the RGN polypeptide comprises an amino acid sequence shown in SEQ ID NO: 1, 7, 579, 583, 584 or 591.

84. One or more vectors according to claim 82 or 83, wherein the PEgRNA comprises tracrRNA, the tracrRNA comprising a nucleotide sequence having at least 90% sequence identity with any one of SEQ ID NO: 4, 472, 473 and 474.

85. One or more vectors according to any one of claims 82 to 84, wherein the PEgRNA comprises tracrRNA, the tracrRNA comprising a nucleotide sequence having at least 95% sequence identity with any one of SEQ ID NO: 4, 472, 473 and 474.

86. One or more vectors according to any one of claims 82 to 85, wherein the PEgRNA comprises tracrRNA, the tracrRNA comprising the nucleotide sequence of any one of SEQ ID NO: 4, 472, 473 and 474.

87. One or more vectors according to any one of claims 77 to 81, wherein the PEgRNA comprises CRISPR RNA, the CRISPR RNA comprising a nucleotide sequence containing any one of SEQ ID NO: 5, 475 and 476 or a CRISPR RNA (crRNA) repeat sequence differing from any one of SEQ ID NO: 5, 475 and 476 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:

2.

88. One or more vectors according to any one of claims 77 to 81 and 87, wherein the PEgRNA comprises CRISPR RNA, the CRISPR RNA comprising a CRISPR RNA (crRNA) repeat sequence containing a nucleotide sequence of any one of SEQ ID NO: 5, 475 and 476, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO:

2.

89. One or more vectors according to claim 87 or 88, wherein the PEgRNA comprises tracrRNA, the tracrRNA comprising a nucleotide sequence having at least 90% sequence identity with any one of SEQ ID NO: 6, 477, 478, 479 and 480.

90. One or more vectors according to claim 87 or 88, wherein the PEgRNA comprises tracrRNA, the tracrRNA comprising a nucleotide sequence having at least 95% sequence identity with any one of SEQ ID NO: 6, 477, 478, 479 and 480.

91. One or more vectors according to any one of claims 87 to 90, wherein the PEgRNA comprises tracrRNA, the tracrRNA comprising the nucleotide sequence of any one of SEQ ID NO: 6, 477, 478, 479 and 480.

92. One or more vectors according to any one of claims 77 to 81, wherein the PEgRNA comprises CRISPR RNA, the CRISPR RNA comprising a nucleotide sequence containing SEQ ID NO: 571 or a CRISPR RNA (crRNA) repeat sequence differing from SEQ ID NO: 571 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:

565.

93. One or more vectors according to any one of claims 77 to 81 and 92, wherein the PEgRNA comprises CRISPR RNA, the CRISPR RNA comprises a CRISPR RNA (crRNA) repeat sequence containing the nucleotide sequence of SEQ ID NO: 571, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO:

565.

94. One or more vectors according to claim 92 or 93, wherein the PEgRNA comprises tracrRNA, the tracrRNA comprising a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:

572.

95. One or more vectors according to any one of claims 92 to 94, wherein the PEgRNA comprises tracrRNA, the tracrRNA comprising a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:

572.

96. One or more vectors according to any one of claims 92 to 95, wherein the PEgRNA comprises tracrRNA, the tracrRNA comprising the nucleotide sequence of SEQ ID NO:

572.

97. One or more vectors according to any one of claims 77 to 81, wherein the PEgRNA comprises CRISPR RNA, the CRISPR RNA comprising a nucleotide sequence containing SEQ ID NO: 573 or 1180 or a CRISPR RNA (crRNA) repeat sequence differing from SEQ ID NO: 573 or 1180 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:

566.

98. One or more vectors according to any one of claims 77 to 81 and 97, wherein the PEgRNA comprises CRISPR RNA, the CRISPR RNA comprises a CRISPR RNA (crRNA) repeat sequence containing the nucleotide sequence of SEQ ID NO: 573 or 1180, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO:

566.

99. One or more vectors according to claim 97 or 98, wherein the PEgRNA comprises tracrRNA, the tracrRNA comprising a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 574 or 1181.

100. One or more vectors according to any one of claims 97 to 99, wherein the PEgRNA comprises tracrRNA, the tracrRNA comprising a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 574 or 1181.

101. One or more vectors according to any one of claims 97 to 100, wherein the PEgRNA comprises tracrRNA, the tracrRNA comprising the nucleotide sequence of SEQ ID NO: 574 or 1181.

102. One or more vectors according to any one of claims 76 to 101, wherein the one or more vectors further comprises a nucleotide sequence encoding a nick guide RNA.

103. One or more vectors according to any one of claims 76 to 102, wherein the one or more vectors further comprises a nucleotide sequence encoding a dominant-negative MLH1.

104. One or more vectors according to any one of claims 76 to 103, wherein the one or more vectors are adeno-associated virus (AAV) vectors.

105. A cell comprising one or more polynucleotides according to any one of claims 1 to 75 or one or more carriers according to any one of claims 76 to 104.

106. The cell according to claim 105, wherein the cell is a prokaryotic cell.

107. The cell according to claim 105, wherein the cell is a eukaryotic cell.

108. The cell of claim 107, wherein the eukaryotic cell is a mammalian cell.

109. The cell of claim 108, wherein the mammalian cell is a human cell.

110. The cell of claim 108, wherein the human cell is an immune cell.

111. The cell of claim 108, wherein the human cell is a stem cell.

112. The cell according to claim 111, wherein the stem cell is an induced pluripotent stem cell.

113. The cell according to claim 107, wherein the eukaryotic cell is an insect cell or a bird cell.

114. The cell according to claim 107, wherein the eukaryotic cell is a fungal cell.

115. The cell according to claim 107, wherein the eukaryotic cell is a plant cell.

116. A plant or plant part comprising the plant cells according to claim 115.

117. A method for preparing PE, comprising culturing cells according to claim 105 under conditions expressing said PE.

118. The method of claim 117, further comprising purifying the PE.

119. The method of claim 117, wherein the cell further expresses one or more of the RGN polypeptides capable of binding to the PE to form a ribonucleoprotein complex guide RNA.

120. The method of claim 119, further comprising purifying the ribonucleoprotein complex.

121. A polymerase editor (PE) comprising a DNA polymerase and an RNA-guided nuclease (RGN) polypeptide, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, 2, 7, 565, 566, 579, 583, 584, 591 or 1165-1179.

122. The PE of claim 121, wherein the RGN polypeptide comprises an amino acid sequence having the following elements: a) Has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and includes: i) S at position corresponding to amino acid position 1020 in SEQ ID NO: 1; ii) G at position 1021 corresponding to amino acid position in SEQ ID NO: 1; iii) N at position 1022 corresponding to amino acid position in SEQ ID NO: 1; iv) K at position corresponding to amino acid position 1023 in SEQ ID NO: 1; and v) E at position corresponding to amino acid position 1025 in SEQ ID NO: 1; b) Having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 579, wherein the RGN polypeptide contains Q at position corresponding to amino acid position 1023 in SEQ ID NO: 579; c) Having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 584, wherein the RGN polypeptide contains a T at position 1021 corresponding to amino acid position 1021 in SEQ ID NO: 584 and an N at position 1023 corresponding to amino acid position 1023 in SEQ ID NO: 584; d) Having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 583, wherein the RGN polypeptide contains N at position 1021 corresponding to amino acid position 1023 in SEQ ID NO: 583; or e) Having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 591, wherein the RGN polypeptide comprises: i) Q at position 975 corresponding to amino acid position in SEQ ID NO: 591; ii) A at position A corresponding to amino acid position 1020 in SEQ ID NO: 591; iii) G at position 1022 corresponding to amino acid position in SEQ ID NO: 591; and iv) Q at position 1023 corresponding to amino acid position in SEQ ID NO:

591.

123. The PE according to claim 121, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, 2, 7, 565, 566, 579, 583, 584, 591 or 1165-1179.

124. The PE according to claim 121 or 123, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1, 2, 7, 565, 566, 579, 583, 584, 591 or 1165-1179.

125. The PE according to any one of claims 121 to 124, wherein the DNA polymerase and the RGN polypeptide are two independent polypeptides.

126. The PE of claim 125, wherein the DNA polymerase, the RGN polypeptide, or both the DNA polymerase and the RGN polypeptide are operatively fused with one or more nuclear localization signals (NLS).

127. The PE according to claim 126, wherein the one or more NLS are selected from the group consisting of SEQ ID NO: 107-109, 2086-2101, 2118 and 2119.

128. The PE according to claim 126 or 127, wherein the DNA polymerase, the RGN polypeptide, or both the DNA polymerase and the RGN polypeptide are operatively fused to the NLS at their N-terminus, C-terminus, or both their N-terminus and C-terminus.

129. The PE according to any one of claims 126 to 128, wherein the DNA polymerase, the RGN polypeptide, or both the DNA polymerase and the RGN polypeptide are further operably linked to one or more adapter sequences, the one or more adapter sequences being intermediate between the NLS and the DNA polymerase, the RGN polypeptide, or both the DNA polymerase and the RGN polypeptide.

130. The PE according to any one of claims 121 to 124, wherein the DNA polymerase is operatively fused with the RGN polypeptide.

131. The PE of claim 130, wherein the PE comprises the DNA polymerase fused at the amino terminus of the RGN polypeptide.

132. The PE of claim 130, wherein the PE comprises the DNA polymerase fused at the carboxyl terminus of the RGN polypeptide.

133. The PE according to any one of claims 130 to 132, wherein the PE is operatively fused with one or more NLS.

134. The PE according to claim 133, wherein the NLS is selected from the group consisting of SEQ ID NO: 107-109, 2086-2101, 2118 and 2119.

135. The PE according to claim 133 or 134, wherein the PE is operatively fused with the NLS at its N-terminus, C-terminus, within the PE, or in a combination thereof.

136. The PE according to any one of claims 131 to 135, wherein the PE is further operable to be fused with one or more connector sequences.

137. The PE of claim 136, wherein at least one of the one or more adapter sequences is between the DNA polymerase and the RGN polypeptide.

138. The PE according to claim 137, wherein the one or more connector sequences have the formula -(SGGS) x -NLS m -(SGGS) y -NLS n -(SGGS) z - where x, y, or z are 0, 1, 2, 3, or 4 respectively; and where m or n are 0 or 1 respectively.

139. The PE of claim 137, wherein the one or more linker sequences between the DNA polymerase and the RGN polypeptide comprise one or more copies of the amino acid sequence SGGS.

140. The PE according to any one of claims 137 to 139, wherein the one or more linker sequences between the DNA polymerase and the RGN polypeptide have a length of 42 amino acids.

141. The PE according to any one of claims 137 to 140, wherein the one or more adapter sequences between the DNA polymerase and the RGN polypeptide contain at least one NLS.

142. The PE of claim 141, wherein the one or more adapter sequences between the DNA polymerase and the RGN polypeptide comprise two NLS.

143. The PE according to any one of claims 137 to 142, wherein the one or more adapter sequences between the DNA polymerase and the RGN polypeptide comprise the sequence shown in SEQ ID NO: 110, 366, 603, 605, 2120 or 2126.

144. The PE of claim 130, wherein the PE comprises the DNA polymerase inserted into the RGN polypeptide.

145. The PE according to claim 144, wherein the DNA polymerase is inserted into the adapter domain 2, wedge domain, RuvC domain, HNH domain, Rec-2 domain or PAM interaction domain of the RGN polypeptide, or wherein the DNA polymerase is inserted between the adapter domain 2, wedge domain, RuvC domain, HNH domain, Rec-2 domain or PAM interaction domain of the RGN polypeptide and another domain located at the 5' or 3' of the adapter domain 2, wedge domain, RuvC domain, HNH domain, Rec-2 domain or PAM interaction domain.

146. The PE according to claim 145, wherein the RuvC domain is a RuvCIII domain.

147. The PE according to claim 145, wherein the DNA polymerase is inserted into the adapter domain 2, wedge domain or RuvCIII domain of the RGN polypeptide.

148. The PE of claim 144, wherein the DNA polymerase is inserted into the RGN polypeptide immediately following an amino acid selected from the group consisting of: a) The amino acid position corresponding to position 678 of SEQ ID NO: 2; b) The amino acid position corresponding to position 736 of SEQ ID NO: 2; c) The amino acid position corresponding to position 922 of SEQ ID NO: 2; d) The amino acid position corresponding to position 642 of SEQ ID NO: 1, 7, 579, 583, 584 or 591; e) The amino acid position 772 corresponding to SEQ ID NO: 1, 7, 579, 583, 584 or 591; f) The amino acid position corresponding to position 739 of SEQ ID NO: 565; g) The amino acid position corresponding to position 744 of SEQ ID NO: 565; h) The amino acid position corresponding to position 680 of SEQ ID NO: 566; i) The amino acid position corresponding to position 785 of SEQ ID NO: 566; and j) The amino acid position corresponding to position 910 of SEQ ID NO:

566.

149. The PE according to any one of claims 144 to 148, wherein the PE is operatively fused with one or more NLSs.

150. The PE according to claim 149, wherein the NLS is selected from the group consisting of SEQ ID NO: 107-109, 2086-2101, 2118 and 2119.

151. The PE according to claim 149 or 150, wherein the PE is operatively fused with the NLS at its N-terminus, C-terminus, within the PE, or in a combination thereof.

152. The PE according to any one of claims 149 to 151, wherein the PE is further operably connected to one or more connector sequences.

153. The PE of claim 152, wherein at least one of the one or more adapter sequences is between the DNA polymerase and the fragment of the RGN polypeptide.

154. The PE of claim 153, wherein the one or more linker sequences between the DNA polymerase and the RGN polypeptide comprise one or more copies of the amino acid sequence SGGS.

155. The PE according to any one of claims 121 to 154, wherein when bound to guide RNA (gRNA), the RGN polypeptide is capable of binding to a target sequence in a target polynucleotide in an RNA-guided sequence-specific manner, wherein the target sequence comprises a target strand and a non-target strand, and wherein the gRNA is capable of hybridizing with the target strand of the target sequence.

156. The PE of claim 155, wherein the RGN polypeptide recognizes a protospacer adjacent motif (PAM) at the 3' of the target sequence.

157. The PE according to claim 156, wherein: a) The RGN polypeptide containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1 recognizes the PAM having a common nucleotide sequence shown in NNNNCY; b) The RGN polypeptide containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 7 recognizes the PAM having a common nucleotide sequence shown in NNNNCC; c) The RGN polypeptide containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 2 recognizes the PAM having a common nucleotide sequence shown in NNRYA; d) The RGN polypeptide containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 565 recognizes the PAM having a common nucleotide sequence shown as NNGRR; or e) The RGN polypeptide containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 566 recognizes the PAM having a common nucleotide sequence shown in NNGG; f) The RGN polypeptide containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 579 or 583 recognizes PAM having a common nucleotide sequence shown in NNNNC; g) The RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 584 recognizes PAM having a common nucleotide sequence shown in NNNNCM; or h) The RGN polypeptide containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 591 recognizes PAM having a common nucleotide sequence shown in NNNNCT.

158. The PE according to any one of claims 155 to 157, wherein the RGN polypeptide is capable of cleaving the target polynucleotide upon binding.

159. The PE according to claim 158, wherein the RGN polypeptide is capable of producing double-strand breaks.

160. The PE of claim 158, wherein the RGN polypeptide is capable of generating single-chain breaks.

161. The PE of claim 160, wherein the RGN polypeptide comprises an RGN nickase, the RGN nickase comprising at least one mutated HNH domain having reduced or eliminated nuclease activity.

162. The PE according to claim 160, wherein the RGN polypeptide does not contain an HNH domain.

163. The PE according to claim 162, wherein the HNH domain of the RGN polypeptide has been replaced by the DNA polymerase.

164. The PE of claim 163, wherein the DNA polymerase is operatively fused to one or more adapter sequences.

165. The PE of claim 164, wherein at least one of the one or more adapter sequences is between the DNA polymerase and the RGN polypeptide.

166. The PE according to claim 161, wherein the RGN nickase comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 8, 106, 365, 567 or 568.

167. The PE according to claim 161 or 166, wherein the RGN nickase comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 8, 106, 365, 567 or 568.

168. The PE according to any one of claims 161, 166 and 167, wherein the RGN nickase comprises the amino acid sequence shown in SEQ ID NO: 8, 106, 365, 567 or 568.

169. The PE according to claim 161, wherein the HNH domain comprises at least two mutations that reduce nuclease activity.

170. The PE according to claim 169, wherein the RGN nickase comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 367, 368, 369, 569 or 570.

171. The PE according to claim 169 or 170, wherein the RGN nickase comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 367, 368, 369, 569 or 570.

172. The PE according to any one of claims 169 to 171, wherein the RGN nickase comprises the amino acid sequence shown in SEQ ID NO: 367, 368, 369, 569 or 570.

173. The PE according to any one of claims 155 to 157, wherein the RGN polypeptide has no nuclease activity.

174. The PE according to any one of claims 121 to 173, wherein the DNA polymerase is a reverse transcriptase (RT).

175. The PE according to claim 174, wherein the RT is Moroni murine leukemia virus RT (MMLV RT).

176. The PE according to claim 174, wherein the RT has an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NO: 174 and 2127-2131.

177. The PE according to claim 174 or 176, wherein the RT has an amino acid sequence having at least 95% sequence identity with any one of SEQ ID NO: 174 and 2127-2131.

178. The PE according to any one of claims 174, 176 and 177, wherein the RT has an amino acid sequence of any one of SEQ ID NO: 174 and 2127-2131.

179. The PE according to claim 174, wherein the RT lacks the RNase H domain.

180. The PE according to claim 179, wherein the RT is truncated after the amino acid at the position corresponding to D497 in SEQ ID NO:

174.

181. The PE according to claim 179 or 180, wherein the RT has an amino acid sequence having at least 90% sequence identity with SEQ ID NO:

175.

182. The PE according to any one of claims 179 to 181, wherein the RT has an amino acid sequence having at least 95% sequence identity with SEQ ID NO:

175.

183. The PE according to any one of claims 179 to 182, wherein the RT has the amino acid sequence shown in SEQ ID NO:

175.

184. The PE according to any one of claims 121 to 183, wherein the PE has an amino acid sequence having at least 90% sequence identity or at least 95% sequence identity with any one of SEQ ID NO: 9-11, 111-130, 137-173, 364, 774-787, 790-814, 817-901, 926, 1143-1155, 1164, 1182-1185, 1189-2085 and 2121-2125.

185. The PE according to any one of claims 121 to 184, wherein the PE has an amino acid sequence having at least 90% sequence identity or at least 95% sequence identity with any one of SEQ ID NO: 364, 1208, 1209, 1210, 1221, 1224, 1225, 1226, 1337, 1361, 1371, 1425, 1432, 1443, 1444, 1445, 1447, 1448, 1516, 1577, 1592, 1622, 1640, 1658, 1666, 1682, 1683, 1692, 1694, 1696 and 1718.

186. The PE according to any one of claims 121 to 185, wherein the PE has SEQ ID NO: 9-11, 111-130, 137-173, 364, 774-787, 790-814, 817-901, 926, 1143-1155, 1164, 1182-1185, 1189-2085, 2121-2125, 1208, 1209, 1210, 1221, 1224, 1225, 12 The amino acid sequence of any one of the following: 26, 1337, 1361, 1371, 1425, 1432, 1443, 1444, 1445, 1447, 1448, 1516, 1577, 1592, 1622, 1640, 1658, 1666, 1682, 1683, 1692, 1694, 1696, and 1718.

187. A ribonucleoprotein (RNP) complex comprising PE according to any one of claims 121 to 186 and PEgRNA bound to the RGN polypeptide, wherein the PEgRNA comprises a primer binding site (PBS) and a DNA synthesis template.

188. The RNP complex of claim 187, wherein the PEgRNA comprises a spacer region that hybridizes with a eukaryotic target sequence.

189. The RNP complex of claim 188, wherein the eukaryotic target sequence comprises a mammalian target sequence.

190. A PE system for modifying one or more target sequences in a target polynucleotide, the system comprising: a) One or more PEgRNAs, or one or more polynucleotides comprising one or more nucleotide sequences encoding said one or more PEgRNAs, wherein said one or more PEgRNAs comprise an extension arm, wherein said extension arm comprises a primer binding site (PBS) and a DNA synthesis template sequence; and b) The PE according to any one of claims 121 to 186; The one or more PEgRNAs thereon are capable of binding to the RGN polypeptide of the PE.

191. The PE system of claim 190, wherein each of the one or more PEgRNAs is capable of hybridizing with the target strand of the target sequence and forming a complex with the RGN polypeptide to guide the RGN polypeptide to bind to the target sequence.

192. The PE system according to claim 190 or 191, wherein the spacer region hybridizes with the eukaryotic target sequence.

193. The PE system of claim 192, wherein the eukaryotic target sequence comprises a mammalian target sequence.

194. The PE system according to any one of claims 190 to 193, wherein the extended arm is located at the 3' end of the PEgRNA.

195. The PE system according to any one of claims 190 to 194, wherein the DNA synthesis template sequence is 19, 22, 23, 24, 25, 26, 29, 30, 32, 34, 37, 38, 39, 40, 42 or 46 nucleotides in length.

196. The PE system according to any one of claims 190 to 195, wherein the DNA synthesis template sequence comprises an RT template sequence (RTT).

197. The PE system according to any one of claims 190 to 196, wherein the primer binding site is 9, 11, 12, 13 or 15 nucleotides in length.

198. The PE system according to any one of claims 190 to 197, wherein the PEgRNA comprises CRISPRRNA, the CRISPRRNA comprising a nucleotide sequence containing any one of SEQ ID NO: 3, 470 and 471 or a CRISPR RNA (crRNA) repeat sequence differing from any one of SEQ ID NO: 3, 470 and 471 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, 7, 579, 583, 584 or 591.

199. The PE system according to any one of claims 190 to 198, wherein the PEgRNA comprises CRISPRRNA, the CRISPRRNA comprising a CRISPRRNA (crRNA) repeat sequence containing a nucleotide sequence of any one of SEQ ID NO: 3, 470 and 471, wherein the RGN polypeptide comprises an amino acid sequence shown in SEQ ID NO: 1, 7, 579, 583, 584 or 591.

200. The PE system according to claim 198 or 199, wherein the PEgRNA comprises tracrRNA, the tracrRNA comprising a nucleotide sequence having at least 90% sequence identity with any one of SEQ ID NO: 4, 472, 473 and 474.

201. The PE system according to any one of claims 198 to 200, wherein the PEgRNA comprises tracrRNA, the tracrRNA comprising a nucleotide sequence having at least 95% sequence identity with any one of SEQ ID NO: 4, 472, 473 and 474.

202. The PE system according to any one of claims 198 to 201, wherein the PEgRNA comprises tracrRNA, the tracrRNA comprising the nucleotide sequence of any one of SEQ ID NO: 4, 472, 473 and 474.

203. The PE system according to any one of claims 190 to 197, wherein the PEgRNA comprises CRISPRRNA, the CRISPRRNA comprising a nucleotide sequence containing any one of SEQ ID NO: 5, 475 and 476 or a CRISPR RNA (crRNA) repeat sequence differing from any one of SEQ ID NO: 5, 475 and 476 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:

2.

204. The PE system according to any one of claims 190 to 197 and 203, wherein the PEgRNA comprises CRISPR RNA, the CRISPR RNA comprising a CRISPR RNA (crRNA) repeat sequence containing the nucleotide sequence of any one of SEQ ID NO: 5, 475 and 476, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO:

2.

205. The PE system according to claim 203 or 204, wherein the PEgRNA comprises tracrRNA, the tracrRNA comprising a nucleotide sequence having at least 90% sequence identity with any one of SEQ ID NO: 6, 477, 478, 479 and 480.

206. The PE system according to any one of claims 203 to 205, wherein the PEgRNA comprises tracrRNA, the tracrRNA comprising a nucleotide sequence having at least 95% sequence identity with any one of SEQ ID NO: 6, 477, 478, 479 and 480.

207. The PE system according to any one of claims 203 to 206, wherein the PEgRNA comprises tracrRNA, and the tracrRNA comprises the nucleotide sequence of any one of SEQ ID NO: 6, 477, 478, 479 and 480.

208. The PE system according to any one of claims 190 to 197, wherein the PEgRNA comprises CRISPRRNA, the CRISPRRNA comprising a nucleotide sequence containing SEQ ID NO: 571 or a CRISPR RNA (crRNA) repeat sequence differing from SEQ ID NO: 571 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:

565.

209. The PE system according to any one of claims 190 to 197 and 208, wherein the PEgRNA comprises CRISPR RNA, the CRISPR RNA comprising a CRISPR RNA (crRNA) repeat sequence containing the nucleotide sequence of SEQ ID NO: 571, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO:

565.

210. The PE system according to claim 208 or 209, wherein the PEgRNA comprises tracrRNA, the tracrRNA comprising a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:

572.

211. The PE system according to any one of claims 208 to 210, wherein the PEgRNA comprises tracrRNA, the tracrRNA comprising a nucleotide sequence having at least 95% sequence identity with SEQ ID NO:

572.

212. The PE system according to any one of claims 208 to 211, wherein the PEgRNA comprises tracrRNA, the tracrRNA comprising the nucleotide sequence of SEQ ID NO:

572.

213. The PE system according to any one of claims 190 to 197, wherein the PEgRNA comprises CRISPRRNA, the CRISPRRNA comprising a nucleotide sequence containing SEQ ID NO: 573 or 1180 or a CRISPR RNA (crRNA) repeat sequence differing from SEQ ID NO: 573 or 1180 by 1 to 5 nucleotides, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:

566.

214. The PE system according to any one of claims 190 to 197 and 213, wherein the PEgRNA comprises CRISPR RNA, the CRISPR RNA comprising a CRISPR RNA (crRNA) repeat sequence containing the nucleotide sequence of SEQ ID NO: 573 or 1180, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO:

566.

215. The PE system according to claim 213 or 214, wherein the PEgRNA comprises tracrRNA, the tracrRNA comprising a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 574 or 1181.

216. The PE system according to any one of claims 213 to 215, wherein the PEgRNA comprises tracrRNA, the tracrRNA comprising a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 574 or 1181.

217. The PE system according to any one of claims 213 to 216, wherein the PEgRNA comprises tracrRNA, the tracrRNA comprising the nucleotide sequence of SEQ ID NO: 574 or 1181.

218. The PE system according to any one of claims 190 to 217, wherein the polynucleotide encoding the one or more PEgRNAs and the polynucleotide encoding the PE are on a single vector.

219. The PE system according to any one of claims 190 to 217, wherein the polynucleotide encoding the one or more PEgRNAs and the polynucleotide encoding the PE are on separate vectors.

220. The PE system according to any one of claims 190 to 217, wherein the polynucleotide encoding the DNA polymerase and the polynucleotide encoding the RGN polypeptide are on separate vectors.

221. The PE system according to any one of claims 190 to 217, wherein the polynucleotide encoding the one or more PEgRNAs, the polynucleotide encoding the DNA polymerase, and the polynucleotide encoding the RGN polypeptide are all on separate vectors.

222. The PE system according to any one of claims 190 to 217, wherein the polynucleotide encoding the PE is an RNA polynucleotide.

223. The PE system according to claim 222, wherein the RNA polynucleotide is mRNA.

224. The PE system of claim 222, wherein the RNA polynucleotide is circRNA.

225. The PE system according to any one of claims 190 to 224, wherein the PE system further comprises nick guide RNA or a polynucleotide comprising a nucleotide sequence encoding nick guide RNA.

226. The PE system according to any one of claims 190 to 225, wherein the PE system further comprises a dominant-negative MLH1 or a polynucleotide comprising a nucleotide sequence encoding a dominant-negative MLH1.

227. A cell comprising PE according to any one of claims 121 to 186, an RNP complex according to any one of claims 187 to 189, or a PE system according to any one of claims 190 to 226.

228. The cell of claim 227, wherein the cell is a prokaryotic cell.

229. The cell of claim 227, wherein the cell is a eukaryotic cell.

230. The cell of claim 229, wherein the eukaryotic cell is a mammalian cell.

231. The cell of claim 230, wherein the mammalian cell is a human cell.

232. The cell of claim 231, wherein the human cell is an immune cell.

233. The cell of claim 231, wherein the human cell is a stem cell.

234. The cell according to claim 233, wherein the stem cell is an induced pluripotent stem cell.

235. The cell of claim 229, wherein the eukaryotic cell is an insect cell or a bird cell.

236. The cell of claim 229, wherein the eukaryotic cell is a fungal cell.

237. The cell of claim 229, wherein the eukaryotic cell is a plant cell.

238. A plant or plant part comprising the plant cells according to claim 237.

239. A pharmaceutical composition comprising one or more polynucleotides according to any one of claims 1 to 75, one or more carriers according to any one of claims 76 to 104, PE according to any one of claims 121 to 186, an RNP complex according to any one of claims 187 to 189, a cell according to any one of claims 107 to 112 and 229 to 234, or a PE system according to any one of claims 190 to 226, and a pharmaceutically acceptable carrier.

240. A method for modifying a target polynucleotide comprising a target sequence, the method comprising delivering a PE system according to any one of claims 190 to 226 to the target sequence or a cell comprising the target sequence, wherein the method produces the modified target polynucleotide, and wherein components of the PE system are delivered simultaneously or sequentially to the target sequence or the cell.

241. The method of claim 240, wherein the modified target polynucleotide comprises a heterologous DNA insertion in the target polynucleotide.

242. The method of claim 240, wherein the modified target polynucleotide comprises a target polynucleotide missing at least one nucleotide.

243. The method of claim 240, wherein the modified target polynucleotide comprises at least one nucleotide mutation in the target polynucleotide.

244. A method for modifying one or more target sequences in a target polynucleotide, the method comprising: a) The RNP complex is assembled under conditions suitable for the formation of the ribonucleoprotein (RNP) complex by combining the following: i) PEgRNA containing an extension arm, wherein the extension arm includes a primer binding site (PBS) and a DNA synthesis template; and ii) PE according to any one of claims 121 to 186; as well as b) Contact the target polynucleotide or a cell containing the target polynucleotide with the assembled RNP complex; Thus, the PE is used to modify the one or more target sequences.

245. The method according to any one of claims 240 to 244, wherein the method is performed in vitro, in vivo, or ex vivo.

246. The method according to any one of claims 240 to 244, wherein the target polynucleotide is intracellular.

247. The method of claim 246, wherein the cell is a eukaryotic cell.

248. The method of claim 247, wherein the eukaryotic cell is a mammalian cell.

249. A cell produced by the method of claim 246, wherein the target polynucleotide has been modified at the target sequence.

250. The cell of claim 249, wherein the cell is a prokaryotic cell.

251. The cell of claim 249, wherein the cell is a eukaryotic cell.

252. The cell of claim 251, wherein the eukaryotic cell is a mammalian cell.

253. The cell of claim 252, wherein the mammalian cell is a human cell.

254. The cell of claim 253, wherein the human cell is an immune cell.

255. The cell of claim 253, wherein the human cell is a stem cell.

256. The cell of claim 255, wherein the stem cell is an induced pluripotent stem cell.

257. The cell of claim 249, wherein the eukaryotic cell is an insect cell or a bird cell.

258. The cell according to claim 249, wherein the eukaryotic cell is a fungal cell.

259. The cell according to claim 249, wherein the eukaryotic cell is a plant cell.

260. A plant or plant part comprising the plant cells according to claim 259.

261. A pharmaceutical composition comprising cells and a pharmaceutically acceptable carrier according to any one of claims 251 to 256.

262. A method for treating a subject suffering from a disease, condition, or symptom, or at risk of developing a disease, condition, or symptom, the method comprising: The subject is given one or more polynucleotides according to any one of claims 1 to 75, one or more carriers according to any one of claims 76 to 104, PE according to any one of claims 121 to 186, RNP complex according to any one of claims 187 to 189, PE system according to any one of claims 190 to 226, cells according to any one of claims 107 to 112, 229 to 234 and 251 to 256, or a pharmaceutical composition according to claim 239 or 261.

263. The method of claim 262, wherein the disease, symptom, or condition is related to a mutation, and the treatment comprises correcting the mutation.

264. Use of one or more polynucleotides according to any one of claims 1 to 75, one or more carriers according to any one of claims 76 to 104, PE according to any one of claims 121 to 186, RNP complex according to any one of claims 187 to 189, PE system according to any one of claims 190 to 226, cell according to any one of claims 107 to 112, 229 to 234 and 251 to 256, or pharmaceutical composition according to claim 239 or 261, for the treatment of a subject suffering from a disease, condition or symptom, or at risk of developing a disease, condition or symptom.

265. The use according to claim 264, wherein the disease, symptom, or condition is related to a mutation, and the treatment comprises correcting the mutation.

266. Use of one or more polynucleotides according to any one of claims 1 to 75, one or more carriers according to any one of claims 76 to 104, PE according to any one of claims 121 to 186, RNP complex according to any one of claims 187 to 189, PE system according to any one of claims 190 to 226, or cell according to any one of claims 107 to 112, 229 to 234 and 251 to 256, or pharmaceutical composition according to claim 239 or 261, for the preparation of a medicament suitable for treating a disease, condition or symptom.

267. The use according to claim 266, wherein the disease, symptom, or condition is related to a mutation, and an effective amount of the drug corrects the mutation.

268. A polynucleotide comprising a nucleotide sequence encoding an RNA-guided nuclease (RGN) polypeptide, said RGN polypeptide comprising an amino acid sequence having the following: a) Having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, wherein the RGN polypeptide comprises: i) S at position corresponding to amino acid position 1020 in SEQ ID NO: 1; ii) G at position 1021 corresponding to amino acid position in SEQ ID NO: 1; iii) N at position 1022 corresponding to amino acid position in SEQ ID NO: 1; iv) K at position corresponding to amino acid position 1023 in SEQ ID NO: 1; and v) E at position corresponding to amino acid position 1025 in SEQ ID NO: 1; b) Having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 579, wherein the RGN polypeptide contains Q at position corresponding to amino acid position 1023 in SEQ ID NO: 579; c) Having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 584, wherein the RGN polypeptide contains a T at position 1021 corresponding to amino acid position 1021 in SEQ ID NO: 584 and an N at position 1023 corresponding to amino acid position 1023 in SEQ ID NO: 584; d) Having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 583, wherein the RGN polypeptide contains N at position 1021 corresponding to amino acid position 1023 in SEQ ID NO: 583; or e) Having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 591, wherein the RGN polypeptide comprises: i) Q at position 975 corresponding to amino acid position in SEQ ID NO: 591; ii) A at position A corresponding to amino acid position 1020 in SEQ ID NO: 591; iii) G at position 1022 corresponding to amino acid position in SEQ ID NO: 591; and iv) Q at position 1023 corresponding to amino acid position in SEQ ID NO:

591.

269. The polynucleotide of claim 268, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, 579, 583, 584 or 591.

270. The polynucleotide of claim 268 or 269, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1, 579, 583, 584 or 591.

271. The polynucleotide of any one of claims 268 to 270, wherein the polynucleotide encoding the RGN polypeptide comprises mRNA, the mRNA further comprising a heterologous 5' untranslated region (UTR) and / or a heterologous 3' UTR.

272. The polynucleotide according to any one of claims 268 to 271, wherein the polynucleotide encoding the RGN polypeptide is operatively linked to a promoter heterologous to the polynucleotide.

273. The polynucleotide according to any one of claims 268 to 272, wherein when bound to guide RNA (gRNA), the RGN polypeptide is capable of binding to a target sequence in the target polynucleotide in an RNA-guided sequence-specific manner, wherein the target sequence comprises a target strand and a non-target strand, and wherein the gRNA is capable of hybridizing with the target strand of the target sequence.

274. The polynucleotide of claim 273, wherein the RGN polypeptide recognizes a protospacer adjacent motif (PAM) at the 3' of the target sequence.

275. The polynucleotide of claim 274, wherein the RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1 recognizes a PAM having a common nucleotide sequence shown in NNNNCY.

276. The polynucleotide of claim 274, wherein the RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 579 or 583 recognizes a PAM having a common nucleotide sequence shown in NNNNC.

277. The polynucleotide of claim 274, wherein the RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 584 recognizes a PAM having a common nucleotide sequence shown in NNNNCM.

278. The polynucleotide of claim 274, wherein the RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 591 recognizes a PAM having a common nucleotide sequence shown in NNNNCT.

279. The polynucleotide according to any one of claims 273 to 278, wherein the RGN polypeptide is capable of cleaving the target polynucleotide upon binding.

280. The polynucleotide of claim 279, wherein the RGN polypeptide is capable of producing double-strand breaks.

281. The polynucleotide of claim 279, wherein the RGN polypeptide is capable of producing single-strand breaks.

282. The polynucleotide according to any one of claims 273 to 278, wherein the RGN polypeptide is free of nuclease activity.

283. The polynucleotide according to any one of claims 268 to 282, wherein the RGN polypeptide is operatively fused with a heterologous polypeptide.

284. The polynucleotide of claim 283, wherein the heterologous polypeptide is a deaminase.

285. The polynucleotide of claim 284, wherein the deaminase is cytosine deaminase or adenine deaminase.

286. The polynucleotide of claim 283, wherein the heterologous polypeptide is a detectable label.

287. The polynucleotide of claim 283, wherein the heterologous polypeptide is an expression regulator.

288. The polynucleotide of any one of claims 268 to 287, wherein the RGN polypeptide is operatively fused with one or more nuclear localization signals.

289. The polynucleotide of any one of claims 268 to 288, wherein the nucleotide sequence encoding the RGN polypeptide is codon-optimized for expression in eukaryotic cells.

290. The polynucleotide of claim 289, wherein the eukaryotic cell is a mammalian cell.

291. The polynucleotide according to any one of claims 268 to 290, wherein the polynucleotide is a separated polynucleotide.

292. A carrier comprising a polynucleotide according to any one of claims 268 to 291.

293. The vector of claim 292, wherein the vector further comprises at least one nucleotide sequence encoding guide RNA (gRNA).

294. The vector according to claim 293, wherein the gRNA comprises CRISPR RNA (crRNA), the CRISPR RNA comprising a nucleotide sequence containing any one of SEQ ID NO: 3, 470 and 471 or a crRNA repeat sequence differing from any one of SEQ ID NO: 3, 470 and 471 by 1 to 5 nucleotides.

295. The vector of claim 294, wherein the gRNA comprises tracrRNA, the tracrRNA comprising a nucleotide sequence having at least 90% sequence identity with any one of SEQ ID NO: 4, 472, 473 and 474.

296. The vector according to any one of claims 292 to 295, wherein the vector is an adeno-associated virus (AAV) vector.

297. A cell comprising a polynucleotide according to any one of claims 268 to 291 or a carrier according to any one of claims 292 to 296.

298. The cell of claim 297, wherein the cell is a prokaryotic cell.

299. The cell of claim 297, wherein the cell is a eukaryotic cell.

300. The cell of claim 299, wherein the eukaryotic cell is a mammalian cell.

301. The cell of claim 300, wherein the mammalian cell is a human cell.

302. The cell of claim 301, wherein the human cell is an immune cell.

303. The cell of claim 301, wherein the human cell is a stem cell.

304. The cell of claim 303, wherein the stem cell is an induced pluripotent stem cell.

305. The cell of claim 299, wherein the eukaryotic cell is an insect cell or a bird cell.

306. The cell of claim 299, wherein the eukaryotic cell is a fungal cell.

307. The cell according to claim 299, wherein the eukaryotic cell is a plant cell.

308. A plant or plant part comprising the plant cells according to claim 307.

309. A method for preparing an RGN polypeptide, comprising culturing cells according to claim 297 under conditions expressing the RGN polypeptide.

310. A method for preparing an RGN polypeptide, comprising introducing a heteropolynucleotide into a cell, said heteropolynucleotide comprising a nucleotide sequence encoding an RNA-guided nuclease (RGN) polypeptide, said RGN polypeptide comprising an amino acid sequence having the following: a) Having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, wherein the RGN polypeptide comprises: i) S at position corresponding to amino acid position 1020 in SEQ ID NO: 1; ii) G at position 1021 corresponding to amino acid position in SEQ ID NO: 1; iii) N at position 1022 corresponding to amino acid position in SEQ ID NO: 1; iv) K at position corresponding to amino acid position 1023 in SEQ ID NO: 1; and v) E at position corresponding to amino acid position 1025 in SEQ ID NO: 1; b) Having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 579, wherein the RGN polypeptide contains Q at position corresponding to amino acid position 1023 in SEQ ID NO: 579; c) Having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 584, wherein the RGN polypeptide contains a T at position 1021 corresponding to amino acid position 1021 in SEQ ID NO: 584 and an N at position 1023 corresponding to amino acid position 1023 in SEQ ID NO: 584; d) Having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 583, wherein the RGN polypeptide contains N at position 1021 corresponding to amino acid position 1023 in SEQ ID NO: 583; or e) Having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 591, wherein the RGN polypeptide comprises: i) Q at position 975 corresponding to amino acid position in SEQ ID NO: 591; ii) A at position A corresponding to amino acid position 1020 in SEQ ID NO: 591; iii) G at position 1022 corresponding to amino acid position in SEQ ID NO: 591; and iv) Q at position corresponding to amino acid position 1023 in SEQ ID NO: 591; and The cells were cultured under conditions expressing the RGN polypeptide.

311. The method of claim 310, wherein the heteropolynucleotide comprising the nucleotide sequence encoding an RNA-guided nuclease (RGN) polypeptide comprises mRNA, the mRNA further comprising a hetero 5' untranslated region (UTR) and / or a hetero 3' UTR.

312. The method of claim 310 or 311, wherein when bound to guide RNA (gRNA), the RGN polypeptide is capable of binding to a target sequence in a target polynucleotide in an RNA-guided, sequence-specific manner, wherein the target sequence comprises a target strand and a non-target strand, and wherein the gRNA is capable of hybridizing with the target strand of the target sequence.

313. The method according to any one of claims 310 to 312, further comprising purifying the RGN polypeptide.

314. The method according to any one of claims 310 to 312, wherein the cell further expresses one or more guide RNAs capable of binding to the RGN polypeptide to form the RGN ribonucleoprotein complex.

315. The method of claim 314, further comprising purifying the RGN ribonucleoprotein complex.

316. An RNA-guided nuclease (RGN) polypeptide, wherein the RGN polypeptide comprises an amino acid sequence having the following: a) Having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, wherein the RGN polypeptide comprises: i) S at position corresponding to amino acid position 1020 in SEQ ID NO: 1; ii) G at position 1021 corresponding to amino acid position in SEQ ID NO: 1; iii) N at position 1022 corresponding to amino acid position in SEQ ID NO: 1; iv) K at position corresponding to amino acid position 1023 in SEQ ID NO: 1; and v) E at position corresponding to amino acid position 1025 in SEQ ID NO: 1; b) Having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 579, wherein the RGN polypeptide contains Q at position corresponding to amino acid position 1023 in SEQ ID NO: 579; c) Having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 584, wherein the RGN polypeptide contains a T at position 1021 corresponding to amino acid position 1021 in SEQ ID NO: 584 and an N at position 1023 corresponding to amino acid position 1023 in SEQ ID NO: 584; d) Having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 583, wherein the RGN polypeptide contains N at position 1021 corresponding to amino acid position 1023 in SEQ ID NO: 583; or e) Having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 591, wherein the RGN polypeptide comprises: i) Q at position 975 corresponding to amino acid position in SEQ ID NO: 591; ii) A at position A corresponding to amino acid position 1020 in SEQ ID NO: 591; iii) G at position 1022 corresponding to amino acid position in SEQ ID NO: 591; and iv) Q at position 1023 corresponding to amino acid position in SEQ ID NO:

591.

317. The RGN polypeptide of claim 316, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, 579, 583, 584 or 591.

318. The RGN polypeptide according to claim 316 or 317, wherein the RGN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1, 579, 583, 584 or 591.

319. The RGN polypeptide according to any one of claims 316 to 318, wherein the RGN polypeptide is operably fused with a heterologous polypeptide.

320. The RGN polypeptide according to any one of claims 316 to 319, wherein when bound to guide RNA (gRNA), the RGN polypeptide is capable of binding to a target sequence in a target polynucleotide in an RNA-guided sequence-specific manner, wherein the target sequence comprises a target strand and a non-target strand, and wherein the gRNA is capable of hybridizing with the target strand of the target sequence.

321. The RGN polypeptide of claim 320, wherein the RGN polypeptide recognizes a protospacer adjacent motif (PAM) at the 3' of the target sequence.

322. The RGN polypeptide of claim 321, wherein the RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1 recognizes PAM having a common nucleotide sequence shown as NNNNCY.

323. The RGN polypeptide of claim 321, wherein the RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 579 or 583 recognizes PAM having a common nucleotide sequence shown in NNNNC.

324. The RGN polypeptide of claim 321, wherein the RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 584 recognizes PAM having a common nucleotide sequence shown in NNNNCM.

325. The RGN polypeptide of claim 321, wherein the RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 591 recognizes PAM having a common nucleotide sequence shown in NNNNCT.

326. An RNA-guided nuclease (RGN) polypeptide comprising a protospacer adjacent motif (PAM) interaction domain that recognizes a PAM site containing a nucleotide sequence represented by NNNNCY, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence represented by SEQ ID NO:

1.

327. An RNA-guided nuclease (RGN) polypeptide comprising a protospacer adjacent motif (PAM) interaction domain that recognizes a PAM site comprising a nucleotide sequence represented by NNNNC, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence represented by SEQ ID NO: 479 or 583.

328. An RNA-guided nuclease (RGN) polypeptide comprising a protospacer adjacent motif (PAM) interaction domain that recognizes a PAM site comprising a nucleotide sequence shown in NNNNCM, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:

584.

329. An RNA-guided nuclease (RGN) polypeptide comprising a protospacer adjacent motif (PAM) interaction domain that recognizes a PAM site comprising a nucleotide sequence shown in NNNNCT, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:

591.

330. The RGN polypeptide according to any one of claims 316 to 329, wherein the RGN polypeptide is capable of cleaving the target polynucleotide upon binding.

331. The RGN polypeptide of claim 330, wherein cleavage of the RGN polypeptide results in double-strand breaks.

332. The RGN polypeptide of claim 330, wherein cleavage of the RGN polypeptide results in single-chain breaks.

333. The RGN polypeptide according to any one of claims 316 to 329, wherein the RGN polypeptide has no nuclease activity.

334. The RGN polypeptide according to any one of claims 316 to 333, wherein the RGN polypeptide is operably fused with a heterologous polypeptide.

335. The RGN polypeptide according to claim 334, wherein the heterologous polypeptide is a deaminase.

336. The RGN polypeptide according to claim 335, wherein the deaminase is cytosine deaminase or adenine deaminase.

337. The RGN polypeptide of claim 334, wherein the heterologous polypeptide is a detectable label.

338. The RGN polypeptide according to claim 334, wherein the heterologous polypeptide is an expression regulator.

339. The RGN polypeptide according to any one of claims 316 to 338, wherein the RGN polypeptide is operatively fused with one or more nuclear localization signals.

340. The RGN polypeptide according to any one of claims 316 to 339, wherein the RGN polypeptide is an isolated RGN polypeptide.

341. A ribonucleoprotein (RNP) complex comprising an RGN polypeptide according to any one of claims 316 to 340 and a guide RNA bound to said RGN polypeptide.

342. The RNP complex of claim 341, wherein the guide RNA and the RGN polypeptide are not found to be complexed with each other in nature.

343. The RNP complex according to claim 341 or 342, wherein the guide RNA comprises a spacer region that hybridizes with a eukaryotic target sequence.

344. The RNP complex of claim 343, wherein the eukaryotic target sequence comprises a mammalian target sequence.

345. An RNA-guided nuclease (RGN) system for binding one or more target sequences, said system comprising: a) One or more guide RNAs (gRNAs), or one or more polynucleotides comprising one or more nucleotide sequences encoding said one or more gRNAs, wherein each of said one or more gRNAs comprises CRISPR RNA (crRNA), said CRISPR RNA comprising a crRNA repeat sequence and a spacer region; and b) An RGN polypeptide containing an amino acid sequence having the following: i) A polynucleotide having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, or containing a nucleotide sequence encoding the RGN polypeptide, wherein the RGN polypeptide comprises: A) S at position corresponding to amino acid position 1020 in SEQ ID NO: 1; B) G at position 1021 corresponding to amino acid position in SEQ ID NO: 1; C) N at position 1022 corresponding to amino acid position in SEQ ID NO: 1; D) K at position corresponding to amino acid position 1023 in SEQ ID NO: 1; and E) E at position corresponding to amino acid position 1025 in SEQ ID NO: 1; ii) having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 579, wherein the RGN polypeptide contains Q at position corresponding to amino acid position 1023 in SEQ ID NO: 579; iii) having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 584, wherein the RGN polypeptide contains a T at position 1021 corresponding to amino acid position 1021 in SEQ ID NO: 584 and an N at position 1023 corresponding to amino acid position 1023 in SEQ ID NO: 584; iv) having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 583, wherein the RGN polypeptide contains N at position 1021 corresponding to amino acid position 1023 in SEQ ID NO: 583; or v) The amino acid sequence shown in SEQ ID NO: 591 has at least 90% sequence identity, wherein the RGN polypeptide comprises: A) Q at position 975 corresponding to amino acid position in SEQ ID NO: 591; B) Position A at the location corresponding to amino acid position 1020 in SEQ ID NO: 591; C) G at position 1022 corresponding to amino acid position in SEQ ID NO: 591; and D) Q at position 1023 corresponding to amino acid position in SEQ ID NO:

591.

346. The RGN system of claim 345, wherein each of the one or more gRNAs is capable of hybridizing with the target strand of the target sequence and forming a complex with the RGN polypeptide to guide the RGN polypeptide to bind to the target sequence.

347. The RGN system of claim 345 or 346, wherein the spacer region is hybridized with a eukaryotic target sequence.

348. The RGN system of claim 347, wherein the eukaryotic target sequence comprises a mammalian target sequence.

349. The RGN system according to any one of claims 345 to 348, wherein at least one of the one or more nucleotide sequences encoding the one or more gRNAs and the nucleotide sequence encoding the RGN polypeptide is operatively linked to a promoter heterologous to the nucleotide sequences.

350. The RGN system according to any one of claims 345 to 349, wherein the polynucleotide comprising a nucleotide sequence encoding the RGN polypeptide comprises mRNA, the mRNA further comprising a heterologous 5' untranslated region (UTR) and / or a heterologous 3' UTR.

351. The RGN system according to any one of claims 345 to 350, wherein the one or more gRNAs comprise a crRNA repeat sequence, the crRNA repeat sequence comprising a nucleotide sequence of any one of SEQ ID NO: 3, 470 and 471 or differing from any one of SEQ ID NO: 3, 470 and 471 by 1 to 5 nucleotides.

352. The RGN system of claim 351, wherein the gRNA comprises tracrRNA, the tracrRNA comprising a nucleotide sequence having at least 90% sequence identity with any one of SEQ ID NO: 4, 472, 473 and 474.

353. The RGN system according to any one of claims 345 to 352, wherein the RGN polypeptide and the one or more gRNAs are not found to be complexed with each other in nature.

354. The RGN system according to any one of claims 345 to 353, wherein the target sequence is a eukaryotic target sequence.

355. The RGN system according to any one of claims 345 to 354, wherein the RGN peptide recognizes a protospacer adjacent motif (PAM) at the 3' of the target sequence.

356. The RGN system of claim 355, wherein the RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1 recognizes a PAM having a common nucleotide sequence shown in NNNNCY.

357. The RGN system according to any one of claims 345 to 356, wherein the system directs the cleavage of the target polynucleotide.

358. The RGN system of claim 357, wherein the cutting results in double-chain breakage.

359. The RGN system of claim 357, wherein the cutting results in single-chain breakage.

360. The RGN system according to any one of claims 345 to 356, wherein the RGN polypeptide has no nuclease activity.

361. The RGN system according to any one of claims 345 to 360, wherein the RGN polypeptide is operatively fused with a heterologous polypeptide.

362. The RGN system according to claim 361, wherein the heterologous polypeptide is a deaminase.

363. The RGN system according to claim 362, wherein the deaminase is cytosine deaminase or adenine deaminase.

364. The RGN system of claim 361, wherein the heterologous polypeptide is a detectable label.

365. The polynucleotide of claim 361, wherein the heterologous polypeptide is an expression regulator.

366. The RGN system according to any one of claims 345 to 365, wherein the RGN polypeptide is operatively fused with one or more nuclear localization signals.

367. The RGN system according to any one of claims 345 to 366, wherein the polynucleotide comprising the nucleotide sequence encoding the RGN polypeptide is codon-optimized for expression in eukaryotic cells.

368. The RGN system according to any one of claims 345 to 367, wherein the system further comprises one or more donor polynucleotides.

369. A cell comprising the RGN polypeptide according to any one of claims 316 to 340, the RNP complex according to any one of claims 341 to 344, or the RGN system according to any one of claims 345 to 368.

370. The cell of claim 369, wherein the cell is a prokaryotic cell.

371. The cell of claim 369, wherein the cell is a eukaryotic cell.

372. The cell of claim 371, wherein the eukaryotic cell is a mammalian cell.

373. The cell of claim 372, wherein the mammalian cell is a human cell.

374. The cell of claim 373, wherein the human cell is an immune cell.

375. The cell of claim 373, wherein the human cell is a stem cell.

376. The cell of claim 375, wherein the stem cell is an induced pluripotent stem cell.

377. The cell of claim 371, wherein the eukaryotic cell is an insect cell or a bird cell.

378. The cell according to claim 371, wherein the eukaryotic cell is a fungal cell.

379. The cell according to claim 371, wherein the eukaryotic cell is a plant cell.

380. A plant or plant part comprising the plant cells according to claim 379.

381. A pharmaceutical composition comprising a polynucleotide according to any one of claims 268 to 291, a carrier according to any one of claims 292 to 296, an RGN polypeptide according to any one of claims 316 to 340, an RNP complex according to any one of claims 341 to 344, a cell according to any one of claims 299 to 304 and 371 to 376 or an RGN system according to any one of claims 345 to 368, and a pharmaceutically acceptable carrier.

382. A method for binding a target sequence in a target polynucleotide, comprising delivering an RGN system according to any one of claims 345 to 368 to the target sequence or a cell containing the target sequence.

383. The method of claim 382, ​​wherein the RGN polypeptide or the one or more gRNAs further comprises a detectable marker, thereby allowing detection of the target sequence.

384. The method of claim 382, ​​wherein the RGN polypeptide or the one or more gRNAs further comprises an expression regulator to regulate the expression of a target gene containing the target sequence.

385. A method for cleaving and / or modifying a target polynucleotide containing a target sequence, the method comprising delivering an RGN system according to any one of claims 345 to 368 to the target sequence or a cell containing the target sequence.

386. The method of claim 385, wherein the modified target polynucleotide comprises a heterologous DNA insertion in the target polynucleotide.

387. The method of claim 385, wherein the modified target polynucleotide comprises a target polynucleotide missing at least one nucleotide.

388. The method of claim 385, wherein the modified target polynucleotide comprises at least one nucleotide mutation in the target polynucleotide.

389. A method for binding one or more target sequences in a target polynucleotide to an RNA-guided nuclease (RGN) polypeptide, the method comprising: a) The RNP complex is assembled under conditions suitable for the formation of the ribonucleoprotein (RNP) complex by combining the following: i) Guide RNA (gRNA); and ii) An RGN polypeptide comprising an amino acid sequence having the following: A) Having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, wherein the RGN polypeptide comprises: I) S at position corresponding to amino acid position 1020 in SEQ ID NO: 1; II) G at position 1021 corresponding to amino acid position in SEQ ID NO: 1; III) N at position 1022 corresponding to amino acid position in SEQ ID NO: 1; IV) K at position corresponding to amino acid position 1023 in SEQ ID NO: 1; and V) E at position corresponding to amino acid position 1025 in SEQ ID NO: 1; B) Having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 579, wherein the RGN polypeptide contains Q at position corresponding to amino acid position 1023 in SEQ ID NO: 579; C) Having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 584, wherein the RGN polypeptide contains a T at position 1021 corresponding to amino acid position 1021 in SEQ ID NO: 584 and an N at position 1023 corresponding to amino acid position 1023 in SEQ ID NO: 584; D) The amino acid sequence has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 583, wherein the RGN polypeptide contains N at position 1021 corresponding to amino acid position 1023 in SEQ ID NO: 583; or E) Having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 591, wherein the RGN polypeptide comprises: I) Q at position 975 corresponding to amino acid position in SEQ ID NO: 591; II) Position A at the location corresponding to amino acid position 1020 in SEQ ID NO: 591; III) G at position 1022 corresponding to amino acid position in SEQ ID NO: 591; and IV) Q at position 1023 corresponding to amino acid position in SEQ ID NO: 591; and b) Contact the target polynucleotide or a cell containing the target polynucleotide with the assembled RNP complex; This allows one or more target sequences to bind to the RGN polypeptide.

390. The method according to any one of claims 382 to 389, wherein the method is performed in vitro, in vivo or ex vivo.

391. The method according to any one of claims 382 to 389, wherein the target polynucleotide is intracellular.

392. The method according to claim 391, wherein the cell is a eukaryotic cell.

393. The method of claim 392, wherein the eukaryotic cell is a mammalian cell.

394. A cell produced by the method according to any one of claims 391 to 393, wherein the target polynucleotide has been cleaved and / or modified at the target sequence.

395. The cell according to claim 394, wherein the cell is a prokaryotic cell.

396. The cell of claim 394, wherein the cell is a eukaryotic cell.

397. The cell of claim 396, wherein the eukaryotic cell is a mammalian cell.

398. The cell of claim 397, wherein the mammalian cell is a human cell.

399. The cell of claim 398, wherein the human cell is an immune cell.

400. The cell of claim 398, wherein the human cell is a stem cell.

401. The cell of claim 400, wherein the stem cell is an induced pluripotent stem cell.

402. The cell of claim 396, wherein the eukaryotic cell is an insect cell or a bird cell.

403. The cell according to claim 396, wherein the eukaryotic cell is a fungal cell.

404. The cell of claim 396, wherein the eukaryotic cell is a plant cell.

405. A plant or plant part comprising plant cells according to claim 404.

406. A pharmaceutical composition comprising cells and a pharmaceutically acceptable carrier according to any one of claims 396 to 401.

407. A method for treating a subject suffering from a disease, condition, or symptom, or at risk of developing a disease, condition, or symptom, the method comprising: The subject is administered the polynucleotide according to any one of claims 268 to 291, the carrier according to any one of claims 292 to 296, the RGN polypeptide according to any one of claims 316 to 340, the RNP complex according to any one of claims 341 to 344, the RGN system according to any one of claims 345 to 368, the cell according to any one of claims 299 to 304, 371 to 376 and 396 to 401, or the pharmaceutical composition according to claim 381 or 406.

408. The method of claim 407, wherein the disease, symptom, or condition is related to a mutation, and the treatment comprises correcting the mutation.

409. Use of a polynucleotide according to any one of claims 268 to 291, a carrier according to any one of claims 292 to 296, an RGN polypeptide according to any one of claims 316 to 340, an RNP complex according to any one of claims 341 to 344, an RGN system according to any one of claims 345 to 368, a cell according to any one of claims 299 to 304, 371 to 376 and 396 to 401, or a pharmaceutical composition according to claim 381 or 406, for the treatment of a subject suffering from a disease, condition or symptom, or at risk of developing a disease, condition or symptom.

410. The use according to claim 409, wherein the disease, condition, or symptom is related to a mutation, and the treatment comprises correcting the mutation.

411. Use of a polynucleotide according to any one of claims 268 to 291, a carrier according to any one of claims 292 to 296, an RGN polypeptide according to any one of claims 316 to 340, an RNP complex according to any one of claims 341 to 344, an RGN system according to any one of claims 345 to 368, a cell according to any one of claims 299 to 304, 371 to 376 and 396 to 401, or a pharmaceutical composition according to claim 381 or 406, for the preparation of a medicament suitable for treating a disease, condition or symptom.

412. The use according to claim 411, wherein the disease, condition, or symptom is related to a mutation, and an effective amount of the drug corrects the mutation.

413. A dual polymerase editing (DPE) system comprising: a) A first polymerase editing (PE) system comprising: a first PE or a polynucleotide encoding thereof; and a first PEgRNA, the first PEgRNA comprising a first DNA synthesis template or a polynucleotide encoding thereof; and b) A second PE system comprising: a second PE or a polynucleotide encoding thereof; and a second PEgRNA, the second PEgRNA comprising a second DNA synthesis template or a polynucleotide encoding thereof; The first PEgRNA and the second PEgRNA bind to opposite strands of the target DNA, wherein the first DNA synthesis template encodes a first single-stranded DNA sequence and the second DNA synthesis template encodes a second single-stranded DNA sequence, wherein the first single-stranded DNA sequence and the second single-stranded DNA sequence are at least partially complementary to each other and form a substitution sequence; The first PE, the second PE, or both the first PE and the second PE are PEs according to any one of claims 121 to 186.

414. The DPE system of claim 413, wherein the first PE is the same as the second PE.

415. The DPE system of claim 413, wherein the first PE and the second PE are different from each other.

416. A dual polymerase editing (DPE) system comprising: a PE or a polynucleotide encoding the PE; a first PEgRNA containing a first DNA synthesis template or a polynucleotide encoding the PE; and a second PEgRNA containing a second DNA synthesis template or a polynucleotide encoding the second PEgRNA. The first PEgRNA and the second PEgRNA bind to opposite strands of the target DNA, wherein the first DNA synthesis template encodes a first single-stranded DNA sequence and the second DNA synthesis template encodes a second single-stranded DNA sequence, wherein the first single-stranded DNA sequence and the second single-stranded DNA sequence are at least partially complementary to each other and form a substitution sequence; The PE therein is the PE according to any one of claims 121 to 186.

417. The DPE system according to any one of claims 413 to 416, wherein the replacement sequence comprises a first recombinase site.

418. The DPE system of claim 417, wherein the DPE system further comprises donor DNA, wherein the donor DNA comprises a second recombinase site.

419. The DPE system of claim 418, wherein the DPE system further comprises a recombinase that recognizes the first recombinase site and the second recombinase site.

420. A cell comprising the DPE system according to any one of claims 413 to 419.

421. The cell of claim 420, wherein the cell is a prokaryotic cell.

422. The cell of claim 420, wherein the cell is a eukaryotic cell.

423. The cell according to claim 422, wherein the eukaryotic cell is a mammalian cell.

424. The cell of claim 423, wherein the mammalian cell is a human cell.

425. The cell of claim 424, wherein the human cell is an immune cell.

426. The cell of claim 424, wherein the human cell is a stem cell.

427. The cell of claim 426, wherein the stem cell is an induced pluripotent stem cell.

428. The cell according to claim 422, wherein the eukaryotic cell is an insect cell or a bird cell.

429. The cell according to claim 422, wherein the eukaryotic cell is a fungal cell.

430. The cell according to claim 422, wherein the eukaryotic cell is a plant cell.

431. A plant or plant part comprising plant cells according to claim 430.

432. A pharmaceutical composition comprising the DPE system according to any one of claims 413 to 419 or the cell and pharmaceutically acceptable carrier according to any one of claims 423 to 427.

433. A method for modifying a target polynucleotide comprising a target sequence, comprising delivering a DPE system according to any one of claims 413 to 419 to the target sequence or a cell comprising the target sequence, wherein the method produces the modified target polynucleotide, and wherein components of the DPE system are delivered simultaneously or sequentially to the target sequence or the cell.

434. The method of claim 433, wherein the method is performed in vitro, in vivo or ex vivo.

435. The method of claim 433 or 434, wherein the target polynucleotide is intracellular.

436. The method of claim 435, wherein the cell is a eukaryotic cell.

437. The method of claim 436, wherein the eukaryotic cell is a mammalian cell.

438. A cell produced by the method of claim 435, wherein the target polynucleotide has been modified at the target sequence.

439. The cell of claim 438, wherein the cell is a prokaryotic cell.

440. The cell of claim 438, wherein the cell is a eukaryotic cell.

441. The cell of claim 440, wherein the eukaryotic cell is a mammalian cell.

442. The cell of claim 441, wherein the mammalian cell is a human cell.

443. The cell of claim 442, wherein the human cell is an immune cell.

444. The cell of claim 442, wherein the human cell is a stem cell.

445. The cell of claim 444, wherein the stem cell is an induced pluripotent stem cell.

446. The cell of claim 440, wherein the eukaryotic cell is an insect cell or a bird cell.

447. The cell of claim 440, wherein the eukaryotic cell is a fungal cell.

448. The cell of claim 440, wherein the eukaryotic cell is a plant cell.

449. A plant or plant part comprising plant cells according to claim 448.

450. A pharmaceutical composition comprising cells and a pharmaceutically acceptable carrier according to any one of claims 440 to 445.

451. A method for treating a subject suffering from a disease, condition, or symptom, or at risk of developing a disease, condition, or symptom, the method comprising: The subject is given the DPE system according to any one of claims 413 to 419, the cells according to any one of claims 423 to 427 and 440 to 445, or the pharmaceutical composition according to claim 432 or 450.

452. The method of claim 451, wherein the disease, symptom, or condition is related to a mutation, and the treatment comprises correcting the mutation.

453. Use of a DPE system according to any one of claims 413 to 419, a cell according to any one of claims 423 to 427 and 440 to 445, or a pharmaceutical composition according to claim 432 or 450, for the treatment of a subject suffering from a disease, condition, or symptom, or at risk of developing a disease, condition, or symptom.

454. The use according to claim 453, wherein the disease, symptom, or condition is related to a mutation, and the treatment comprises correcting the mutation.

455. Use of a DPE system according to any one of claims 413 to 419, a cell according to any one of claims 423 to 427 and 440 to 445, or a pharmaceutical composition according to claim 432 or 450, for the preparation of a medicament suitable for treating a disease, condition, or symptom.

456. The use according to claim 455, wherein the disease, condition, or symptom is related to a mutation, and an effective amount of the drug corrects the mutation.

Images