RNA-guided nucleases and active fragments and variants thereof, and methods of use

Abstract

Compositions and methods are provided for binding to a target sequence of interest. The compositions find use in cleaving or modifying a target sequence of interest, visualizing a target sequence of interest, and modifying the expression of a sequence of interest. The compositions include an RNA-guided nuclease (RGN) polypeptide, a CRISPR RNA, a trans-activated CRISPR RNA, a guide RNA, and nucleic acid molecules encoding them. Vectors and host cells containing the nucleic acid molecules are also provided. Additionally provided is an RGN system for binding to a target sequence of interest, comprising an RNA-guided nuclease polypeptide and one or more guide RNAs.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 302,271, filed January 24, 2022, and U.S. Provisional Patent Application No. 63 / 386,061, filed December 5, 2022, each of which is incorporated by reference in its entirety.

[0002] Reference to sequence listing submitted electronically as an XML file This application contains a Sequence Listing that has been submitted via the USPTO Patent Center in xml format and is incorporated herein by reference in its entirety. The xml copy, created on January 23, 2023, is named L103438_1260WO_Seq_List.xml and is 905KB in size.

[0003] The present invention relates to the fields of molecular biology and gene editing. [Background technology]

[0004] Targeted genome editing or modification is rapidly becoming an important tool for basic and applied research. Early methods involve engineering nucleases such as meganucleases, zinc finger fusion proteins, or TALENs, which require the generation of chimeric nucleases with engineered, programmable, sequence-specific DNA-binding domains specific for each particular target sequence. RNA-guided nucleases, such as the CRISPR-associated (Cas) proteins of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas bacterial system, enable targeting of specific sequences by complexing the nuclease with a guide RNA that specifically hybridizes to the specific target sequence. Producing target-specific guide RNAs is less costly and more efficient than generating chimeric nucleases for each target sequence. Such RNA-guided nucleases can be used to selectively edit genomes by introducing sequence-specific double-strand breaks that are repaired via error-prone non-homologous end joining (NHEJ) and introduce mutations at specific genomic locations. Alternatively, heterologous DNA can be introduced into genomic sites by homology-directed repair. RNA-guided nucleases (RGNs) can also be used for base editing when fused to deaminases. Summary of the Invention

[0005] Compositions and methods for binding to a target sequence of interest are provided. The compositions find use in cleaving or modifying a target sequence of interest, detecting a target sequence of interest, and modifying the expression of a sequence of interest. The compositions include an RNA-guided nuclease (RGN) polypeptide, a CRISPR RNA (crRNA), a trans-activating CRISPR RNA (tracrRNA), a guide RNA (gRNA), nucleic acid molecules encoding them, and vectors and host cells containing the nucleic acid molecules. Also provided is an RGN system for binding to a target sequence of interest, the RGN system comprising an RNA-guided nuclease polypeptide and one or more guide RNAs. Thus, methods disclosed herein relate to binding to a target sequence of interest, and in some embodiments, to cleaving or modifying the target sequence of interest. The target sequence of interest can be modified, for example, as a result of non-homologous end joining, homology-directed repair with an introduced donor sequence, or base editing.

[0006] In a first aspect, the present disclosure provides a nucleic acid molecule comprising a polynucleotide encoding an RNA-guided nuclease (RGN) polypeptide, wherein the polynucleotide comprises a nucleotide sequence encoding an RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOS: 1-12, and wherein the RGN polypeptide is capable of RNA-guided sequence-specific binding to a target DNA sequence when bound to a guide RNA (gRNA) capable of hybridizing to the target DNA sequence. In some embodiments, the RGN polypeptide comprises an amino acid sequence having 90% sequence identity to SEQ ID NOS: 6. In some embodiments of the above aspect, the RGN polypeptide comprises an amino acid sequence having 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 100% sequence identity to any one of SEQ ID NOS: 1-12. In some embodiments, the RGN polypeptide comprises an amino acid sequence having 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 100% sequence identity to SEQ ID NO:6.

[0007] In some embodiments of the above aspects, the polynucleotide encoding the RGN polypeptide is operably linked to a promoter heterologous to the polynucleotide.

[0008] In some embodiments of the above aspects, the RGN polypeptide is capable of cleaving the target DNA sequence upon binding.

[0009] In some embodiments of the above aspects, the RGN polypeptide is capable of generating a double-strand break.

[0010] In some embodiments of the above aspects, the RGN polypeptide is capable of generating a single-strand break.

[0011] In some embodiments of the above aspects, the RGN polypeptide is nuclease inactive or is a nickase.

[0012] In some embodiments of the above aspects, the RGN polypeptide is operably linked to a prime editing polypeptide. In some embodiments, the prime editing polypeptide comprises a DNA polymerase. In some embodiments, the prime editing polypeptide comprises a reverse transcriptase. In some embodiments of the above aspects, the RGN polypeptide is operably linked to a base-editing polypeptide. In some embodiments, the base-editing polypeptide is a deaminase. In some embodiments, the deaminase is a cytosine deaminase or a cytidine deaminase. In some embodiments, the deaminase has at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 377-448. In some embodiments of the above aspects, the deaminase has 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 100% sequence identity to any one of SEQ ID NOs: 377-448.

[0013] In some embodiments of the above aspects, the RGN polypeptide comprises one or more nuclear localization signals.

[0014] In some embodiments of the above aspects, the RGN polypeptide is codon-optimized for expression in eukaryotic cells.

[0015] In some embodiments of the above aspects, the target DNA sequence is located adjacent to a protospacer adjacent motif (PAM).

[0016] In another aspect, the present disclosure provides a vector comprising the above-described nucleic acid molecule.

[0017] In some embodiments of the above aspects, the vector further comprises at least one nucleotide sequence encoding a gRNA capable of hybridizing to a target DNA sequence.

[0018] In some embodiments of the above aspects, the guide RNA is selected from the group consisting of: a) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 13 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 25, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1; b) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 14 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 26, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2; c) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 15. a) a guide RNA comprising a CRISPR RNA and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 27, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 3; b) a guide RNA comprising a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 16 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 28, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 4; c) a guide RNA comprising a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 17 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 29, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 5; and f) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 18. a guide RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 19; and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 30, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 6;a guide RNA comprising a CRISPR RNA and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 31, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 7; h) a guide RNA comprising a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 20 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 32, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 8; i) a guide RNA comprising a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 21 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 33, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 9; j) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 22. a guide RNA comprising a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:23 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:35, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:11; and a guide RNA comprising a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:24 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:36, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:12.

[0019] In some embodiments of the above aspects, the guide RNA is a CRISPR comprising a CRISPR repeat sequence having 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 100% sequence identity to SEQ ID NO: 13. a) a guide RNA comprising an RGN polypeptide comprising an amino acid sequence having 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 100% sequence identity to SEQ ID NO: 25; and a tracrRNA having 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 100% sequence identity to SEQ ID NO: 1; b) a CRISPR comprising CRISPR repeats having 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 100% sequence identity to SEQ ID NO: 14. a guide RNA comprising an RGN polypeptide comprising an amino acid sequence having 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 100% sequence identity to SEQ ID NO:26; and a tracrRNA having 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 100% sequence identity to SEQ ID NO:26; c) a CRISPR comprising CRISPR repeats having 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 100% sequence identity to SEQ ID NO:15.a guide RNA comprising an RGN polypeptide comprising an amino acid sequence having 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 100% sequence identity to SEQ ID NO: 27; and a tracrRNA having 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 100% sequence identity to SEQ ID NO: 3; d) a CRISPR comprising CRISPR repeats having 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 100% sequence identity to SEQ ID NO: 16. a guide RNA comprising an RGN polypeptide comprising an amino acid sequence having 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 100% sequence identity to SEQ ID NO:28; e) a CRISPR comprising CRISPR repeats having 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 100% sequence identity to SEQ ID NO:17;a guide RNA comprising an RGN polypeptide comprising an amino acid sequence having 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 100% sequence identity to SEQ ID NO: 29; f) a CRISPR comprising a CRISPR repeat sequence having 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 100% sequence identity to SEQ ID NO: 18; a guide RNA comprising an RGN polypeptide comprising an amino acid sequence having 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 100% sequence identity to SEQ ID NO: 30; and a tracrRNA having 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 100% sequence identity to SEQ ID NO: 6; g) a CRISPR comprising CRISPR repeats having 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 100% sequence identity to SEQ ID NO: 19.a guide RNA comprising an RGN polypeptide comprising an RGN polypeptide and a tracrRNA having 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 100% sequence identity to SEQ ID NO: 31, wherein the RGN polypeptide comprises an amino acid sequence having 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 100% sequence identity to SEQ ID NO: 7; h) a CRISPR comprising CRISPR repeats having 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 100% sequence identity to SEQ ID NO: 20; and a tracrRNA having 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 100% sequence identity to SEQ ID NO:32, wherein the RGN polypeptide comprises an amino acid sequence having 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 100% sequence identity to SEQ ID NO:8; i) a CRISPR comprising CRISPR repeats having 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 100% sequence identity to SEQ ID NO:21;a guide RNA comprising an RGN polypeptide comprising an RGN polypeptide and a tracrRNA having 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 100% sequence identity to SEQ ID NO: 33, wherein the RGN polypeptide comprises an amino acid sequence having 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 100% sequence identity to SEQ ID NO: 9; j) a CRISPR comprising CRISPR repeats having 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 100% sequence identity to SEQ ID NO: 22; a guide RNA comprising an RGN polypeptide comprising an amino acid sequence having 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 100% sequence identity to SEQ ID NO: 34; and a tracrRNA having 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 100% sequence identity to SEQ ID NO: 10; k) a CRISPR comprising CRISPR repeats having 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 100% sequence identity to SEQ ID NO: 23.and l) a tracrRNA having 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 100% sequence identity to SEQ ID NO:35, wherein the RGN polypeptide comprises an amino acid sequence having 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 100% sequence identity to SEQ ID NO:11; and l) a CRISPR comprising CRISPR repeats having 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 100% sequence identity to SEQ ID NO:24. and a tracrRNA having 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 100% sequence identity to SEQ ID NO:36, wherein the RGN polypeptide comprises an amino acid sequence having 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 100% sequence identity to SEQ ID NO:12.

[0020] In some embodiments of the above aspects, the gRNA is a single guide RNA, hi some embodiments, the single guide RNA further comprises an extension comprising an editing template for prime editing.

[0021] In some embodiments of the above aspects, the gRNA is a dual guide RNA.

[0022] In another aspect, the present disclosure provides a cell comprising the above-described nucleic acid molecule or the above-described vector. In yet another aspect, the present disclosure provides a method for producing an RGN polypeptide, comprising culturing the above-described cell under conditions in which the RGN polypeptide is expressed.

[0023] In another aspect, the disclosure provides a method for producing an RGN polypeptide, the method comprising: introducing into a cell a heterologous nucleic acid molecule comprising a nucleotide sequence encoding an RNA-guided nuclease (RGN) polypeptide comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-12, wherein the RGN polypeptide is capable of RNA-guided sequence-specific binding to a target DNA sequence when bound to a guide RNA (gRNA) that can hybridize to the target DNA sequence; and culturing the cell under conditions in which the RGN polypeptide is expressed. In some embodiments, the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 6. In some embodiments, the RGN polypeptide comprises an amino acid sequence having 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 100% sequence identity to any one of SEQ ID NOs: 1-12. In some embodiments, the RGN polypeptide comprises an amino acid sequence having 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 100% sequence identity to SEQ ID NO:6.

[0024] In some embodiments of the above aspects, the method further comprises purifying the RGN polypeptide.

[0025] In some embodiments of the above aspects, the cell further expresses one or more guide RNAs capable of binding to an RGN polypeptide to form an RGN ribonucleoprotein complex.

[0026] In some embodiments of the above aspects, the method further comprises purifying the RGN ribonucleoprotein complex.

[0027] In yet another aspect, the disclosure provides an RNA-guided nuclease (RGN) polypeptide, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-12, and wherein the RGN polypeptide is capable of RNA-guided sequence-specific binding to a target DNA sequence of a DNA molecule when bound to a guide RNA (gRNA) that can hybridize to the target DNA sequence. In some embodiments, the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 6. In some embodiments, the RGN polypeptide comprises an amino acid sequence having 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 100% sequence identity to any one of SEQ ID NOs: 1-12. In some embodiments, the RGN polypeptide comprises an amino acid sequence having 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 100% sequence identity to SEQ ID NO: 6. In some embodiments of the above aspects, the RGN polypeptide is an isolated RGN polypeptide.

[0028] In some embodiments of the above aspects, the RGN polypeptide is capable of cleaving the target DNA sequence upon binding.

[0029] In some embodiments of the above aspects, cleavage by the RGN polypeptide generates a double-stranded break.

[0030] In some embodiments of the above aspects, cleavage by the RGN polypeptide generates a single-strand break.

[0031] In some embodiments of the above aspects, the RGN polypeptide is nuclease inactive or is a nickase.

[0032] In some embodiments of the above aspects, the RGN polypeptide is operably linked to a prime editing polypeptide. In some embodiments, the prime editing polypeptide comprises a DNA polymerase. In some embodiments, the prime editing polypeptide comprises a reverse transcriptase.

[0033] In some embodiments of the above aspects, the RGN polypeptide is operably linked to a base-editing polypeptide. In some embodiments, the base-editing polypeptide is a deaminase. In some embodiments, the deaminase is a cytosine deaminase or a cytidine deaminase. In some embodiments, the deaminase has at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 377-448. In some embodiments, the deaminase has 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 100% sequence identity to any one of SEQ ID NOs: 377-448.

[0034] In some embodiments of the above aspects, the target DNA sequence is located adjacent to a protospacer adjacent motif (PAM).

[0035] In some embodiments of the above aspects, the RGN polypeptide comprises one or more nuclear localization signals.

[0036] In another aspect, the disclosure provides a ribonucleoprotein (RNP) complex comprising the above-described RGN polypeptide and a guide RNA bound to the RGN polypeptide. In some embodiments, the guide RNA is a single-guide RNA. In some embodiments, the single-guide RNA comprises an extension comprising an editing template for prime editing. In yet another aspect, the disclosure provides a system for binding to a target DNA sequence of a DNA molecule, the system comprising: a) one or more polynucleotides comprising one or more nucleotide sequences encoding one or more guide RNAs (gRNAs) capable of hybridizing to the target DNA sequence; and b) an RNA-guided nuclease (RGN) polypeptide comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-12, or a polynucleotide comprising a nucleotide sequence encoding the RGN polypeptide, wherein the one or more guide RNAs are capable of hybridizing to the target DNA sequence, and the one or more guide RNAs are capable of forming a complex with the RGN polypeptide to guide the RGN polypeptide to bind to the target DNA sequence of the DNA molecule.

[0037] In some embodiments of the above aspects, the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:6.

[0038] In some embodiments of the above aspects, at least one of the nucleotide sequence encoding the one or more guide RNAs and the nucleotide sequence encoding the RGN polypeptide is operably linked to a promoter heterologous to the nucleotide sequence.

[0039] In some embodiments of the above aspects, the nucleotide sequence encoding one or more guide RNAs and the nucleotide sequence encoding the RGN polypeptide are located on a single vector.

[0040] In yet another aspect, the present disclosure provides a system for binding to a target DNA sequence of a DNA molecule, the system comprising: a) one or more guide RNAs capable of hybridizing to the target DNA sequence, or one or more polynucleotides comprising one or more nucleotide sequences encoding one or more guide RNAs (gRNAs); and b) an RNA-guided nuclease (RGN) polypeptide comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-12, wherein the one or more guide RNAs are capable of hybridizing to the target DNA sequence, and the one or more guide RNAs are capable of forming a complex with the RGN polypeptide to guide the RGN polypeptide to bind to the target DNA sequence of the DNA molecule.

[0041] In some embodiments of the above aspects, the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:6.

[0042] In some embodiments of the above aspects, the RGN polypeptide comprises an amino acid sequence having 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 100% sequence identity to any one of SEQ ID NOs: 1-12. In some embodiments, the RGN polypeptide comprises an amino acid sequence having 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 100% sequence identity to SEQ ID NO: 6.

[0043] In some embodiments of the above aspects, at least one of the nucleotide sequences encoding the one or more guide RNAs is operably linked to a promoter heterologous to the nucleotide sequence.

[0044] In some embodiments of the above aspects, the RGN polypeptide and the one or more guide RNAs are not found complexed to each other in nature.

[0045] In some embodiments of the above aspects, the target DNA sequence is a eukaryotic target DNA sequence.

[0046] In some embodiments of the above aspects, the gRNA is a single guide RNA (sgRNA). In some embodiments, the sgRNA comprises an extension that includes an editing template for prime editing.

[0047] In some embodiments of the above aspects, the gRNA is a dual guide RNA.

[0048] In some embodiments of the above aspects, the gRNA is a) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 13 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 25, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1; or b) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 14 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 26. a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 15 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 27, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 3; a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 16 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 28, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 3; a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity with SEQ ID NO: 17 and a tracrRNA having at least 90% sequence identity with SEQ ID NO: 29, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 5; and a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity with SEQ ID NO: 18 and a tracrRNA having at least 90% sequence identity with SEQ ID NO: 29, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 5. gRNA comprising a CRISPR repeat sequence and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 30, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 6; gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 19 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 31, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 7;h) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:20 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:32, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:8; i) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:21 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:33, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:9; j) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:22 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:34. a) a gRNA comprising an RGN polypeptide having an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 10; b) a gRNA comprising a CRISPR repeat sequence with at least 90% sequence identity to SEQ ID NO: 23 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 35, wherein the RGN polypeptide has an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 11; and c) a gRNA comprising a CRISPR repeat sequence with at least 90% sequence identity to SEQ ID NO: 24 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 36, wherein the RGN polypeptide has an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 12. In some embodiments, the gRNA comprises a CRISPR repeat sequence with at least 90% sequence identity to SEQ ID NO: 18 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 30.

[0049] In some embodiments of the above aspects, the gRNA comprises: a) a CRISPR repeat sequence having 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 100% sequence identity to SEQ ID NO: 13; and a tracr repeat sequence having 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 100% sequence identity to SEQ ID NO: 25. and a gRNA comprising an RGN polypeptide having an amino acid sequence having 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 100% sequence identity to SEQ ID NO: 1; b) a gRNA comprising an RGN polypeptide having 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 100% sequence identity to SEQ ID NO: 14. and a tracrRNA having 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 100% sequence identity to SEQ ID NO:26, wherein the RGN polypeptide has 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 100% sequence identity to SEQ ID NO:2. c) a CRISPR repeat sequence having 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 100% sequence identity to SEQ ID NO: 15; and a tracrRNA having 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 100% sequence identity to SEQ ID NO: 27.wherein the RGN polypeptide comprises an amino acid sequence having 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 100% sequence identity to SEQ ID NO:3; d) a gRNA comprising 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 100% sequence identity to SEQ ID NO:16. and a tracrRNA having 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 100% sequence identity to SEQ ID NO:28, wherein the RGN polypeptide has 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 100% sequence identity to SEQ ID NO:4. e) a gRNA comprising an amino acid sequence having 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 100% sequence identity to SEQ ID NO: 17; and f) a CRISPR repeat sequence having 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 100% sequence identity to SEQ ID NO: 29. and f) a tracrRNA having the following sequence identity: wherein the RGN polypeptide comprises an amino acid sequence having 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 100% sequence identity to SEQ ID NO: 5; f) a tracrRNA having the following sequence identity:or 100% sequence identity to SEQ ID NO:30; and a tracrRNA having 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 100% sequence identity to SEQ ID NO:30, wherein the RGN polypeptide has 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 100% sequence identity to SEQ ID NO:6. g) a gRNA comprising an amino acid sequence with 9% or 100% sequence identity to SEQ ID NO: 19; g) a CRISPR repeat sequence with 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 100% sequence identity to SEQ ID NO: 31; a gRNA comprising a tracrRNA having 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 100% sequence identity to SEQ ID NO:7, wherein the RGN polypeptide comprises an amino acid sequence having 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 100% sequence identity to SEQ ID NO:20; a gRNA comprising a CRISPR repeat sequence with 100% sequence identity and a tracrRNA with 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 100% sequence identity to SEQ ID NO:32, wherein the RGN polypeptide has 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 100% sequence identity to SEQ ID NO:8;or 100% sequence identity to SEQ ID NO: 21; i) a CRISPR repeat sequence having 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 100% sequence identity to SEQ ID NO: 21; and and a tracrRNA having identity to SEQ ID NO: 9, wherein the RGN polypeptide comprises an amino acid sequence having 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 100% sequence identity to SEQ ID NO: 9; j) a gRNA comprising a tracrRNA having identity to SEQ ID NO: 22, wherein the RGN polypeptide comprises an amino acid sequence having 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 100% sequence identity to SEQ ID NO: 22; and a tracrRNA having 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 100% sequence identity to SEQ ID NO:34, wherein the RGN polypeptide has at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, k) a gRNA comprising an amino acid sequence having 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 100% sequence identity to SEQ ID NO: 23; and c) a CRISPR repeat sequence having 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 100% sequence identity to SEQ ID NO: 35;or 100% sequence identity to SEQ ID NO: 11, wherein the RGN polypeptide comprises an amino acid sequence having 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 100% sequence identity to SEQ ID NO: 11; and l) a tracrRNA having 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 100% sequence identity to SEQ ID NO: 24. a gRNA comprising an ISPR repeat sequence and a tracrRNA having 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 100% sequence identity to SEQ ID NO:36, wherein the RGN polypeptide comprises an amino acid sequence having 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 100% sequence identity to SEQ ID NO:12. In some embodiments, the gRNA comprises a CRISPR repeat sequence having 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 100% sequence identity to SEQ ID NO: 18, and a tracrRNA having 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 100% sequence identity to SEQ ID NO: 30.

[0050] In some embodiments of the above aspects, the target DNA sequence is located adjacent to a protospacer adjacent motif (PAM).

[0051] In some embodiments of the above aspects, the target DNA sequence is within a cell.

[0052] In some embodiments of the above aspects, one or more guide RNAs are capable of hybridizing to a target DNA sequence, and the guide RNAs are capable of forming a complex with an RGN polypeptide to direct cleavage of the target DNA sequence.

[0053] In some embodiments of the above aspects, the cleavage generates a double-stranded break.

[0054] In some embodiments of the above aspects, the cleavage generates a single-strand break.

[0055] In some embodiments of the above aspects, the RGN polypeptide is nuclease inactive or is a nickase.

[0056] In some embodiments of the above aspects, the RGN polypeptide is operably linked to a prime editing polypeptide. In some embodiments, the prime editing polypeptide comprises a DNA polymerase. In some embodiments, the prime editing polypeptide comprises a reverse transcriptase.

[0057] In some embodiments of the above aspects, the RGN polypeptide is operably linked to the base-editing polypeptide.

[0058] In some embodiments of the above aspects, the base-editing polypeptide is a deaminase. In some embodiments, the deaminase is a cytosine deaminase or a cytidine deaminase. In some embodiments, the deaminase has at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 377-448. In some embodiments, the deaminase has 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 100% sequence identity to any one of SEQ ID NOs: 377-448.

[0059] In some embodiments of the above aspects, the RGN polypeptide comprises one or more nuclear localization signals.

[0060] In some embodiments of the above aspects, the RGN polypeptide is codon-optimized for expression in eukaryotic cells.

[0061] In some embodiments of the above aspects, the system further comprises one or more donor polynucleotides.

[0062] In another aspect, the present disclosure provides a cell comprising the above-described system.

[0063] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising a nucleic acid molecule, a vector, a cell, an RGN polypeptide, an RNP complex, or the system described above, and a pharmaceutically acceptable carrier.

[0064] In yet another aspect, the present disclosure provides a method for binding a DNA molecule to a target DNA sequence, comprising delivering the above-described system to the target DNA sequence or to a cell containing the target DNA sequence.

[0065] In some embodiments of the above aspects, the RGN polypeptide or guide RNA further comprises a detectable label, thereby allowing detection of the target DNA sequence.

[0066] In some embodiments of the above aspects, the guide RNA or RGN polypeptide further comprises an expression regulator, thereby regulating expression of the target DNA sequence or a gene under transcriptional control by the target DNA sequence.

[0067] In another aspect, the present disclosure provides a method for cleaving and / or modifying a target DNA sequence of a DNA molecule, comprising delivering the above-described system to a cell containing the target DNA sequence or DNA molecule, whereby cleavage or modification of the target DNA sequence occurs.

[0068] In some embodiments of the above aspects, the modified target DNA sequence comprises an insertion of heterologous DNA into the target DNA sequence.

[0069] In some embodiments of the above aspects, the modified target DNA sequence comprises a deletion of at least one nucleotide from the target DNA sequence.

[0070] In some embodiments of the above aspects, the modified target DNA sequence comprises a mutation of at least one nucleotide in the target DNA sequence.

[0071] In another aspect, the present disclosure provides a method for binding to a target DNA sequence of a DNA molecule, the method comprising: (a) assembling an RNA-guided nuclease (RGN) ribonucleotide complex by combining one or more guide RNAs capable of hybridizing to the target DNA sequence with an RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOS: 1-12 under conditions suitable for the formation of an RGN ribonucleotide complex; and (b) contacting the target DNA sequence or a cell comprising the target DNA sequence with the assembled RGN ribonucleotide complex, wherein the one or more guide RNAs hybridize to the target DNA sequence, thereby directing the RGN polypeptide to bind to the target DNA sequence. In some embodiments, the RGN polypeptide has at least 90% sequence identity to SEQ ID NO: 6.

[0072] In some embodiments of the above aspects, the RGN polypeptide comprises an amino acid sequence having 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 100% sequence identity to any one of SEQ ID NOs: 1-12. In some embodiments, the RGN polypeptide has 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 100% sequence identity to SEQ ID NO: 6.

[0073] In some embodiments of the above aspects, the method is carried out in vitro, in vivo, or ex vivo.

[0074] In some embodiments of the above aspects, the RGN polypeptide or guide RNA further comprises a detectable label, thereby allowing detection of the target DNA sequence.

[0075] In some embodiments of the above aspects, the guide RNA or RGN polypeptide further comprises an expression regulator, thereby allowing for regulation of expression of the target DNA sequence.

[0076] In some embodiments of the above aspects, the RGN polypeptide is operably linked to a prime editing polypeptide, thereby enabling modification of the target DNA sequence. In some embodiments, the prime editing polypeptide comprises a DNA polymerase. In some embodiments, the prime editing polypeptide comprises a reverse transcriptase.

[0077] In some embodiments of the above aspects, the RGN polypeptide is operably linked to a base-editing polypeptide, thereby allowing for modification of the target DNA sequence. In some embodiments, the base-editing polypeptide comprises a deaminase. In some embodiments, the deaminase is a cytosine deaminase or a cytidine deaminase. In some embodiments of the above aspects, the RGN polypeptide is capable of cleaving the target DNA sequence, thereby allowing for cleavage and / or modification of the target DNA sequence.

[0078] In another aspect, the disclosure provides a method for cleaving and / or modifying a target DNA sequence of a DNA molecule, the method comprising contacting the DNA molecule with: (a) an RNA-guided nuclease (RGN) polypeptide, wherein RGN comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-12; and (b) one or more guide RNAs capable of targeting the RGN of (a) to the target DNA sequence, wherein the one or more guide RNAs hybridize to the target DNA sequence, thereby directing the RGN polypeptide to bind to the target DNA sequence, resulting in cleavage and / or modification of the target DNA sequence. In some embodiments, the RGN polypeptide has at least 90% sequence identity to SEQ ID NO: 6.

[0079] In some embodiments of the above aspects, the RGN polypeptide comprises an amino acid sequence having 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 100% sequence identity to any one of SEQ ID NOs: 1-12. In some embodiments, the RGN polypeptide has 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 100% sequence identity to SEQ ID NO: 6.

[0080] In some embodiments of the above aspects, cleavage by the RGN polypeptide generates a double-stranded break.

[0081] In some embodiments of the above aspects, cleavage by the RGN polypeptide generates a single-strand break.

[0082] In some embodiments of the above aspects, the RGN polypeptide is nuclease inactive or is a nickase.

[0083] In some embodiments of the above aspects, the RGN polypeptide is operably linked to a prime editing polypeptide, thereby enabling modification of the target DNA sequence. In some embodiments, the prime editing polypeptide comprises a DNA polymerase. In some embodiments, the prime editing polypeptide comprises a reverse transcriptase.

[0084] In some embodiments of the above aspects, the RGN polypeptide is operably linked to the base-editing polypeptide.

[0085] In some embodiments of the above aspects, the base-editing polypeptide is a deaminase. In some embodiments, the deaminase is a cytosine deaminase or a cytidine deaminase. In some embodiments, the deaminase has at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 377-448. In some embodiments, the deaminase has 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 100% sequence identity to any one of SEQ ID NOs: 377-448.

[0086] In some embodiments of the above aspects, the modified target DNA sequence comprises an insertion of heterologous DNA into the target DNA sequence.

[0087] In some embodiments of the above aspects, the modified target DNA sequence comprises a deletion of at least one nucleotide from the target DNA sequence.

[0088] In some embodiments of the above aspects, the modified target DNA sequence comprises a mutation of at least one nucleotide in the target DNA sequence.

[0089] In some embodiments of the above aspects, the target DNA sequence is located adjacent to a protospacer adjacent motif (PAM).

[0090] In some embodiments of the above aspects, the target DNA sequence is a eukaryotic target DNA sequence.

[0091] In some embodiments of the above aspects, the gRNA is a single guide RNA (sgRNA). In some embodiments, the sgRNA comprises an extension that includes an editing template for prime editing.

[0092] In some embodiments of the above aspects, the gRNA is a dual guide RNA.

[0093] In some embodiments of the above aspects, the RGN, guide RNA, and tracrRNA are selected from the group consisting of: a) an RGN having at least 90% sequence identity to SEQ ID NO: 1, a guide RNA comprising a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO: 13, and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 25; b) an RGN having at least 90% sequence identity to SEQ ID NO: 2, a guide RNA comprising a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO: 14, and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 26. c) a tracrRNA having at least 90% sequence identity with SEQ ID NO:3, a guide RNA comprising a crRNA repeat sequence having at least 90% sequence identity with SEQ ID NO:15, and a tracrRNA having at least 90% sequence identity with SEQ ID NO:27, d) a RGN having at least 90% sequence identity with SEQ ID NO:4, a guide RNA comprising a crRNA repeat sequence having at least 90% sequence identity with SEQ ID NO:16, and a tracrRNA having at least 90% sequence identity with SEQ ID NO:28, e) a tracrRNA having at least 90% sequence identity with SEQ ID NO:5 f) an RGN having at least 90% sequence identity with SEQ ID NO: 6, a guide RNA comprising a crRNA repeat sequence having at least 90% sequence identity with SEQ ID NO: 18, and a tracrRNA having at least 90% sequence identity with SEQ ID NO: 30; g) an RGN having at least 90% sequence identity with SEQ ID NO: 7, a guide RNA comprising a crRNA repeat sequence having at least 90% sequence identity with SEQ ID NO: 19, and a tracrRNA having at least 90% sequence identity with SEQ ID NO: 29; h) a guide RNA comprising a crRNA repeat sequence having at least 90% sequence identity with SEQ ID NO: 8, and a tracrRNA having at least 90% sequence identity with SEQ ID NO: 31; h) a guide RNA comprising a RGN having at least 90% sequence identity with SEQ ID NO: 8, a crRNA repeat sequence having at least 90% sequence identity with SEQ ID NO: 20, and a tracrRNA having at least 90% sequence identity with SEQ ID NO: 32; i) a guide RNA comprising a RGN having at least 90% sequence identity with SEQ ID NO: 9, and a crRNA repeat sequence having at least 90% sequence identity with SEQ ID NO: 21;and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 33; j) an RGN having at least 90% sequence identity to SEQ ID NO: 10, a guide RNA comprising a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO: 22, and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 34; k) an RGN having at least 90% sequence identity to SEQ ID NO: 11, a guide RNA comprising a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO: 23, and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 35; and l) an RGN having at least 90% sequence identity to SEQ ID NO: 12, a guide RNA comprising a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO: 24, and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 36. In some embodiments, the RGN has at least 90% sequence identity to SEQ ID NO:6, the guide RNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO:18, and the tracrRNA has at least 90% sequence identity to SEQ ID NO:30.

[0094] In some embodiments of the above aspects, the RGN, guideRNA, and tracrRNA are: a) an RGN having 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 100% sequence identity to SEQ ID NO: 1; an RGN having 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 100% sequence identity to SEQ ID NO: 13; a) a guide RNA comprising a crRNA repeat sequence having the same sequence identity as SEQ ID NO:25, and a tracrRNA having 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 100% sequence identity to SEQ ID NO:25; b) a RGN having 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 100% sequence identity to SEQ ID NO:2; a) a guide RNA comprising a crRNA repeat sequence having 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 100% sequence identity to SEQ ID NO: 14, and a tracrRNA having 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 100% sequence identity to SEQ ID NO: 26; a guide RNA comprising a crRNA repeat sequence having 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 100% sequence identity to SEQ ID NO: 15; a guide RNA comprising a crRNA repeat sequence having 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 100% sequence identity to SEQ ID NO: 27;d) a tracrRNA having at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:4; d) a RGN having 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 100% sequence identity to SEQ ID NO:16; e) a guide RNA comprising a crRNA repeat sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:28; and f) a tracrRNA having 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 100% sequence identity to SEQ ID NO:5. a guide RNA comprising a crRNA repeat sequence having 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 100% sequence identity to SEQ ID NO: 17; and a guide RNA comprising a crRNA repeat sequence having 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 100% sequence identity to SEQ ID NO: 29. f) a tracrRNA having 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 100% sequence identity to SEQ ID NO: 6; f) a RGN having 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 100% sequence identity to SEQ ID NO: 18;g) a guide RNA comprising a crRNA repeat sequence with 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 100% sequence identity to SEQ ID NO: 30; g) a tracrRNA with 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 100% sequence identity to SEQ ID NO: 7; a guide RNA comprising a crRNA repeat sequence having 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 100% sequence identity to SEQ ID NO: 19; and a tracrRNA having 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 100% sequence identity to SEQ ID NO: 31. ) an RGN having 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 100% sequence identity to SEQ ID NO:8, a guide RNA comprising a crRNA repeat sequence having 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 100% sequence identity to SEQ ID NO:20, and a guide RNA having 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 100% sequence identity to SEQ ID NO:32; i) a tracrRNA having 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 100% sequence identity with SEQ ID NO: 9; i) an RGN having 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 100% sequence identity with SEQ ID NO: 21;j) a guide RNA comprising a crRNA repeat sequence with at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 33; j) a tracrRNA with 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 100% sequence identity to SEQ ID NO: 10; a guide RNA comprising a crRNA repeat sequence having 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 100% sequence identity to SEQ ID NO:22; and a guide RNA comprising a crRNA repeat sequence having 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 100% sequence identity to SEQ ID NO:34. k) a tracrRNA having at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 11; k) a RGN having 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 100% sequence identity with SEQ ID NO: 23; l) a guide RNA comprising a crRNA repeat sequence with 9% or 100% sequence identity, and a tracrRNA with 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 100% sequence identity to SEQ ID NO: 35; and l) a tracrRNA with 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 100% sequence identity to SEQ ID NO: 12.or 100% sequence identity to SEQ ID NO:24; a guide RNA comprising a crRNA repeat sequence that has 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 100% sequence identity to SEQ ID NO:24; and a tracrRNA that has 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 100% sequence identity to SEQ ID NO:36. In some embodiments, the RGN has 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 100% sequence identity to SEQ ID NO:6, the guide RNA comprises a crRNA repeat sequence having 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 100% sequence identity to SEQ ID NO:18, and the tracrRNA has 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 100% sequence identity to SEQ ID NO:30.

[0095] In some embodiments of the above aspects, the target DNA sequence is within a cell.

[0096] In some embodiments of the above aspects, the method further comprises culturing the cells under conditions in which the RGN polypeptide is expressed and cleaves and modifies the target DNA sequence to produce a DNA molecule comprising the modified target DNA sequence, and selecting for cells comprising the modified target DNA sequence.

[0097] In another aspect, the present disclosure provides a cell comprising a target DNA sequence modified by the above-described method.

[0098] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising the cells described above and a pharmaceutically acceptable carrier.

[0099] In yet another aspect, the present disclosure provides a method of treating a disease, disorder, or condition, comprising administering to a subject in need of treatment for the disease, disorder, or condition an effective amount of the pharmaceutical composition described above.

[0100] In some embodiments of the above aspects, the disease is associated with a causative mutation and the effective amount of the pharmaceutical composition corrects or eliminates the causative mutation.

[0101] In some embodiments of the above aspects, the subject is at risk of developing the disease, disorder, or condition.

[0102] In another aspect, the present disclosure provides a nucleic acid molecule comprising a CRISPR RNA (crRNA) or a polynucleotide encoding the crRNA, wherein the crRNA comprises a spacer sequence and a CRISPR repeat sequence, the CRISPR repeat sequence comprising a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 13-24, and a guide RNA comprising the crRNA and a trans-activating CRISPR RNA (tracrRNA) hybridized to the CRISPR repeat sequence of the crRNA can hybridize to a target DNA sequence in a sequence-specific manner via the spacer sequence of the crRNA when the guide RNA is bound to an RNA-guided nuclease (RGN) polypeptide. In some embodiments, the CRISPR repeat sequence comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 18.

[0103] In some embodiments of the above aspects, the polynucleotide encoding the crRNA is operably linked to a promoter heterologous to the polynucleotide.

[0104] In some embodiments of the above aspects, the CRISPR repeat sequence comprises a nucleotide sequence having 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 100% sequence identity to any one of SEQ ID NOs: 13-24. In some embodiments, the CRISPR repeat sequence comprises a nucleotide sequence having at least 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 100% sequence identity to SEQ ID NO: 18.

[0105] In another aspect, the present disclosure provides a vector comprising a nucleic acid molecule comprising a polynucleotide encoding the above-described crRNA.

[0106] In some embodiments of the above aspects, the vector further comprises a polynucleotide encoding a tracrRNA.

[0107] In some embodiments of the above aspects, the tracrRNA is selected from the group consisting of: a) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 25, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 13; b) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 26, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 14; c) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 27, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 14; a tracrRNA having at least 90% sequence identity with SEQ ID NO: 15, a tracrRNA having at least 90% sequence identity with SEQ ID NO: 28, and a CRISPR repeat sequence having at least 90% sequence identity with SEQ ID NO: 16, a tracrRNA having at least 90% sequence identity with SEQ ID NO: 29, and a CRISPR repeat sequence having at least 90% sequence identity with SEQ ID NO: 17, and a tracrRNA having at least 90% sequence identity with SEQ ID NO: 30. a tracrRNA having at least 90% sequence identity with SEQ ID NO: 18; g) a tracrRNA having at least 90% sequence identity with SEQ ID NO: 31, wherein the CRISPR repeat sequence has at least 90% sequence identity with SEQ ID NO: 19; h) a tracrRNA having at least 90% sequence identity with SEQ ID NO: 32, wherein the CRISPR repeat sequence has at least 90% sequence identity with SEQ ID NO: 20; i) a tracrRNA having at least 90% sequence identity with SEQ ID NO: 33. a tracrRNA having at least 90% sequence identity with SEQ ID NO: 34, wherein the CRISPR repeat sequence has at least 90% sequence identity with SEQ ID NO: 22; a tracrRNA having at least 90% sequence identity with SEQ ID NO: 35, wherein the CRISPR repeat sequence has at least 90% sequence identity with SEQ ID NO: 23;and l) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 36, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 24. In some embodiments, the tracrRNA has at least 90% sequence identity to SEQ ID NO: 30, and the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 18.

[0108] In some embodiments of the above aspects, the tracrRNA is a tracrRNA that: a) has 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 100% sequence identity to SEQ ID NO:25, and the CRISPR repeat sequence is 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 100% sequence identity to SEQ ID NO:13; a) a tracrRNA having 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 100% sequence identity to SEQ ID NO:26, wherein the CRISPR repeat sequence has 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 100% sequence identity to SEQ ID NO:14; c) a tracrRNA having 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 100% sequence identity to SEQ ID NO:27, wherein the CRISPR repeat sequence has 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 100% sequence identity to SEQ ID NO:15. d) a tracrRNA having 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 100% sequence identity to SEQ ID NO:28, wherein the CRISPR repeat sequence has 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 100% sequence identity to SEQ ID NO:16;e) a tracrRNA having 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 100% sequence identity to SEQ ID NO:29, wherein the CRISPR repeat sequence has 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 100% sequence identity to SEQ ID NO:17; acrRNA, f) a tracrRNA having 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 100% sequence identity to SEQ ID NO: 30, wherein the CRISPR repeat sequence has 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 100% sequence identity to SEQ ID NO: 18. g) a tracrRNA having 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 100% sequence identity to SEQ ID NO: 31, wherein the CRISPR repeat sequence has 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 100% sequence identity to SEQ ID NO: 19; h) a tracrRNA having 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 100% sequence identity to SEQ ID NO: 32, wherein the CRISPR repeat sequence has 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 100% sequence identity to SEQ ID NO: 20; i) a tracrRNA having 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 100% sequence identity to SEQ ID NO: 33, wherein the CRISPR repeat sequence has 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 100% sequence identity to SEQ ID NO: 21; j) a tracrRNA having a sequence k) a tracrRNA having 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 100% sequence identity to SEQ ID NO: 34, wherein the CRISPR repeat sequence has 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 100% sequence identity to SEQ ID NO: 22; 5, wherein the CRISPR repeat sequence has 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 100% sequence identity to SEQ ID NO: 23; and l) a tracrRNA with SEQ ID NO: 36. a tracrRNA having 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 100% sequence identity, wherein the CRISPR repeat sequence has 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 100% sequence identity to SEQ ID NO:24.In some embodiments, the tracrRNA has 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 100% sequence identity to SEQ ID NO:30, and the CRISPR repeat sequence has 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 100% sequence identity to SEQ ID NO:18.

[0109] In some embodiments of the above aspects, the vector further comprises a polynucleotide encoding an RGN polypeptide.

[0110] In some embodiments of the above aspects, the RGN polypeptide is selected from the group consisting of: a) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 1, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 13, and the tracrRNA has at least 90% sequence identity to SEQ ID NO: 25; b) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 2, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 14, and the tracrRNA has at least 90% sequence identity to SEQ ID NO: 26. c) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO:3, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO:15 and the tracrRNA has at least 90% sequence identity to SEQ ID NO:27; d) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO:4, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO:16 and the tracrRNA has at least 90% sequence identity to SEQ ID NO: e) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO:5, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO:17 and the tracrRNA has at least 90% sequence identity to SEQ ID NO:29; f) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO:6, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO:18 and the tracrRNA has at least 90% sequence identity to SEQ ID NO:29. g) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 7, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 19, and the tracrRNA has at least 90% sequence identity to SEQ ID NO: 31; h) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 8, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 20, and the tracrRNA has at least 90% sequence identity to SEQ ID NO:i) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 32; i) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 9, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 21 and the tracrRNA has at least 90% sequence identity to SEQ ID NO: 33; j) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 10, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 22 and the tracrRNA has at least 90% sequence identity to SEQ ID NO: 34; k) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 11, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 23 and the tracrRNA has at least 90% sequence identity to SEQ ID NO: 35, and l) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 12, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 24 and the tracrRNA has at least 90% sequence identity to SEQ ID NO: 36. In some embodiments, the RGN polypeptide has at least 90% sequence identity to SEQ ID NO: 6, the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 18, and the tracrRNA has at least 90% sequence identity to SEQ ID NO: 30.

[0111] In some embodiments of the above aspects, the RGN polypeptide is: a) an RGN polypeptide having 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 100% sequence identity to SEQ ID NO: 1; and wherein the CRISPR repeat sequence is 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 100% identical to SEQ ID NO: 13. has 100% sequence identity to SEQ ID NO:25, and the tracrRNA has 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 100% sequence identity to SEQ ID NO:25; b) an RGN polypeptide having 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 100% sequence identity to SEQ ID NO:2. a polypeptide, wherein the CRISPR repeat sequence has 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 100% sequence identity to SEQ ID NO:14, and the tracrRNA has 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 100% sequence identity to SEQ ID NO:26; c) an RGN polypeptide having 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 100% sequence identity to SEQ ID NO:3, wherein the CRISPR repeat sequence has 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 100% sequence identity to SEQ ID NO:15, and the tracrRNA isd) an RGN polypeptide having 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 100% sequence identity to SEQ ID NO: 27; d) an RGN polypeptide having 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 100% sequence identity to SEQ ID NO: 4, wherein the CRISPR repeat sequence is e) an RGN polypeptide having 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 100% sequence identity with SEQ ID NO: 16, and the tracrRNA has 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 100% sequence identity with SEQ ID NO: 28; , at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 17, wherein the CRISPR repeat sequence has 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 100% sequence identity to SEQ ID NO: 17, and the tracrRNA has at least 91%, at least 92%, at least f) an RGN polypeptide having 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 100% sequence identity to SEQ ID NO: 6, wherein the CRISPR repeat sequence is 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 100% sequence identity to SEQ ID NO: 18;a) an RGN polypeptide having at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 30, wherein the tracrRNA has 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 100% sequence identity with SEQ ID NO: 30; b) an RGN polypeptide having 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 100% sequence identity with SEQ ID NO: 7; an RGN polypeptide having at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, wherein the CRISPR repeat sequence has 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 100% sequence identity to SEQ ID NO: 19; and a tracrRNA having 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 100% sequence identity to SEQ ID NO: 31. h) an RGN polypeptide having 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 100% sequence identity to SEQ ID NO:8, wherein the CRISPR repeat sequence is 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 100% identical to SEQ ID NO:20. , at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 32, and the tracrRNA has 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 100% sequence identity to SEQ ID NO: 32; i) an RGN polypeptide having at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, with SEQ ID NO: 9;an RGN polypeptide having at least 99% or 100% sequence identity, wherein the CRISPR repeat sequence has 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 100% sequence identity to SEQ ID NO:21; and a tracrRNA having at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:33. j) an RGN polypeptide having 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 100% sequence identity to SEQ ID NO: 10, wherein the CRISPR repeat sequence is 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 100% identical to SEQ ID NO: 22; k) an RGN polypeptide having 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 100% sequence identity with SEQ ID NO: 34, and the tracrRNA has 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 100% sequence identity with SEQ ID NO: 11; an RGN polypeptide comprising: a CRISPR repeat sequence having 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 100% sequence identity to SEQ ID NO:23; and a tracrRNA having 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 100% sequence identity to SEQ ID NO:35;and l) an RGN polypeptide having 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 100% sequence identity to SEQ ID NO: 12, wherein the CRISPR repeat sequence has 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 100% sequence identity to SEQ ID NO: 24, and the tracrRNA has 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 100% sequence identity to SEQ ID NO: 36. In some embodiments, the RGN polypeptide has 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 100% sequence identity to SEQ ID NO:6; the CRISPR repeat sequence has 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 100% sequence identity to SEQ ID NO:18; and the tracrRNA has 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 100% sequence identity to SEQ ID NO:30.

[0112] In another aspect, the present disclosure provides a nucleic acid molecule comprising a trans-activating CRISPR RNA (tracrRNA) or a polynucleotide encoding the tracrRNA, the tracrRNA comprising a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOS: 25-36, wherein the guide RNA comprising the tracrRNA and the crRNA comprising a spacer sequence and a CRISPR repeat sequence, wherein the tracrRNA hybridizes to the CRISPR repeat sequence of the crRNA, and the guide RNA can hybridize to a target DNA sequence in a sequence-specific manner via the spacer sequence of the crRNA when the guide RNA is bound to an RNA-guided nuclease (RGN) polypeptide. In some embodiments, the tracrRNA comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 30.

[0113] In some embodiments of the above aspects, the polynucleotide encoding the tracrRNA is operably linked to a promoter heterologous to the polynucleotide.

[0114] In some embodiments of the above aspects, the tracrRNA comprises a nucleotide sequence having 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 100% sequence identity to any one of SEQ ID NOs: 25-36. In some embodiments, the tracrRNA comprises a nucleotide sequence having at least 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 100% sequence identity to SEQ ID NO: 30.

[0115] In another aspect, the present disclosure provides a vector comprising a nucleic acid molecule comprising a polynucleotide encoding the above-described tracrRNA.

[0116] In yet another aspect, the present disclosure provides a single guide RNA comprising a nucleic acid molecule comprising the above-described crRNA and a nucleic acid molecule comprising the above-described tracrRNA.

[0117] In yet another aspect, the present disclosure provides a double guide RNA comprising a nucleic acid molecule comprising the above-described crRNA and a nucleic acid molecule comprising the above-described tracrRNA.

[0118] In another aspect, the present disclosure provides a cell comprising the nucleic acid molecule, single guide RNA, double guide RNA, or vector described above. [Brief explanation of the drawings]

[0119] [Figure 1] The editing rates of LPG10134, LPG10136, LPG10138, LPG10139, LPG10141, LPG10145, LPG10146, LPG10147, LPG10150, LPG10155, LPG10159, and LPG10160 on multiple genes are provided. Plasmids containing an expression cassette for RGN and guide RNA were co-transfected into HEK293T cells. [Figure 2] We demonstrate that the single guide RNA (sgRNA) SGN002661, together with the LPG10136 RNA-guided nuclease (RGN), provides greater than 75% editing of the T-cell receptor alpha (TRAC) gene as measured by flow cytometry when RGN and sgRNA are delivered as a ribonucleoprotein (RNP) complex in activated human peripheral blood mononuclear cells (PBMCs). B2M: beta-2-microglobulin. [Figure 3A] Demonstrating that increasing the concentration of RNP improves editing. The figure shows the editing efficiency of increasing concentrations of RNP composed of LPG10145 and various sgRNAs at a protein:RNA ratio of 1:2, as measured by flow cytometry of beta-2-microglobulin (B2M) or T-cell receptor alpha (TRAC) levels in activated human PBMCs. [Figure 3B]We demonstrate that increasing the concentration of RNP improves editing. The figure shows the editing efficiency of increasing concentrations of RNP composed of LPG10145 and various sgRNAs at a protein:RNA ratio of 1:2, as measured by flow cytometry of beta-2-microglobulin (B2M) or T-cell receptor alpha (TRAC) levels in activated human PBMCs. The dose-dependent editing activity increases with increasing amounts of RNP containing sgRNAs with two different spacer lengths (20 or 25 nucleotides). [Figure 4] We demonstrate that LPG10145 can edit targets within multiple genes. LPG10145 was delivered as RNP into T cells by nucleofection. All guides showed a coefficient of variation (CV) of less than 50%. DETAILED DESCRIPTION OF THE INVENTION

[0120] Many modifications and other embodiments of the inventions described herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. It is to be understood, therefore, that the invention is not limited to the particular embodiments disclosed, and that modifications and other embodiments are intended to be included within the scope of the accompanying embodiments. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0121] I. Overview RNA-guided nucleases (RGNs) allow targeted manipulation of specific site(s) within the genome and are useful in the context of gene targeting for therapeutic and research applications. In various organisms, including mammals, RNA-guided nucleases have been used for genome manipulation, for example, by stimulating non-homologous end joining and homologous recombination. The compositions and methods described herein are useful for creating single- or double-strand breaks in polynucleotides, modifying polynucleotides, detecting specific sites within polynucleotides, or modifying the expression of specific genes.

[0122] The RNA-guided nucleases disclosed herein can alter gene expression by modifying target sequences. In certain embodiments, the RNA-guided nuclease is guided to a target sequence by a guide RNA (gRNA) as part of a clustered regularly interspaced short palindromic repeats (CRISPR) RNA-guided nuclease system. The guide RNA forms a complex with the RNA-guided nuclease to guide the RNA-guided nuclease to bind to the target sequence, and in some embodiments, introduces a single- or double-stranded break in the target sequence, so that the RGN is considered "RNA-guided." After the target sequence is cleaved, the break can be repaired so that the DNA sequence of the target sequence is modified during the repair process. Thus, provided herein is a method for modifying a target sequence in the DNA of a host cell using an RNA-guided nuclease. For example, an RNA-guided nuclease can be used to modify a target sequence in a genomic locus of a eukaryotic or prokaryotic cell.

[0123] II. RNA-guided nucleases The present specification provides an RNA-guided nuclease. The term RNA-guided nuclease (RGN) refers to a polypeptide that binds to a specific target nucleotide sequence in a sequence-specific manner, complexes with a polypeptide, and is guided to the target nucleotide sequence by a guide RNA molecule that hybridizes with the target sequence. Although RNA-guided nucleases can cleave the target sequence upon binding, the term RNA-guided nuclease also encompasses nuclease-inactive RNA-guided nucleases that can bind to but not cleave the target sequence. Cleavage of the target sequence by an RNA-guided nuclease can result in single-strand or double-strand cleavage. An RNA-guided nuclease that can cleave only one strand of a double-stranded nucleic acid molecule is referred to herein as a nickase.

[0124] RNA-guided nucleases disclosed herein include LPG10134, LPG10136, LPG10138, LPG10139, LPG10141, LPG10145, LPG10146, LPG10147, LPG10150, LPG10155, LPG10159, and LPG10160 RNA-guided nucleases set forth as SEQ ID NOs: 1 to 12, respectively, as well as active fragments or variants thereof that retain the ability to bind to target nucleotide sequences in an RNA-guided sequence-specific manner. In some of these embodiments, the active fragment or variant of LPG10134, LPG10136, LPG10138, LPG10139, LPG10141, LPG10145, LPG10146, LPG10147, LPG10150, LPG10155, LPG10159, or LPG10160 RGN is capable of cleaving a single-stranded or double-stranded target sequence. In some embodiments, an active variant of LPG10134, LPG10136, LPG10138, LPG10139, LPG10141, LPG10145, LPG10146, LPG10147, LPG10150, LPG10155, LPG10159, or LPG10160 RGN comprises an amino acid 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 more sequence identity to the amino acid sequence set forth as any one of SEQ ID NOs: 1-12. In certain embodiments, an active fragment of LPG10134, LPG10136, LPG10138, LPG10139, LPG10141, LPG10145, LPG10146, LPG10147, LPG10150, LPG10155, LPG10159, or LPG10160 RGN comprises at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, or more consecutive amino acid residues of the amino acid sequence set forth as any one of SEQ ID NOs: 1-12.The RNA-guided nucleases provided herein may comprise at least one nuclease domain (e.g., DNase domain, RNase domain) and at least one RNA recognition domain and / or RNA binding domain to interact with the guide RNA. Additional domains that may be found in the RNA-guided nucleases provided herein include, but are not limited to, a DNA-binding domain, a helicase domain, a protein-protein interaction domain, and a dimerization domain. In certain embodiments, the RNA-guided nucleases provided herein may comprise at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with one or more of the DNA-binding domain, the helicase domain, the protein-protein interaction domain, and the dimerization domain.

[0125] The target nucleotide sequence is bound by the RNA-guided nuclease provided herein and hybridizes with a guide RNA that associates with the RNA-guided nuclease. Then, if the polypeptide has nuclease activity, the target sequence can be cleaved by the RNA-guided nuclease. The term "cleaving" or "cleavage" refers to the hydrolysis of at least one phosphodiester bond in the backbone of the target nucleotide sequence, which can result in either a single-stranded break or a double-stranded break within the target sequence. The RGNs of the present disclosure can cleave nucleotides within a polynucleotide, functioning as an endonuclease, or can be exonucleases, removing consecutive nucleotides from the ends (5' and / or 3' ends) of a polynucleotide. In other embodiments, the disclosed RGNs can cleave nucleotides of a target sequence within any position of a polynucleotide, thus functioning as both an endonuclease and an exonuclease. Cleavage of a target polynucleotide by the RGNs of the present disclosure can result in a staggered break or a blunt end.

[0126] The RNA-guided nuclease of the present disclosure can be a wild-type sequence derived from a bacterial or archaeal species. Alternatively, the RNA-guided nuclease can be a variant or fragment of a wild-type polypeptide. The wild-type RGN can be modified, for example, to change the nuclease activity or change the PAM specificity. In some embodiments, the RNA-guided nuclease does not exist in nature.

[0127] In certain embodiments, the RNA-guided nuclease functions as a nickase and cleaves only one strand of the target nucleotide sequence. Such RNA-guided nucleases have a single functional nuclease domain. In certain embodiments, the nickase can cleave either the positive or negative strand. In some of these embodiments, the additional nuclease domain is mutated to reduce or eliminate nuclease activity.

[0128] In other embodiments, the RNA-guided nuclease completely lacks nuclease activity and is referred to herein as nuclease-inactive or nuclease-inactive. Any method known in the art for introducing mutations into amino acid sequences, such as PCR-mediated mutagenesis and site-directed mutagenesis, can be used to generate a nickase or nuclease-inactive RGN. See, for example, U.S. Patent Publication No. 2014 / 0068797 and U.S. Patent No. 9,790,490 (each of which is incorporated by reference in its entirety). A non-limiting example of a nickase is the LPG10145 D16A nickase set forth herein as SEQ ID NO: 551. Nickases containing mutations in the RuvC domain and having a functional HNH domain are useful for base editing, where the nickase is fused to a base-editing polypeptide such as a deaminase. Another non-limiting example of a nickase is the LPG10145 H611A nickase set forth herein as SEQ ID NO: 552. Nickases containing mutations in the HNH domain and having a functional RuvC domain are useful for prime editing, where the nickase is fused to a prime editing polypeptide such as a reverse transcriptase. A non-limiting example of a nuclease-inactive RGN is the LPG10145 D16A H611A sequence set forth as SEQ ID NO: 553.

[0129] RNA-guided nucleases lacking nuclease activity can be used to deliver fusion polypeptides, polynucleotides, or small molecule payloads to specific genomic locations. In some of these embodiments, the RGN polypeptide or guide RNA can be fused to a detectable label to enable detection of specific sequences. As a non-limiting example, nuclease-inactive RGN can be fused to a detectable label (e.g., a fluorescent protein) to target specific sequences associated with disease, enabling detection of the disease-associated sequence.

[0130] Alternatively, nuclease-inactive RGN can be targeted to a specific genomic location to alter the expression of a desired sequence. In some embodiments, binding of the nuclease-inactive RNA-guided nuclease to the target sequence results in decreased expression of the target sequence or a gene under transcriptional control by the target sequence by preventing binding of RNA polymerase or a transcription factor within the target genomic region. In other embodiments, the RGN (e.g., nuclease-inactive RGN) or its complexed guide RNA further comprises an expression regulator that, upon binding to the target sequence, serves to repress or activate expression of the target sequence or a gene under transcriptional control by the target sequence. In some of these embodiments, the expression regulator regulates the expression of the target sequence or regulatory gene via an epigenetic mechanism.

[0131] In other embodiments, nuclease-inactive RGN or RGN with nickase activity can be targeted to specific genomic locations and modify the sequence of a target polynucleotide through fusion to a base-editing polypeptide, such as a deaminase polypeptide or an active variant or fragment thereof, that directly chemically modifies (e.g., deamidates) nucleobases (resulting in the conversion of one nucleobase to another). The base-editing polypeptide can be fused to the RGN at its N-terminus or C-terminus. Furthermore, the base-editing polypeptide can be fused to the RGN via a peptide linker. Non-limiting examples of deaminase polypeptides useful in such compositions and methods include cytosine deaminase or adenine deaminase (Gaudelli et al. (2017) Nature 106:111-112). 551:464-471, U.S. Patent Publication Nos. 2017 / 0121693 and 2018 / 0073012, and WO 2018 / 027078, or any of the deaminases disclosed in WO 2020 / 139873, U.S. Provisional Patent Applications Nos. 63 / 077,089, filed September 11, 2020, 63 / 146,840, filed February 8, 2021, and 63 / 164,273, filed March 22, 2021, and International Application No. PCT / US2021 / 049853, filed September 10, 2021 (each of which is incorporated by reference in its entirety). In one embodiment, a deaminase polypeptide useful in such compositions and methods is a cytosine deaminase or adenine deaminase comprising an amino acid sequence selected from any one of SEQ ID NOs: 377-448. In one embodiment, a deaminase polypeptide useful 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 more identity to any one of the amino acid sequences set forth as SEQ ID NOs: 377-448.In some embodiments, deaminase polypeptides useful in such compositions and methods of the present disclosure are deaminases disclosed in Table 17 of WO 2020 / 139873, which is incorporated herein by reference in its entirety. Additionally, it is known in the art that certain fusion proteins between RGN and a base editing enzyme (e.g., cytosine deaminase) can also include at least one uracil-stabilizing polypeptide that increases the mutation rate of cytidine, deoxythymidine, or cytosine to thymidine, deoxythymidine, or thymine in a nucleic acid molecule by the deaminase. Non-limiting examples of uracil-stabilizing polypeptides include those disclosed in U.S. Provisional Application No. 63 / 052,175, filed July 15, 2020, and International Application No. PCT / US2021 / 041809, filed July 15, 2021, each of which is incorporated herein by reference in its entirety, which contain USP2 (SEQ ID NO: 325) and a uracil glycosylase inhibitor (UGI) domain (SEQ ID NO: 315), which may increase base editing efficiency. Thus, a fusion protein may contain an RGN or a variant thereof described herein, a deaminase, and, optionally, at least one uracil-stabilizing polypeptide, such as UGI or USP2. In certain embodiments, the RGN fused to the base-editing polypeptide is a nickase that cleaves DNA strands not acted upon by the base-editing polypeptide (e.g., a deaminase).

[0132] In some embodiments, RGN can be fused to a prime editing polypeptide. Prime editing is a versatile and precise genome editing method that uses a nucleic acid-programmable DNA-binding protein that functions in association with a polymerase to directly write new genetic information into specific DNA sites. The prime editing system uses RGN, a nickase, and the system is programmed with a prime editing (PE) guide RNA ("PEgRNA"). PEgRNA is a guide RNA that identifies the target sequence and provides a template for polymerization of a replacement strand containing the edit by an extension engineered onto the guide RNA (e.g., at the 5' or 3' end, or an internal portion of the guide RNA). The RGN nickase / prime editing polypeptide fusion is guided to the target sequence by the PEgRNA and nicks the target strand upstream of the edited sequence and upstream of the PAM, creating a 3' flap on the target strand. The PEGRNA contains a primer binding site (PBS) complementary to the 3' flap of the target strand. In some embodiments, the PBS is 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 length. In certain embodiments, the pegRNA comprises a PBS at least 5 (e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 28, 19, or 20) nucleotides in length. In some embodiments, the pegRNA may comprise a PBS at least 8 nucleotides in length. Hybridization of the PBS with the 3' flap of the target strand allows for polymerization of a replacement strand containing the edit, using the extension of the pegRNA as a template. The extension of the pegRNA can be formed from RNA or DNA. In the case of RNA extension, the polymerase of the prime editor can be an RNA-dependent DNA polymerase (such as reverse transcriptase).In the case of DNA extension, the polymerase of the prime editor can be a DNA-dependent DNA polymerase.

[0133] The replacement strand containing the desired edit (e.g., a single nucleobase substitution) shares the same sequence as the target strand of the target sequence being edited (except for including the desired edit). Through DNA repair and / or replication mechanisms, the target strand of the target sequence is replaced by a newly synthesized replacement strand containing the desired edit. In some cases, prime editing can be considered a "search-and-replace" genome editing technique because the prime editor not only searches for and locates the desired target sequence to be edited, but also simultaneously encodes the replacement strand containing the desired edit that is installed in place of the corresponding target strand of the target sequence. Thus, in some embodiments, the guide RNA of the present disclosure includes an extension that contains an editing template for prime editing. In some embodiments, the prime editing polypeptide that can be fused to an RGN comprises a DNA polymerase. In certain embodiments, the DNA polymerase is a reverse transcriptase. In some embodiments, the RGN is a nickase.

[0134] An RNA-guided nuclease fused to a polypeptide or domain can be separated or joined by a linker. As used herein, the term "linker" refers to a chemical group or molecule that connects two molecules or moieties, such as the binding domain and cleavage domain of a nuclease. In some embodiments, a linker joins the gRNA-binding domain of an RNA-guided nuclease with a base-editing polypeptide such as a deaminase. In some embodiments, a linker joins a nuclease-inactive RGN with a deaminase. Typically, the linker is positioned between or adjacent to two groups, molecules, or other moieties and is connected to each via a covalent bond, thus connecting the two. In some embodiments, the linker is an amino acid or multiple amino acids (e.g., a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 5 to 100 amino acids in length, e.g., 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, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated.

[0135] The RNA-guided nuclease of the present disclosure can include at least one nuclear localization signal (NLS) to enhance the transport of RGN to the cell nucleus. Nuclear localization signals are known in the art and generally comprise a stretch of basic amino acids (see, e.g., Lange et al., J. Biol. Chem. (2007) 282:5101-5105). In some embodiments, the RGN comprises two, three, four, five, six, or more nuclear localization signals. The nuclear localization signal(s) can be heterologous NLSs. Non-limiting examples of nuclear localization signals useful for the RGN of the present disclosure are the nuclear localization signals of SV40 large T antigen, nucleoplasmin, and c-Myc (see, e.g., Ray et al. (2015) Bioconjug Chem 26(6):1004-7). In certain embodiments, RGN comprises the NLS sequence set forth as SEQ ID NO: 316 or 317. RGN can comprise one or more NLS sequences at its N-terminus, C-terminus, or both the N-terminus and C-terminus. For example, RGN can comprise two NLS sequences in the N-terminal region and four NLS sequences in the C-terminal region.

[0136] Other localization signal sequences known in the art that localize polypeptides to specific subcellular location(s) can also be used to target RGN, including, but not limited to, plastid localization sequences, mitochondrial localization sequences, and dual targeting signal sequences that target both plastids and mitochondria (e.g., Nassoury and Morse (2005) Biochim Biophys Acta 1743:5-19; Kunze and Berger (2015) Front Physiol dx.doi.org / 10.3389 / fphys.2015.00259; Herrmann and Neupert (2003) IUBMB Life 55:219-225; Soll (2002) Curr Opin Plant Biol 5:529-535; Carrie and Small (2013) Biochim Biophys Acta 1833:253-259; Carrie et al. (2009) FEBS J 276:1187-1195, Silva-Filho (2003) Curr Opin Plant Biol 6:589-595, Peeters and Small (2001) Biochim Biophys Acta 1541:54-63, Murcha et al. (2014) J Exp Bot 65:6301-6335, Mackenzie (2005) Trends Cell Biol 15:548-554, Glaser et al. (1998) Plant Mol Biol 38:311-338).

[0137] In certain embodiments, the RNA-guided nuclease of the present disclosure comprises at least one cell-penetrating domain that facilitates cellular uptake of RGN. Cell-penetrating domains are known in the art and generally comprise a stretch of positively charged amino acid residues (i.e., polycationic cell-penetrating domains), alternating polar and non-polar amino acid residues (i.e., amphipathic cell-penetrating domains), or hydrophobic amino acid residues (i.e., hydrophobic cell-penetrating domains) (see, e.g., Milletti F. (2012) Drug Discov Today 17:850-860). A non-limiting example of a cell-penetrating domain is the trans-activating transcription activator (TAT) from human immunodeficiency virus 1.

[0138] The nuclear localization signal, the plasmodium localization signal, the mitochondrial localization signal, the dual-targeting localization signal, and / or the cell-penetrating domain can be located at the amino-terminus (N-terminus), the carboxyl-terminus (C-terminus), or an internal position of the RNA-guided nuclease.

[0139] The RGN of the present disclosure can be fused directly or indirectly via a linker peptide to an effector domain, such as a cleavage domain, a deaminase domain, or an expression regulator domain. Such domains can be located at the N-terminus, C-terminus, or internal position of the RNA-guided nuclease. In some embodiments, the RGN component of the fusion protein is a nuclease-inactive RGN or a nickase.

[0140] In some embodiments, the RGN fusion protein comprises a cleavage domain, which is any domain capable of cleaving polynucleotides (i.e., RNA, DNA, or RNA / DNA hybrids), including, but not limited to, restriction endonucleases and homing endonucleases, such as Type IIS endonucleases (e.g., FokI) (see, e.g., Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388; Linn et al. (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993).

[0141] In other embodiments, the RGN fusion protein comprises a deaminase domain that deaminase a nucleobase, resulting in the conversion of one nucleobase to another, and includes a cytosine deaminase or an adenine deaminase (e.g., Gaudelli et al. (2017) Nature 551:464-471, U.S. Patent Publication Nos. 2017 / 0121693 and 2018 / 0073012, U.S. Patent No. 9,840,699, and International Publication No. WO 2018 / 027078, or WO 2020 / 139873, U.S. Provisional Patent Application Nos. 63 / 077,089 filed September 11, 2020, 63 / 146,840 filed February 8, 2021, and 63 / 164,273 filed March 22, 2021, and International Application No. PCT / US2021 / 049853 filed September 10, 2021 (each of which is incorporated by reference in its entirety).

[0142] In some embodiments, the effector domain of an RGN fusion protein can be an expression regulator domain, which is a domain that serves to upregulate or downregulate transcription. The expression regulator domain can be an epigenetic modification domain, a transcriptional repressor domain, or a transcriptional activation domain.

[0143] In some of these embodiments, the expression regulator of the RGN fusion protein comprises an epigenetic modification domain that covalently modifies DNA or histone proteins, altering histone and / or chromosomal structure without altering the DNA sequence, resulting in altered gene expression (i.e., up- or down-regulation). Non-limiting examples of epigenetic modifications include acetylation or methylation of lysine residues, arginine methylation, serine and threonine phosphorylation, lysine ubiquitination and sumoylation of histone proteins, and methylation and hydroxymethylation of cytosine residues in DNA. Non-limiting examples of epigenetic modification domains include histone acetyltransferase domains, histone deacetylase domains, histone methyltransferase domains, histone demethylase domains, DNA methyltransferase domains, and DNA demethylase domains.

[0144] In other embodiments, the expression modulator of the fusion protein comprises a transcriptional control element and / or a transcriptional regulatory protein, such as a transcriptional repressor domain, that interacts with RNA polymerase and transcription factors to reduce or terminate transcription of at least one gene. Transcriptional repressor domains are known in the art and include, but are not limited to, Sp1-like repressor, IκB, and Krueppel-associated box (KRAB) domains.

[0145] In yet other embodiments, the expression modulator of the fusion protein comprises a transcription activation domain that interacts with transcriptional control elements and / or transcription regulatory proteins, such as RNA polymerase and transcription factors, to increase or activate transcription of at least one gene. Transcription activation domains are known in the art and include, but are not limited to, herpes simplex virus VP16 activation domain and NFAT activation domain.

[0146] The RGN polypeptide of the present disclosure can include a detectable label or purification tag. The detectable label or purification tag can be located at the N-terminus, C-terminus, or internal position of the RNA-guided nuclease, either directly or indirectly via a linker peptide. In some of these embodiments, the RGN component of the fusion protein is a nuclease-inactive RGN. In other embodiments, the RGN component of the fusion protein is an RGN with nickase activity.

[0147] A detectable label is a molecule that can be visualized or otherwise observed. Detectable labels may be fused to RGN as a fusion protein (e.g., a fluorescent protein) or may be small molecules conjugated to RGN polypeptides that can be detected visually or by other means. Detectable labels that can be fused to RGN of the present disclosure as a fusion protein include any detectable protein domain, including, but not limited to, fluorescent proteins or protein domains that can be detected with a specific antibody. 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 labels include: 3 H and 35 Examples of radioactive labels include S.

[0148] RGN polypeptides can also include a purification tag, which is any molecule that can be used to isolate a protein or fusion protein from a mixture (e.g., a biological sample, culture medium). Non-limiting examples of purification tags include biotin, myc, maltose-binding protein (MBP), glutathione-S-transferase (GST), and 3X FLAG tags.

[0149] III. Guide RNA The present disclosure provides guide RNAs and polynucleotides encoding them. The term "guide RNA" refers to a nucleotide sequence that has sufficient complementarity with a target nucleotide sequence to hybridize with the target sequence and direct the sequence-specific binding of an associated RNA-guided nuclease to the target nucleotide sequence. Thus, each guide RNA of an RGN is one or more RNA molecules (generally one or two) that can bind to the RGN and guide the RGN to bind to a specific target nucleotide sequence, and in those embodiments in which the RGN has nickase or nuclease activity, also cleaves the target nucleotide sequence. Generally, guide RNAs include CRISPR RNAs (crRNAs) and trans-activating CRISPR RNAs (tracrRNAs). Natural guide RNAs, including both crRNAs and tracrRNAs, generally comprise two separate RNA molecules that hybridize to each other via the repeat sequence of the crRNA and the anti-repeat sequence of the tracrRNA.

[0150] Naturally occurring direct repeat sequences within a CRISPR array generally range in length from 28 to 37 base pairs, but their length can vary from about 23 bp to about 55 bp. Spacer sequences within a CRISPR array generally range in length from about 32 to about 38 bp, but their length can be from about 21 bp to about 72 bp. Each CRISPR array generally contains fewer than 50 units of CRISPR repeat spacer sequences. CRISPRs are transcribed as part of a long transcript called the primary CRISPR transcript, which comprises the majority of the CRISPR array. The primary CRISPR transcript is cleaved by Cas proteins to produce crRNA, or in some cases, produces a pre-crRNA that is further processed into mature crRNA by additional Cas proteins. The mature crRNA contains a spacer sequence and a CRISPR repeat sequence. In some embodiments, where the pre-crRNA is processed into mature (or processed) crRNA, maturation involves the removal of about one to about six or more 5', 3', or 5' and 3' nucleotides. For the purposes of genome editing or targeting of a specific target nucleotide sequence of interest, these nucleotides that are removed during maturation of the pre-crRNA molecule are not required to generate or design the guide RNA.

[0151] CRISPR RNA (crRNA) comprises a spacer sequence and a CRISPR repeat sequence. A "spacer sequence" is a nucleotide sequence that directly hybridizes to a target nucleotide sequence of interest. The spacer sequence is engineered to be fully or partially complementary to the target sequence of interest. In various embodiments, the spacer sequence can comprise from about 8 nucleotides to about 30 nucleotides, or more. For example, the spacer 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 length. In some embodiments, the spacer 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 length. In some embodiments, the spacer sequence is about 10 to about 26 nucleotides in length, or about 12 to about 30 nucleotides in length. In some embodiments, the spacer sequence is 19 to 27 nucleotides in length. In certain embodiments, particularly when the RGN has the amino acid sequence set forth as SEQ ID NO: 6, or an active variant or fragment thereof, the spacer sequence is 21 to 24 nucleotides in length. In certain embodiments, the spacer sequence is about 30 nucleotides in length. In some embodiments, the spacer sequence is 30 nucleotides in length.In some embodiments, the degree of complementarity between a spacer sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is 50% to 99% or more, including, but not limited to, about 50% or more, about 60% or more, about 70% or more, about 75% or more, about 80% or more, about 81% or more, about 82% or more, about 83% or more, about 84% or more, about 85% or more, about 86% or more, about 87% or more, about 88% or more, about 89% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, or more. In certain embodiments, the degree of complementarity between a spacer sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, 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 certain embodiments, the spacer sequence does not contain secondary structure, which can be predicted using any suitable polynucleotide folding algorithm known in the art, including, but not limited to, mFold (see, e.g., Zuker and Stiegler (1981) Nucleic Acids Res. 9:133-148) and RNAfold (see, e.g., Gruber et al. (2008) Cell 106(1):23-24).

[0152] CRISPR RNA repeats comprise a nucleotide sequence that, by itself or in concert with a hybridized tracrRNA, forms a structure recognized by an RGN molecule. In various embodiments, CRISPR RNA repeats can comprise from about 8 nucleotides to about 30 nucleotides, or more. For example, CRISPR repeats can be about 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 length. In certain embodiments, the CRISPR repeat sequences are 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 length. In some embodiments, the degree of complementarity between a CRISPR repeat sequence and its corresponding tracrRNA sequence, when optimally aligned using a suitable alignment algorithm, is about 50% or more, about 60% or more, about 70% or more, about 75% or more, about 80% or more, about 81% or more, about 82% or more, about 83% or more, about 84% or more, about 85% or more, about 86% or more, about 87% or more, about 88% or more, about 89% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, or more.In certain embodiments, the degree of complementarity between a CRISPR repeat sequence and its corresponding tracrRNA sequence, when optimally aligned using a suitable alignment algorithm, 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.

[0153] In certain embodiments, the CRISPR repeat comprises the nucleotide sequence of any one of SEQ ID NOs: 13-24, or an active variant or fragment thereof that, when included within a guide RNA, is capable of directing sequence-specific binding of an associated RNA-guided nuclease provided herein to a target sequence of interest. In certain embodiments, an active CRISPR repeat variant of a 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 more sequence identity to any one of the nucleotide sequences set forth as SEQ ID NOs: 13-24. In certain embodiments, an active CRISPR repeat fragment of a wild-type sequence comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 consecutive nucleotides of any one of the nucleotide sequences set forth as SEQ ID NOs: 13-24.

[0154] In certain embodiments, crRNA does not exist in nature.In some of these embodiments, specific CRISPR repeat sequence is not connected to the spacer sequence that is naturally engineered, and the CRISPR repeat sequence is considered to be heterologous to the spacer sequence.In certain embodiments, the spacer sequence is an engineered sequence that does not exist in nature.

[0155] A transactivating CRISPR RNA or tracrRNA molecule comprises a nucleotide sequence containing a region sufficiently complementary to hybridize to the CRISPR repeat sequence of the crRNA, referred to herein as the anti-repeat region. In some embodiments, the tracrRNA molecule further comprises a region with a secondary structure (e.g., a stem-loop) or forms a secondary structure upon hybridization with its corresponding crRNA. In certain embodiments, the region of the tracrRNA that is fully or partially complementary to the CRISPR repeat sequence is at the 5' end of the molecule, and the 3' end of the tracrRNA contains the secondary structure. This region of secondary structure generally contains several hairpin structures, including a nexus hairpin found adjacent to the anti-repeat sequence. The nexus forms the core of interaction between the guide RNA and RGN and is at the intersection between the guide RNA, RGN, and target DNA. Nexus hairpins often have a conserved nucleotide sequence at the base of the hairpin stem, and the motif UNANNC (SEQ ID NO: 318) is found in many nexus hairpins in tracrRNA. Interestingly, some of the RGNs of the present invention use tracrRNAs that contain non-canonical sequences at the bases of the hairpin stem of their nexus hairpin, including UNANNA, UNANNG, and CNANNC (SEQ ID NOS: 319, 321, and 322, respectively). Often, there is a terminal hairpin at the 3' end of the tracrRNA that can vary in structure and number, but often contains a GC-rich Rho-independent transcription terminator hairpin followed by a string of U's at the 3' end. See, e.g., Briner et al. (2014) Molecular Cell 56:333-339; Briner and Barrangou (2016) Cold Spring Harb Protoc; doi:10.1101 / pdb.top090902; and U.S. Publication No. 2017 / 0275648, each of which is incorporated herein by reference in its entirety.

[0156] In various embodiments, the anti-repeat region of the tracrRNA that is fully or partially complementary to the CRISPR repeat sequence comprises from about 8 nucleotides to about 30 nucleotides, or more. For example, the base-paired region between the tracrRNA anti-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 length. In certain embodiments, the base-paired region between the tracrRNA anti-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 length. In some embodiments, the degree of complementarity between a CRISPR repeat sequence and its corresponding tracrRNA anti-repeat sequence, when optimally aligned using a suitable alignment algorithm, is about 50% or more, about 60% or more, about 70% or more, about 75% or more, about 80% or more, about 81% or more, about 82% or more, about 83% or more, about 84% or more, about 85% or more, about 86% or more, about 87% or more, about 88% or more, about 89% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, or more.In certain embodiments, the degree of complementarity between a CRISPR repeat sequence and its corresponding tracrRNA anti-repeat sequence, when optimally aligned using a suitable alignment algorithm, 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.

[0157] In various embodiments, the entire tracrRNA can comprise from about 60 nucleotides to more than about 210 nucleotides. For example, the tracrRNA can be about 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 length. In certain embodiments, the tracrRNA is 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 length. In certain embodiments, the tracrRNA is about 70 to about 105 nucleotides in length, including about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, 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, about 100, about 101, about 102, about 103, about 104, and about 105 nucleotides in length. In certain embodiments, the tracrRNA is 70 to 105 nucleotides in length, including 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, and 105 nucleotides.

[0158] In certain embodiments, the tracrRNA comprises the nucleotide sequence of any one of SEQ ID NOs: 25-36, or an active variant or fragment thereof that, when included within a guide RNA, is capable of directing sequence-specific binding of a related RNA-guided nuclease provided herein to a target sequence of interest. In certain embodiments, an active tracrRNA repeat variant of a 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 more sequence identity to any one of the nucleotide sequences set forth as SEQ ID NOs: 25-36. In certain embodiments, an active tracrRNA sequence fragment of a wild-type sequence comprises at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more contiguous nucleotides of any one of the nucleotide sequences set forth as SEQ ID NOS:25-36.

[0159] Two polynucleotide sequences can be considered substantially complementary when the two sequences hybridize to each other under stringent conditions. Similarly, when a guide RNA bound to an RGN binds to a target sequence under stringent conditions, the RGN is considered to bind to a particular target sequence in a sequence-specific manner. "Stringent conditions" or "stringent hybridization conditions" refer to conditions under which two polynucleotide sequences hybridize to each other detectably more strongly than other sequences (e.g., at least twice as strongly as background). Stringent conditions are sequence-dependent and will vary in different circumstances. Typically, stringent conditions would be conditions in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion (or other salt), at pH 7.0 to 8.3, and the temperature is 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., more than 50 nucleotides). Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization in a buffer of 30-35% formamide, 1 M NaCl, and 1% SDS (sodium dodecyl sulfate) at 37°C, followed by a wash in 1x-2x SSC (20x SSC = 3.0 M NaCl / 0.3 M trisodium citrate) at 50-55°C. Exemplary medium stringency conditions include hybridization in 40-45% formamide, 1.0 M NaCl, and 1% SDS at 37°C, followed by a wash in 0.5x-1x SSC at 55-60°C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, and 1% SDS at 37°C, followed by a wash in 0.1x SSC at 60-65°C. Optionally, the wash buffer may contain about 0.1% to about 1% SDS. The duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours. The duration of the wash period is at least long enough to reach equilibrium.

[0160] Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched sequence. For DNA-DNA hybrids, Tm can be approximated by the equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284: Tm = 81.5°C + 16.6 (log M) + 0.41 (% GC) - 0.61 (% form) - 500 / L, where M is the molar concentration of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the hybrid length in base pairs. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) of a specific sequence and its complementary strand at a defined ionic strength and pH. However, strictly stringent conditions can utilize hybridization and / or wash temperatures that are 1, 2, 3, or 4° C. below the thermal melting point (Tm). Moderately stringent conditions can utilize hybridization and / or wash temperatures that are 6, 7, 8, 9, or 10° C. below the thermal melting point (Tm). Low stringency conditions can utilize hybridization and / or wash temperatures that are 11, 12, 13, 14, 15, or 20° C. below the thermal melting point (Tm). Using the equations, hybridization and wash compositions, and desired Tm, one of skill in the art will understand that variations in the stringency of hybridization and / or wash solutions are essentially described.Extensive guides to 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. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience, New York). See Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York).

[0161] The term "sequence-specific" can also refer to binding of a target sequence at a frequency greater than binding to randomized background sequences.

[0162] The guide RNA can be a single-guide RNA (sgRNA) or a dual-guide RNA system. A single-guide RNA comprises a crRNA and a tracrRNA on a single molecule of RNA, while a dual-guide RNA system comprises a crRNA and a tracrRNA present on two different RNA molecules hybridized to each other via at least a portion of the CRISPR repeat sequence in the crRNA and at least a portion of the tracrRNA, which may be fully or partially complementary to the CRISPR repeat sequence in the crRNA. In some embodiments where the guide RNA is a single-guide RNA, the crRNA and the tracrRNA are separated by a linker nucleotide sequence. Generally, the linker nucleotide sequence does not contain complementary bases to avoid the formation of secondary structures within or involving the nucleotides of the linker nucleotide sequence. In some embodiments, the linker nucleotide sequence between the crRNA and the 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 length. In certain embodiments, the linker nucleotide sequence of the single guide RNA is at least 4 nucleotides in length. In certain embodiments, the linker nucleotide sequence is the nucleotide sequence set forth as SEQ ID NO: 323.

[0163] Single-guide or dual-guide RNAs can be synthesized chemically or via in vitro transcription. Assays for determining sequence-specific binding between RGN and guide RNA are known in the art, including, but not limited to, in vitro binding assays between expressed RGN and guide RNA, which may be tagged with a detectable label (e.g., biotin) and used in pull-down detection assays in which the guide RNA:RGN complex is captured via the detectable label (e.g., streptavidin beads). A control guide RNA with a sequence or structure unrelated to the guide RNA can be used as a negative control for non-specific binding of RGN to RNA. In certain embodiments, the guide RNA is any one of SEQ ID NOs: 37-48, and the spacer sequence can be any sequence, indicated as a poly-N sequence.

[0164] In certain embodiments, the guide RNA may be introduced into a target cell, organelle, or embryo as an RNA molecule. The guide RNA may be transcribed in vitro or chemically synthesized. In other embodiments, a nucleotide sequence encoding the guide RNA is introduced into a cell, organelle, or embryo. In some of these embodiments, the nucleotide sequence encoding the guide RNA is operably linked to a promoter (e.g., an RNA polymerase III promoter). The promoter may be heterologous to the native promoter or the guide RNA-encoding nucleotide sequence.

[0165] In various embodiments, the guide RNA can be introduced into a target cell, organelle, or embryo as a ribonucleoprotein complex, as described herein, wherein the guide RNA binds to an RNA-guided nuclease polypeptide.

[0166] The guide RNA guides the associated RNA-guided nuclease to a specific target nucleotide sequence of interest through hybridization of the guide RNA to the target nucleotide sequence. The target nucleotide sequence may comprise DNA, RNA, or a combination of both, and may be single-stranded or double-stranded. The target nucleotide sequence may be genomic DNA (i.e., chromosomal DNA), plasmid DNA, or an RNA molecule (e.g., messenger RNA, ribosomal RNA, transfer RNA, microRNA, small interfering RNA). The target nucleotide sequence can be bound (and, in some embodiments, cleaved) by the RNA-guided nuclease in vitro or in a cell. The chromosomal sequence targeted by the RGN may be a nuclear, plastid, or mitochondrial chromosomal sequence. In some embodiments, the target nucleotide sequence is unique in the target genome.

[0167] The target nucleotide sequence is adjacent to a protospacer adjacent motif (PAM). The protospacer adjacent motif is generally within about 1 to about 10 nucleotides of the target nucleotide sequence, including about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 nucleotides from the target nucleotide sequence. In certain embodiments, the PAM is within 1 to 10 nucleotides of the target nucleotide sequence, including 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from the target nucleotide sequence. Unless otherwise specified herein, a given PAM sequence for an RGN of the present disclosure is immediately adjacent to the target nucleotide sequence. The PAM can be 5' or 3' of the target sequence. In some embodiments, the PAM is 3' of the target sequence for an RGN of the present disclosure. Generally, the PAM is a consensus sequence of about 3-4 nucleotides, but in certain embodiments can be 2, 3, 4, 5, 6, 7, 8, 9, or more nucleotides in length. In various embodiments, the PAM sequence recognized by the RGNs of the present disclosure comprises the consensus sequence set forth in any one of SEQ ID NOs: 73-84.

[0168] In certain embodiments, an RNA-guided nuclease having any one of SEQ ID NOs: 1-12, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to a PAM sequence set forth as any one of SEQ ID NOs: 73-84. In some embodiments, the RGN binds to a guide sequence comprising a CRISPR repeat sequence set forth as any one of SEQ ID NOs: 13-24, respectively, or an active variant or fragment thereof, and a tracrRNA sequence set forth as any one of SEQ ID NOs: 25-36, respectively, or an active variant or fragment thereof. RGN systems are further described herein in Examples 1-3 and Tables 1 and 2.

[0169] In some embodiments, the RNA-guided nuclease having SEQ ID NO: 1, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to the PAM sequence shown as SEQ ID NO: 73 when bound to a guide RNA comprising a CRISPR repeat sequence shown as SEQ ID NO: 13, or an active variant or fragment thereof, and a tracrRNA sequence shown as SEQ ID NO: 25, or an active variant or fragment thereof.

[0170] In some embodiments, the RNA-guided nuclease having SEQ ID NO:2, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to the PAM sequence set forth as SEQ ID NO:74 when bound to a guide RNA comprising a CRISPR repeat sequence set forth as SEQ ID NO:14, or an active variant or fragment thereof, and a tracrRNA sequence set forth as SEQ ID NO:26, or an active variant or fragment thereof.

[0171] In some embodiments, the RNA-guided nuclease having SEQ ID NO: 3, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to the PAM sequence set forth as SEQ ID NO: 75 when bound to a guide RNA comprising a CRISPR repeat sequence set forth as SEQ ID NO: 15, or an active variant or fragment thereof, and a tracrRNA sequence set forth as SEQ ID NO: 27, or an active variant or fragment thereof.

[0172] In some embodiments, the RNA-guided nuclease having SEQ ID NO:4, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to the PAM sequence set forth as SEQ ID NO:76 when bound to a guide RNA comprising a CRISPR repeat sequence set forth as SEQ ID NO:16, or an active variant or fragment thereof, and a tracrRNA sequence set forth as SEQ ID NO:28, or an active variant or fragment thereof.

[0173] In some embodiments, the RNA-guided nuclease having SEQ ID NO:5, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to the PAM sequence set forth as SEQ ID NO:77 when bound to a guide RNA comprising a CRISPR repeat sequence set forth as SEQ ID NO:17, or an active variant or fragment thereof, and a tracrRNA sequence set forth as SEQ ID NO:29, or an active variant or fragment thereof.

[0174] In some embodiments, the RNA-guided nuclease having SEQ ID NO:6, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to the PAM sequence set forth as SEQ ID NO:78 when bound to a guide RNA comprising a CRISPR repeat sequence set forth as SEQ ID NO:18, or an active variant or fragment thereof, and a tracrRNA sequence set forth as SEQ ID NO:30, or an active variant or fragment thereof.

[0175] In some embodiments, the RNA-guided nuclease having SEQ ID NO: 7, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to the PAM sequence set forth as SEQ ID NO: 79 when bound to a guide RNA comprising a CRISPR repeat sequence set forth as SEQ ID NO: 19, or an active variant or fragment thereof, and a tracrRNA sequence set forth as SEQ ID NO: 31, or an active variant or fragment thereof.

[0176] In some embodiments, the RNA-guided nuclease having SEQ ID NO:8, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to the PAM sequence set forth as SEQ ID NO:80 when bound to a guide RNA comprising a CRISPR repeat sequence set forth as SEQ ID NO:20, or an active variant or fragment thereof, and a tracrRNA sequence set forth as SEQ ID NO:32, or an active variant or fragment thereof.

[0177] In some embodiments, the RNA-guided nuclease having SEQ ID NO:9, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to the PAM sequence shown as SEQ ID NO:81 when bound to a guide RNA comprising a CRISPR repeat sequence shown as SEQ ID NO:21, or an active variant or fragment thereof, and a tracrRNA sequence shown as SEQ ID NO:33, or an active variant or fragment thereof.

[0178] In some embodiments, the RNA-guided nuclease having SEQ ID NO: 10, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to the PAM sequence set forth as SEQ ID NO: 82 when bound to a guide RNA comprising a CRISPR repeat sequence set forth as SEQ ID NO: 22, or an active variant or fragment thereof, and a tracrRNA sequence set forth as SEQ ID NO: 34, or an active variant or fragment thereof.

[0179] In some embodiments, the RNA-guided nuclease having SEQ ID NO: 11, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to the PAM sequence set forth as SEQ ID NO: 83 when bound to a guide RNA comprising a CRISPR repeat sequence set forth as SEQ ID NO: 23, or an active variant or fragment thereof, and a tracrRNA sequence set forth as SEQ ID NO: 35, or an active variant or fragment thereof.

[0180] In some embodiments, the RNA-guided nuclease having SEQ ID NO: 12, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to the PAM sequence set forth as SEQ ID NO: 84 when bound to a guide RNA comprising a CRISPR repeat sequence set forth as SEQ ID NO: 24, or an active variant or fragment thereof, and a tracrRNA sequence set forth as SEQ ID NO: 36, or an active variant or fragment thereof.

[0181] It is well known in the art that PAM sequence specificity for a given nuclease enzyme is influenced by the promoter used to express the RGN or the enzyme concentration, which can be modified by changing the amount of ribonucleoprotein complex delivered to a cell, organelle, or embryo (see, e.g., Karvelis et al. (2015) Genome Biol 16:253).

[0182] Upon recognizing the corresponding PAM sequence, RGN can cleave the target nucleotide sequence at a specific cleavage site. As used herein, a cleavage site consists of two specific nucleotides within the target nucleotide sequence at which the nucleotide sequence is cleaved by RGN. The cleavage site can include the first and second, second and third, third and fourth, fourth and fifth, fifth and sixth, seventh and eighth, or eighth and ninth nucleotides from the PAM in either the 5' or 3' direction. In some embodiments, the cleavage site can be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or more than 20 nucleotides from the PAM in either the 5' or 3' direction. In some embodiments, particularly in embodiments in which RGN has the amino acid sequence set forth as SEQ ID NO: 6 or an active variant or fragment thereof, the cleavage site of RGN is between the third and fourth nucleotides 5' of the PAM, generating a blunt end. Because RGN cleaves a target nucleotide sequence to produce cross-terminus, in some embodiments, the cleavage site is defined based on a distance of two nucleotides from the PAM on the positive (+) strand of the polynucleotide and a distance of two nucleotides from the PAM on the negative (-) strand of the polynucleotide.

[0183] IV. Nucleotides encoding RNA-guided nucleases, CRISPR RNA, and / or tracrRNA The present disclosure provides polynucleotides comprising the CRISPR RNA, tracrRNA, and / or sgRNA of the present disclosure, as well as polynucleotides comprising nucleotide sequences encoding the RNA-guided nuclease, CRISPR RNA, tracrRNA, and / or sgRNA of the present disclosure. The polynucleotides of the present disclosure include polynucleotides comprising or encoding a CRISPR repeat sequence comprising any one of the nucleotide sequences set forth as SEQ ID NOs: 13-24, or an active variant or fragment thereof that, when included within a guide RNA, can direct sequence-specific binding of an associated RNA-guided nuclease to a target sequence of interest. Also disclosed are polynucleotides comprising or encoding a tracrRNA comprising any one of the nucleotide sequences set forth as SEQ ID NOs: 25-36, or an active variant or fragment thereof that, when included within a guide RNA, can direct sequence-specific binding of an associated RNA-guided nuclease to a target sequence of interest. Also provided are polynucleotides encoding RNA-guided nucleases comprising any one of the amino acid sequences set forth as SEQ ID NOs: 1 to 12, and active fragments or variants thereof that retain the ability to bind to target nucleotide sequences in an RNA-guided sequence-specific manner.

[0184] The use of the terms "polynucleotide" or "nucleic acid molecule" is not intended to limit the present disclosure to polynucleotides comprising DNA. Those skilled in the art will recognize that polynucleotides can include 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 (PNAs), PNA-DNA chimeras, locked nucleic acids (LNAs), and phosphothiolate-linked sequences. Polynucleotides disclosed herein also encompass all forms of sequence, including, but not limited to, single-stranded forms, double-stranded forms, DNA-RNA hybrids, triplex structures, stem and loop structures, etc.

[0185] In some embodiments, the polynucleotide encoding RGN of the present disclosure is an mRNA (messenger RNA) molecule. mRNA refers to any polynucleotide that encodes a polypeptide of interest and can be translated to produce the encoded polypeptide of interest in vitro, in vivo, in situ, or ex vivo. In embodiments, the basic components of an mRNA molecule include at least a coding region, a 5' UTR, a 3' UTR, a 5' cap, and a polyA tail. In embodiments, an RGN-encoding mRNA useful in the methods and compositions of the present disclosure can contain one or more structural and / or chemical modifications or changes that confer useful properties to the polynucleotide. For example, useful properties of an mRNA include a lack of substantial induction of an innate immune response in cells into which the mRNA is introduced. A "structural" feature or modification is one in which two or more linked nucleotides are inserted, deleted, duplicated, inverted, or randomized in an mRNA without significant chemical modification of the nucleotides themselves. Because chemical bonds are necessarily broken and rearranged to result in structural modifications, structural modifications are chemical in nature and are therefore chemical modifications. However, structural modifications will result in different sequences of nucleotides. Chemical modifications of mRNA can include 5-methylcytosine, N1-methyl-pseudouridine, pseudouridine, 2-thiouridine, 4-thiouridine, 5-methoxyuridine, 2'fluoroguanosine, 2'fluorouridine, 5-bromolysine, 5-(2-carbomethoxyvinyl)uridine, 5-[3(1-E-propenylamino)]uridine, α-thiocytidine, N6-methyladenosine, 5-methylcytidine, N4-acetylcytidine, 5-formylcytidine, or combinations thereof in the mRNA.

[0186] Nucleic acid molecules encoding RGN can be codon-optimized for expression in a desired organism. A "codon-optimized" coding sequence is a polynucleotide coding sequence with a frequency of codon usage designed to mimic the frequency of preferred codon usage or transcription conditions of a particular host cell. Expression in a particular host cell or organism is enhanced as a result of changing one or more codons at the nucleic acid level, such that the translated amino acid sequence is unchanged. Nucleic acid molecules can be codon-optimized in whole or in part. Codon tables and other references providing preference information for a wide range of organisms are available in the art (e.g., see Campbell and Gowri (1990) Plant Physiol. 92:1-11 for a review of plant-preferred codon usage). Methods for synthesizing plant-preferred genes or mammalian (e.g., human) codon-optimized coding sequences are available in the art. See, for example, U.S. Patent Nos. 5,380,831 and 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, which are incorporated herein by reference.

[0187] Polynucleotides encoding RGN, crRNA, tracrRNA, and / or sgRNA provided herein can be provided in expression cassettes for in vitro expression or expression in a cell, organelle, embryo, or organism of interest. The cassettes include 5' and 3' regulatory sequences operably linked to the polynucleotides encoding RGN, crRNA, tracrRNA, and / or sgRNA provided herein, allowing for expression of the polynucleotides. The cassettes may further include at least one additional gene or genetic element cotransformed into the organism. When additional genes or elements are included, the components are operably linked. The term "operably linked" is intended to mean a functional linkage between two or more elements. For example, an operable linkage between a promoter and a coding region of interest (e.g., a region encoding RGN, crRNA, tracrRNA, and / or sgRNA) is a functional linkage that allows for expression of the coding region of interest. Operably linked elements may or may not be contiguous. When used to refer to the joining of two protein-coding regions, operably linked means that the coding regions are in the same reading frame. Alternatively, additional gene(s) or element(s) may be provided on multiple expression cassettes. For example, the nucleotide sequence encoding the RGN of the present disclosure may be present on one expression cassette, while the nucleotide sequences encoding the crRNA, tracrRNA, or complete guide RNA may be present on separate expression cassettes. Such expression cassettes are provided with multiple restriction and / or recombination sites for inserting polynucleotides under the transcriptional control of the regulatory regions. The expression cassette may further comprise a selectable marker gene.

[0188] An expression cassette comprises, in the 5'-3' direction of transcription, a transcriptional (and, in some embodiments, translational) initiation region (i.e., promoter), an RGN-, crRNA-, tracrRNA-, and / or sgRNA-encoding polynucleotide of the present invention, and a transcriptional (and, in some embodiments, translational) termination region (i.e., termination region) functional in the organism of interest. The promoter of the present invention can direct or drive expression of a coding sequence in a host cell. Regulatory regions (e.g., promoter, transcriptional regulatory region, and translational termination region) can be endogenous or heterologous to the host cell or to each other. As used herein, "heterologous" with respect to a sequence refers to a sequence that originates from a foreign species or, if from the same species, has been substantially altered in composition and / or genomic locus from its native form by deliberate human intervention. As used herein, a chimeric gene comprises a coding sequence operably linked to a transcriptional initiation region that is heterologous to the coding sequence.

[0189] Convenient termination regions are available from the Ti plasmid of A. tumefaciens, for example the octopine synthase and nopaline synthase termination regions. Also, Guerineau et al. (1991) Mol.Gen.Genet.262:141-144, Proudfoot (1991) Cell 64:671-674, Sanfacon et al. (1991) Genes Dev.5:141-149, Mogen et al. (1990) Plant Cell 2:1261-1272, Munroe et al. (1990) Gene 91:151-158, Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903, and Joshi et al. (1987) Nucleic Acids Res. 15:9627-9639.

[0190] Additional regulatory signals include, but are not limited to, transcription initiation start sites, operators, activators, enhancers, other regulatory elements, ribosome binding sites, start codons, termination signals, etc. See, e.g., U.S. Patent Nos. 5,039,523 and 4,853,331, EPO 0480762A2, Sambrook et al. (1992) Molecular Cloning: A Laboratory Manual, ed. Maniatis et al. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), hereinafter "Sambrook 11," Davis et al., eds. (1980) Advanced Bacterial Genetics (Cold Spring Harbor Laboratory Press), Cold Spring Harbor, NY, and references cited therein.

[0191] In preparing expression cassettes, various DNA fragments may be manipulated to provide DNA sequences in the proper orientation and, if necessary, in the proper reading frame. To this end, adapters or linkers may be employed to join DNA fragments, or other manipulations may be involved to provide convenient restriction sites, remove unnecessary DNA, remove restriction sites, etc. To this end, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions (e.g., transitions and transversions) may be involved.

[0192] Many promoters can be used in the practice of the present invention. The promoter can be selected based on the desired results. The nucleic acid can be combined with a constitutive, inducible, developmental stage-specific, cell type-specific, tissue-preferred, tissue-specific, or other promoter for expression in the organism of interest. See, for example, WO 99 / 43838, as well as 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, 5,608,149, and 5,772,369. See the promoters described in Nos. 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 (incorporated herein by reference).

[0193] For expression in plants, constitutive promoters include the CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812), rice actin (McElroy et al. (1990) Plant Cell 2:163-171), ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689), pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588), and MAS (Velten et al. (1984) EMBOJ. 3:2723-2730).

[0194] Examples of inducible promoters are the Adh1 promoter, which is inducible by hypoxia or cold stress, the Hsp70 promoter, which is inducible by heat stress, and the PPDK promoter and peptocarboxylase promoter, which are inducible by light. Also useful are chemically inducible promoters, such as the safener-induced In2-2 promoter (U.S. Pat. No. 5,364,780), the oxine-induced, tapetum-specific Axig1 promoter (PCT US01 / 22169), which is also active in callus, steroid-responsive promoters (see, e.g., the estrogen-induced ERE promoter and the glucocorticoid-inducible promoters of Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 and McNellis et al. (1998) Plant J. 14(2):247-257), and tetracycline-inducible and tetracycline-repressible promoters (see, e.g., Gatz et al. al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Patent Nos. 5,814,618 and 5,789,156 (incorporated herein by reference).

[0195] Tissue-specific or tissue-preferred promoters can be used to target expression of an expression construct in a specific tissue. In certain embodiments, tissue-specific or tissue-preferred promoters 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" promoter is a promoter that initiates transcription only in a specific tissue. Unlike constitutive expression of a gene, tissue-specific expression is the result of several interacting levels of gene regulation. Therefore, promoters from homologous or closely related plant species may be preferred to achieve efficient and reliable expression of a transgene in a specific tissue. In some embodiments, expression involves a tissue-preferred promoter. A "tissue-preferred" promoter is a promoter that preferentially initiates transcription, but not necessarily entirely or exclusively, in a specific tissue.

[0196] In some embodiments, nucleic acid molecules encoding RGN, crRNA, and / or tracrRNA comprise a cell-type-specific promoter. A "cell-type-specific" promoter is a promoter that drives expression primarily in a particular cell type in one or more organs. Some examples of plant cells in which a cell-type-specific promoter functional in plants may be primarily active include, for example, BETL cells, root vascular cells, leaves, stem cells, and stem cells. A nucleic acid molecule may also comprise a cell-type-preferred promoter. A "cell-type-preferred" promoter is a promoter that primarily, but not necessarily entirely or exclusively, drives expression in a particular cell type in one or more organs. Some examples of plant cells in which a cell-type-preferred promoter functional in plants may be primarily active include, for example, BETL cells, root vascular cells, leaves, stem cells, and stem cells.

[0197] The nucleic acid sequences encoding RGN, crRNA, tracrRNA, and / or sgRNA can be operably linked to a promoter sequence recognized by a phage 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 variation of a 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.

[0198] In certain embodiments, the polynucleotides encoding RGN, crRNA, tracrRNA, and / or sgRNA can also 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. Furthermore, the sequence encoding RGN can also be linked to sequence(s) 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.

[0199] The polynucleotides encoding RGN, crRNA, tracrRNA, and / or sgRNA can be present in a vector or multiple vectors. A "vector" refers to a polynucleotide composition for transferring, delivering, or introducing a nucleic acid into a host cell. Suitable vectors include plasmid vectors, phagemids, cosmids, artificial / minichromosomes, transposons, and viral vectors (e.g., lentiviral vectors, adeno-associated viral vectors, baculoviral vectors). Vectors can contain additional expression control sequences (e.g., enhancer sequences, Kozak sequences, polyadenylation sequences, transcription termination sequences), selectable marker sequences (e.g., antibiotic resistance genes), origins of replication, etc. 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 & Russell, Cold Spring Harbor Press, Cold Spring Harbor, NY, 3rd edition, 2001.

[0200] The vector may contain a selectable marker gene for selecting transformed cells. The selectable marker gene is 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), as well as genes that confer resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinone, and 2,4-dichlorophenoxyacetate (2,4-D).

[0201] In some embodiments, an expression cassette or vector containing a sequence encoding an RGN polypeptide can further include a sequence encoding a crRNA and / or a tracrRNA, or a sequence that combines the crRNA and tracrRNA to create an sgRNA. The sequence(s) encoding the crRNA and / or tracrRNA can be operably linked to at least one transcriptional control sequence for expression of the crRNA and / or tracrRNA in an organism or host cell of interest. For example, a polynucleotide encoding the crRNA and / or tracrRNA can be operably 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 human U6 promoter set forth in SEQ ID NO: 329, and promoters disclosed in U.S. Provisional Application No. 63 / 209,660, filed June 11, 2021, which is incorporated herein by reference in its entirety (including those set forth herein as SEQ ID NOs: 449-458).

[0202] As shown, an expression construct containing a nucleotide sequence encoding an RGN, crRNA, tracrRNA, and / or sgRNA can be used to transform an organism of interest. Methods for transformation involve introducing a nucleotide construct into an organism of interest. By "introducing," we mean introducing a nucleotide construct into a host cell so that the construct has access to the interior of the host cell. The methods of the present invention do not require a specific method for introducing a nucleotide construct into a host organism, so long as the nucleotide construct gains access to the interior of at least one cell of the host organism. The host cell can be a eukaryotic or prokaryotic cell. In certain embodiments, the eukaryotic host cell is a plant cell, a mammalian cell, an avian cell, or an insect cell. In some embodiments, the eukaryotic cell comprising, expressing, or modified by an RGN of the present disclosure is a human cell. In some embodiments, the eukaryotic cell comprising or expressing an RGN of the present disclosure or modified by an RGN of the present disclosure is a cell of hematopoietic origin, such as, but not limited to, an immune cell (i.e., a cell of the innate or adaptive immune system), including 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. In some embodiments, the eukaryotic cell comprising or expressing an RGN of the present disclosure or modified by an RGN of the present disclosure is an ocular cell, a muscle cell (e.g., a skeletal muscle cell), an epithelial cell (e.g., a lung epithelial cell), or a diseased cell (e.g., a tumor cell).

[0203] Methods for introducing nucleotide constructs into plant and other host cells are known in the art and include, but are not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.

[0204] This method results in transformed organisms, including plants, including whole plants, as well as plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, propagules, embryos, and their progeny. Plant cells can be differentiated or undifferentiated (e.g., villi, suspension culture cells, protoplasts, leaf cells, root cells, brown cells, pollen).

[0205] "Transgenic organisms," "transformed organisms," or "stably transformed" organisms, cells, or tissues refer to organisms that incorporate or have incorporated polynucleotides encoding the RGN, crRNA, and / or tracrRNA of the present invention. It is recognized that other exogenous or endogenous nucleic acid sequences or DNA fragments can also be incorporated into host cells. Agrobacterium- and biolistic transformation remain the two predominant approaches for transforming plant cells. However, host cell transformation can also be carried out by infection, transfection, microinjection, electroporation, microprojection, biolistic or particle bombardment, electroporation, silica / carbon fiber, ultrasound-mediated, PEG-mediated, calcium phosphate co-precipitation, polycation DMSO technology, DEAE-dextran procedures, as well as viral, liposome-mediated, and other methods. Viral-mediated introduction of polynucleotides encoding RGN, crRNA, and / or tracrRNA includes retrovirus-, lentivirus-, adenovirus-, and adeno-associated virus-mediated introduction and expression, as well as the use of caulimoviruses, geminiviruses, and RNA plant viruses.

[0206] Transformation protocols and protocols for introducing polypeptide or polynucleotide sequences into plants can vary depending on the type of host cell (e.g., monocotyledonous or dicotyledonous plant cell) targeted for transformation. Methods for transformation are known in the art and include those described in U.S. Patent Nos. 8,575,425, 7,692,068, 8,802,934, and 7,541,517 (each of which is incorporated herein by reference). Also, Rakoczy-Trojanowska, M. (2002) Cell Mol Biol Lett.7:849-858, Jones et al. (2005) Plant Methods 1:5, Rivera et al. (2012) Physics of Life Reviews 9:308-345, Bartlett et al. (2008) Plant Methods 4:1-12, Bates, GW (1999) Methods in Molecular Biology 111:359-366, Binns and Thomashow (1988) Annual Reviews in Microbiology 42:575-606, Christou, P. (1992) The Plant Journal 2:275-281, Christou, P. (1995) Euphytica 85:13-27, Tzfira et. See also Yao et al. (2004) TRENDS in Genetics 20:375-383, Yao et al. (2006) Journal of Experimental Botany 57:3737-3746, Zupan and Zambryski (1995) Plant Physiology 107:1041-1047, Jones et al. (2005) Plant Methods 1:5.

[0207] Transformation can result in stable or transient integration of a nucleic acid into a cell. "Stable transformation" is intended to mean that a nucleotide construct introduced into a host cell is integrated into the genome of the host cell and can be inherited by its progeny. "Transient transformation" is intended to mean that a polynucleotide is introduced into a host cell but is not integrated into the genome of the host cell.

[0208] Methods for chloroplast transformation are known in the art. See, e.g., Svab et al. (1990) Proc. Nail. Acad. Sci. USA 87:8526-8530; Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Svab and Maliga (1993) EMBOJ. 12:601-606. Methods rely on particle gun delivery of DNA containing a selectable marker and targeting the DNA to the plastid genome by homologous recombination. Additionally, plastid transformation can be achieved by transactivation of silent plastid-derived transgenes through tissue-preferential expression of nuclear-encoded and plastid-directed RNA polymerases. Such a system is reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91:7301-7305.

[0209] Transformed cells can be grown into transgenic organisms, such as plants, according to conventional methods. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants can then be grown and pollinated with either the same transformed strain or a different strain, and the resulting hybrids can have constitutive expression of the identified desired phenotypic traits. They can be propagated for two or more generations to ensure that the expression of the desired phenotypic traits is stably maintained and inherited, and then the seeds can be harvested to ensure that expression of the desired phenotypic traits is achieved. Thus, the present invention provides transformed seeds (also referred to as "transgenic seeds") having a nucleotide construct of the present invention, e.g., an expression cassette of the present invention, stably integrated into their genome.

[0210] Alternatively, transformed cells can be introduced into an organism, where the cells can be derived from an organism and the cells transformed in an ex vivo approach.

[0211] The sequences provided herein can be used for the transformation of any plant species, including, but not limited to, monocotyledons or dicotyledons. Examples of plants of interest include, but are not limited to, corn (maize), sorghum, wheat, sunflower, tomato, crucifers, pepper, potato, cotton, rice, soybean, sugar beet, sugarcane, tobacco, barley and rapeseed, Brassica sp., alfalfa, rye, millet, safflower, peanut, sweet potato, cassava, coffee, coconut, pineapple, citrus trees, cocoa, tea, banana, avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond, oat, vegetables, ornamentals, and conifers.

[0212] Vegetables include, but are not limited to, tomatoes, lettuce, green beans, lima beans, peas, and members of the genus Curcumis, such as cucumbers, cantaloupes, and muskmelons. Ornamental plants include, but are not limited to, azaleas, hydrangeas, hibiscus, roses, tulips, daffodils, petunias, carnations, poinsettias, and chrysanthemums. Preferably, the plants of the present invention are crop plants (e.g., corn, sorghum, wheat, sunflowers, tomatoes, crucifers, peppers, potatoes, cotton, rice, soybeans, sugar beets, sugarcane, tobacco, barley, rapeseed, etc.).

[0213] As used herein, the term "plant" includes plant cells, plant protoplasts, plant cell tissue cultures capable of regenerating plants, plant callus, plant mass, and intact plant cells within a plant or plant parts (e.g., embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, grains, ears, cobs, husks, stems, roots, root tips, anthers, etc.). Grain is intended to mean mature seeds produced by commercial growers for purposes other than seed growth or reproduction. Progeny, variants, and mutants of regenerated plants are also included within the scope of the present invention, provided that these parts contain the introduced polynucleotide. Further provided are processed plant products or by-products carrying the sequences disclosed herein, including, for example, soybean meal.

[0214] Polynucleotides encoding RGN, crRNA, and / or tracrRNA or containing crRNA and / or tracrRNA can also be used to transform any prokaryotic species, including, but not limited to, archaea and bacteria (e.g., Bacillus sp., Klebsiella sp., Streptomyces sp., Rhizobium sp., Escherichia sp., Pseudomonas sp., Salmonella sp., Shigella sp., Vibrio sp., Yersinia sp., Mycoplasma sp., Agrobacterium, Lactobacillus sp.).

[0215] Polynucleotides encoding RGN, crRNA, and / or tracrRNA or containing crRNA and / or tracrRNA can be used to transform any eukaryotic species, including, but not limited to, animals (e.g., mammals, insects, fish, birds, and reptiles), fungi, amoebas, algae, and yeast.

[0216] Conventional viral and non-viral gene transfer methods can be used to introduce nucleic acids into mammalian, insect, or avian cells or target tissues. Using such methods, nucleic acids encoding components of the RGN system can be administered to cells in culture or into host organisms. Non-viral vector delivery systems include DNA plasmids, RNA (e.g., transcripts of the vectors described herein), naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to cells. For reviews of gene therapy procedures, see Anderson, Science 256:808-813 (1992); Nabel & Feigner, TIBTECH 11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166 (1993); Dillon, TIBTECH 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 & Perricaudet, British Medical Bulletin 51(1):31-44 (1995); Haddada et al., in Current See Topics in Microbiology and Immunology, Doerfler and Bohm (eds) (1995), and Yu et al., Gene Therapy 1:13-26 (1994).

[0217] Non-viral delivery methods of nucleic acids include lipofection, nucleofection, microinjection, gene guns, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and drug-enhanced uptake of DNA. Lipofection is described, for example, in U.S. Pat. Nos. 5,049,386, 4,946,787, and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides include those described in Feigner, WO91 / 17424, and WO91 / 16024. Delivery can be to cells (in vitro or ex vivo administration) or 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 (e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 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 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.

[0218] The use of RNA or DNA virus-based systems for nucleic acid delivery takes advantage of highly evolved processes for targeting viruses to specific cells in the body and transporting the viral payload to the nucleus. Viral vectors can be administered directly to patients (in vivo) or used to treat cells in vitro, with the modified cells optionally administered to patients (ex vivo). Traditional virus-based systems include retroviral, lentiviral, adenoviral, adeno-associated viral, and herpes simplex viral vectors for gene transfer. Integration into the host genome is possible with retroviral, lentiviral, and adeno-associated viral gene transfer methods, often resulting in long-term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues.

[0219] Retroviral tropism can be altered by incorporating foreign envelope proteins to expand the potential target population of target cells. Lentiviral vectors are retroviral vectors capable of transducing or infecting non-dividing cells and typically produce high viral titers. Therefore, the choice of retroviral gene transfer system will depend on the target tissue. Retroviral vectors consist of cis-acting long terminal repeats (LTRs) capable of packaging up to 6-10 kb of foreign sequence. The minimal cis-acting LTRs are sufficient for vector replication and packaging, which are then used to integrate therapeutic genes into target cells to provide persistent transgene expression. Widely used retroviral vectors include those based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Viral. 66:2731-2739 (1992); Johann et al., J. Viral. 66:1635-1640 (1992); Sommnerfelt et al., Viral. 176:58-59 (1990); Wilson et al., J. Viral. 63:2374-2378 (1989); Miller et al., J. Viral. 65:2220-2224 (1991); PCT / US94 / 05700).

[0220] For applications where transient expression is preferred, adenovirus-based systems may be used. Adenovirus-based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. High titers and expression levels have been obtained using such vectors. This vector can be produced in large quantities in a relatively simple system. Adeno-associated virus ("AAV") vectors may also be used to transduce cells with target nucleic acids, for example, in the in vitro production of nucleic acids and peptides, and in in vivo and ex vivo gene therapy procedures (see, e.g., 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, I. Clin. Invest. 94:1351 (1994)). The construction of recombinant AAV vectors has been described in several publications (U.S. Patent No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al. (e.g., J. et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., I. Viral. 63:03822-3828 (1989)). Packaging cells are typically used to form viral particles capable of infecting host cells. Such cells include 293 cells, which package adenovirus, and ψJ2 or PA317 cells, which package retrovirus.

[0221] Viral vectors used in gene therapy are usually produced by generating cell lines that package nucleic acid vectors into viral particles. The vectors typically contain minimal viral sequences necessary for packaging and subsequent integration into the host, with other viral sequences replaced by an expression cassette for the polynucleotide(s) to be expressed. Missing viral functions are typically supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically contain only the ITR sequences from the AAV genome necessary for packaging and integration into the host genome. Viral DNA is packaged in a cell line containing a helper plasmid encoding other AAV genes, namely rep and cap, but lacking the ITR sequences.

[0222] The cell line can be infected with adenovirus as a helper. The helper virus promotes the replication of AAV vectors and the expression of AAV genes from the helper plasmid. The helper plasmid is not packaged in significant amounts due to the lack of ITR sequences. Contamination by adenovirus can be reduced, for example, by heat treatment, to which adenovirus is more sensitive than AAV. Additional methods for delivering nucleic acids to cells are known to those skilled in the art. See, for example, US2003 / 0087817 (incorporated herein by reference).

[0223] In some embodiments, host cells are transiently or non-transiently transfected with one or more vectors described herein. In some embodiments, cells are transfected as they naturally occur in a subject. In some embodiments, the transfected cells are harvested from a subject. In some embodiments, the cells are derived from a subject, e.g., cells harvested from a cell line. In some embodiments, the cell line may be mammalian, insect, or avian. A wide variety of cell lines for tissue culture are known in the art. Examples of cell lines include 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, A37 5, ARH-77, Calul, SW480, SW620, SKOV3, SK-UT, CaCo2, P388Dl, SEM-K2, WEHI-231, HB56, TIB 55, lurkat, 145.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2, HeLa B, HeLa These include, but are not limited to, T4.COS, COS-1, COS-6, COS-M6A, BS-C-1 monkey kidney epithelium, 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, A2 780cis, 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, HCA 2, HEK-293, HeLa, Hepalclc7, HL-60, HMEC, HT-29, lurkat, lY cells, K562 cells, Ku812, KCL22, KGl, KYOl, LNCap, Ma-Mel 1-48, MC-38, MCF-7, MCF-l0A, MDA-MB-231, MDA-MB-468, MDA-MB-435, MDCKII, MDCKII, MOR / 0.2R, MONO-MAC 6, MTD-lA, MyEnd, NCI-H69 / CPR, NCI-H69 / LX10, NCI-H69 / LX20, NCI-H69 / LX4, NIH-3T3, NALM-1, NW-145, OPCN / OPCT cell line, Peer, PNT-lA / 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 transgenic varieties thereof. Cell lines are available from a variety of sources known to those skilled in the art (see, for example, the American Type Culture Collection (ATCC) (Manassas, Va.)).

[0224] In some embodiments, cells transfected with one or more vectors described herein are used to establish new cell lines containing one or more vector-derived sequences. In some embodiments, cells transiently transfected with components of the RGN system described herein (e.g., by transient transfection of one or more vectors or transfection with RNA) and modified by the activity of the RGN system are used to establish new cell lines comprising cells containing the modifications but lacking any other exogenous sequences. 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 in evaluating one or more test compounds.

[0225] In some embodiments, one or more vectors described herein are used to produce non-human transgenic animals or transgenic plants. In some embodiments, the transgenic animal is a mammal, such as a mouse, rat, hamster, rabbit, cow, or pig. In some embodiments, the transgenic animal is a bird, such as a chicken or duck. In some embodiments, the transgenic animal is an insect, such as a mosquito or tick.

[0226] V. Polypeptide and Polynucleotide Variants and Fragments The present disclosure provides active variants and fragments of naturally occurring (i.e., wild-type) RNA-guided nucleases having the amino acid sequences set forth as any one of SEQ ID NOs: 1-12, and active variants and fragments of naturally occurring CRISPR repeats such as any one of the sequences set forth as SEQ ID NOs: 13-24, and active variants and fragments of naturally occurring tracrRNA such as any one of the sequences set forth as SEQ ID NOs: 25-36, and the polynucleotides encoding them.

[0227] The activity of the variants or fragments may be altered compared to the polynucleotide or polypeptide of interest, but the variants and fragments should retain the function of the polynucleotide or polypeptide of interest. For example, the variants or fragments may have increased activity, decreased activity, a different spectrum of activity, or any other change in activity compared to the polynucleotide or polypeptide of interest.

[0228] Fragments and variants of naturally occurring RGN polypeptides, such as those disclosed herein, retain sequence-specific RNA-guided DNA binding activity. In certain embodiments, fragments and variants of naturally occurring RGN polypeptides, such as those disclosed herein, retain nuclease activity (single- or double-stranded).

[0229] Fragments and variants of naturally occurring CRISPR repeats, such as those disclosed herein, when part of a guide RNA (including tracrRNA), retain the ability to bind to an RNA-guided nuclease (complexed with the guide RNA) in a sequence-specific manner and guide the RNA-guided nuclease to a target nucleotide sequence.

[0230] Naturally occurring fragments and variants of tracrRNA, such as those disclosed herein, when part of a guide RNA (including a CRISPR RNA), retain the ability to guide an RNA-guided nuclease (complexed with the guide RNA) to a target nucleotide sequence in a sequence-specific manner.

[0231] The term "fragment" refers to a portion of a polynucleotide or polypeptide sequence of the present invention. A "fragment" or "biologically active portion" includes a polynucleotide comprising a sufficient number of consecutive nucleotides to retain biological activity (i.e., when contained within a guide RNA, binds to RGN and guides RGN to a target nucleotide sequence in a sequence-specific manner). A "fragment" or "biologically active portion" includes a polypeptide comprising a sufficient number of consecutive amino acid residues to retain biological activity (i.e., when complexed with a guide RNA, binds to a target nucleotide sequence in a sequence-specific manner). Fragments of RGN protein include fragments shorter than the full-length sequence due to the use of alternative downstream start sites. A biologically active portion of an RGN protein can be, for example, a polypeptide comprising 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, or more consecutive amino acid residues of any one of SEQ ID NOS: 1-12. Such biologically active portions can be prepared by recombinant techniques and evaluated for sequence-specific RNA-guided DNA binding activity. Biologically active fragments of CRISPR repeat sequences can comprise at least 8 consecutive amino acids of any one of SEQ ID NOS: 13-24. Biologically active portions of CRISPR repeat sequences can be, for example, polynucleotides comprising 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 consecutive nucleotides of any one of SEQ ID NOS: 13-24.A biologically active portion of a tracrRNA may be a polynucleotide that includes, for example, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more consecutive nucleotides of any one of SEQ ID NOS: 25-36.

[0232] In general, "variant" is intended to mean a substantially similar sequence. With respect to polynucleotides, variants include deletions and / or additions of one or more nucleotides at one or more internal sites within the naturally occurring polynucleotide and / or substitutions of one or more nucleotides at one or more sites within the naturally occurring polynucleotide. As used herein, a "native" or "wild-type" polynucleotide or polypeptide includes a naturally occurring nucleotide sequence or amino acid sequence, respectively. With respect to polynucleotides, conservative variants include sequences that, due to the degeneracy of the genetic code, encode the native amino acid sequence of a gene of interest. Naturally occurring allelic variants such as these can be identified using well-known molecular biology techniques, for example, using polymerase chain reaction (PCR) and hybridization techniques, as outlined below. Variant polynucleotides also include synthetically derived polynucleotides, such as those generated using site-directed mutagenesis, but still encoding a polypeptide or polynucleotide of interest. Generally, variants of a particular polynucleotide 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 more sequence identity to the particular polynucleotide as determined by sequence alignment programs and parameters described elsewhere herein.

[0233] Variants of a particular polynucleotide (i.e., a reference polynucleotide) disclosed herein can also be evaluated by comparing the percent sequence identity between the polypeptide encoded by the variant polynucleotide and the polypeptide encoded by the reference polynucleotide. The percent sequence identity between any two polypeptides can be calculated using sequence alignment programs and parameters described elsewhere herein. When any given pair of polynucleotides disclosed herein is evaluated by comparing the percent sequence identity shared by the two polypeptides they encode, the percent sequence identity between the two encoded polypeptides will be at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity.

[0234] In certain embodiments, a polynucleotide of the present disclosure encodes an RNA-guided nuclease polypeptide comprising 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 more identity to any one of the amino acid sequences set forth as SEQ ID NOs: 1-12.

[0235] Biologically active variants of the RGN polypeptides of the invention may differ by no more than about 1-15 amino acid residues, no more than about 1-10 amino acid residues, e.g., about 6-10 amino acid residues, no more than 5 amino acid residues, no more than 4 amino acid residues, no more than 3 amino acid residues, no more than 2 amino acid residues, or no more than 1 amino acid residue. In some embodiments, the polypeptides may include N- or C-terminal truncations, which may be at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 35 ... Deletions of 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700 amino acids or more may be included.

[0236] In certain embodiments, a polynucleotide of the present disclosure comprises or encodes a CRISPR repeat comprising a nucleotide 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 more identity to any one of the nucleotide sequences set forth as SEQ ID NOs: 13-24.

[0237] The polynucleotides of the present disclosure can comprise or encode a tracrRNA comprising a nucleotide 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 more identity to any one of the nucleotide sequences set forth as SEQ ID NOs:25-36.

[0238] Biologically active variants of CRISPR repeats or tracrRNA of the invention can differ by as few as about 1-15 nucleotides, as few as about 1-10 nucleotides, for example, about 6-10 nucleotides, as few as 5 nucleotides, as few as 4 nucleotides, as few as 3 nucleotides, as few as 2 nucleotides, or as few as 1 nucleotide. In certain embodiments, the polynucleotide can include a 5' or 3' truncation, which can include deletions of 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 95, 100, 105, 110, or more nucleotides from either the 5' or 3' end of the polynucleotide.

[0239] It is recognized that modifications can be made to the RGN polypeptides, CRISPR repeats, and tracrRNA provided herein to create variant proteins and polynucleotides. Human-designed changes can be introduced through the application of site-directed mutagenesis techniques. Alternatively, naturally occurring, unknown, or unidentified polynucleotides and / or polypeptides structurally and / or functionally related to the sequences disclosed herein can also be identified as being within the scope of the present invention. Conservative amino acid substitutions can be made in non-conserved regions that do not alter the function of the RGN protein. Alternatively, modifications can be made that improve the activity of RGN.

[0240] Variant polynucleotides and proteins also encompass sequences and proteins derived from mutagenic and recombinogenic procedures, such as DNA shuffling. In such procedures, one or more of the different RGN proteins disclosed herein (e.g., SEQ ID NOS: 1-12) are manipulated to create new RGN proteins with desired properties. In this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides that share substantial sequence identity and contain sequence regions capable of homologous recombination in vitro or in vivo. For example, using this approach, sequence motifs encoding domains of interest can be shuffled between the RGN sequences provided herein and other known RGN genes to generate sequences with increased K in the case of enzymes. mIt is possible to obtain new genes that encode proteins with improved desired properties, such as: Strategies for such DNA shuffling are known in the art. See, e.g., Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751, Stemmer (1994) Nature 370:389-391, Crameri et al. (1997) Nature Biotech. 15:436-438, Moore et al. (1997) J. Mol. Biol. 272:336-347, Zhang et al. (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509, Crameri et al. (1998) Nature 391:288-291, and U.S. Patent Nos. 5,605,793 and 5,837,458. "Shuffled" nucleic acids are nucleic acids produced by a shuffling procedure, such as any of the shuffling procedures described herein. Shuffled nucleic acids are produced by recombining (physically or virtually) two or more nucleic acids (or character strings), e.g., artificially, and optionally in a recursive manner. Generally, one or more screening steps are used in the shuffling process to identify nucleic acids of interest, and these screening steps can be performed before or after any recombination steps. In some (but not all) shuffling embodiments, it is desirable to perform multiple rounds of recombination before selection to increase the diversity of the pool being screened. The overall process of recombination and selection is optionally repeated recursively. Depending on the context, shuffling can refer to the overall process of recombination and selection, or alternatively, can simply refer to the recombination portion of the overall process.

[0241] As used herein, "sequence identity" or "identity" in the context of two polynucleotide or polypeptide sequences refers to the residues of the two sequences that are the same when aligned for maximum matching over a specified comparison window. When percentage sequence identity is used in reference to proteins, it is recognized that non-identical residue positions often differ by conservative amino acid substitutions (amino acid residues are replaced with other amino acid residues that have similar chemical properties (e.g., charge or hydrophobicity) and therefore do not alter the functional properties of the molecule). When sequences differ by conservative substitutions, the percent sequence identity may be adjusted upward to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity." Means for making this adjustment are well known to those of skill in the art. Typically, this involves scoring conservative substitutions as partial rather than complete mismatches, thereby increasing the percentage sequence identity. Thus, for example, where identical amino acids are given a score of 1 and non-conservative substitutions are given a score of zero, conservative substitutions are given a score between zero and 1. Scoring of conservative substitutions is calculated, for example, as performed in the program PC / GENE (Intelligenetics, Mountain View, California).

[0242] As used herein, "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide sequence within the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity.

[0243] Unless otherwise specified, the sequence identity / similarity values ​​provided herein refer to values ​​obtained using GAP version 10 using the following parameters: % identity and % similarity for nucleotide sequences using a GAP weight of 50 and a length weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for amino acid sequences using a GAP weight of 8 and a length weight of 2, and the BLOSUM62 scoring matrix; or any equivalent program thereof. By "equivalent program" is intended any sequence comparison program that produces alignments with identical nucleotide or amino acid residue matches and identical percent sequence identity for any two sequences in question when compared to corresponding alignments produced by GAP version 10.

[0244] Two sequences are "optimally aligned" when aligned for similarity scoring using a defined amino acid substitution matrix (e.g., BLOSUM62), gap existence penalty, and gap extension penalty to achieve the highest possible score for the pair of sequences. Amino acid substitution matrices and their use in quantifying similarity between two sequences are well known in the art and are described, for example, in Dayhoff et al. (1978) "A model of evolutionary change in proteins." In "Atlas of Protein Sequence and Structure," Vol. 5, Suppl. 3 (ed. MO Dayhoff), pp. 345-352. Natl. Biomed. Res. Found., Washington, DC, and Henikoff et al. (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919. The BLOSUM62 matrix is ​​often used as the default scoring substitution matrix in sequence alignment protocols. A gap existence penalty is imposed for introducing a single amino acid gap into one of the aligned sequences, and a gap extension penalty is imposed for each additional empty amino acid position inserted into an already opened gap. The alignment is defined by the amino acid positions in each sequence where the alignment begins and ends, and optionally by inserting one or more gaps in one or both sequences to achieve the highest possible score. While optimal alignment and scoring can be achieved manually, this process is facilitated by the use of computer-implemented alignment algorithms, such as gapped BLAST 2.0, described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402 and made publicly available at the National Center for Biotechnology Information Website (www.ncbi.nlm.nih.gov).Optimal alignments containing multiple alignments can be prepared using, for example, PSI-BLAST, available through www.ncbi.nlm.nih.gov and described by Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402.

[0245] For an amino acid sequence that is optimally aligned with a reference sequence, the amino acid residue "corresponds" to the position in the reference sequence with which the residue is paired in the alignment. The "position" is indicated by a number that sequentially identifies each amino acid in the reference sequence based on its position relative to the N-terminus. Due to deletions, insertions, truncations, fusions, etc., which must be considered when determining optimal alignment, the number of amino acid residues in a test sequence, determined by simple counting from the N-terminus, is generally not necessarily the same as the number of their corresponding positions in the reference sequence. For example, if there is a deletion in the aligned test sequence, there will be no amino acid corresponding to the position in the reference sequence at the deletion site. If there is 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 a truncation or fusion, there may be a stretch of amino acids in the reference or aligned sequence that does not correspond to any amino acid in the corresponding sequence.

[0246] VI. Antibodies Antibodies against RGN polypeptides or ribonucleoproteins, including those of the present invention, including those having any one of the amino acid sequences set forth as SEQ ID NOS: 1-12 or active variants or fragments thereof, are also encompassed. Methods for producing antibodies are well known in the art (see, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, and U.S. Pat. No. 4,196,265). These antibodies can be used in kits for detecting and isolating RGN polypeptides or ribonucleoproteins. Thus, the present disclosure provides kits containing antibodies that specifically bind to the polypeptides or ribonucleoproteins described herein, including, for example, polypeptides having any one of the amino acid sequences set forth as SEQ ID NOS: 1-12.

[0247] VII. SYSTEMS AND RIBONUCLEOPROTEIN COMPLEXES FOR BINDING TO TARGET SEQUENCES OF INTEREST AND METHODS OF MAKING THEM The present disclosure provides a system for binding to a target sequence of interest, comprising at least one guide RNA or a nucleotide sequence encoding the same, and at least one RNA-guided nuclease or a nucleotide sequence encoding the same. The guide RNA hybridizes to the target sequence of interest and forms a complex with an RGN polypeptide, thereby guiding the RGN polypeptide to bind to the target sequence. In some of these embodiments, the RGN comprises any one of the amino acid sequences set forth as SEQ ID NOS: 1-12, or an active variant or fragment thereof. In various embodiments, the guide RNA comprises a CRISPR repeat sequence comprising any one of the nucleotide sequences set forth as SEQ ID NOS: 13-24, or an active variant or fragment thereof. In certain embodiments, the guide RNA comprises a tracrRNA comprising any one of the nucleotide sequences set forth as SEQ ID NOS: 25-36, or an active variant or fragment thereof. The guide RNAs of the system can be single-guide RNAs or dual-guide RNAs. In certain embodiments, the system comprises an RNA-guided nuclease that is heterologous to the guide RNA, and the RGN and guide RNA are not found complexed to each other (i.e., bound to each other) in nature.

[0248] The system for binding a target sequence of interest provided herein can be a ribonucleoprotein complex, which is at least one molecule of RNA bound to at least one protein. The ribonucleoprotein complex provided herein comprises at least one guide RNA as the RNA component and an RNA-guided nuclease as the protein component. Such a ribonucleoprotein complex can be purified from cells or organisms that naturally express an RGN polypeptide and have been engineered to express a specific guide RNA specific for a target sequence of interest. Alternatively, the ribonucleoprotein complex can be purified from cells or organisms transformed with a polynucleotide encoding an RGN polypeptide and a guide RNA and cultured under conditions that allow expression of the RGN polypeptide and the guide RNA. Thus, methods for producing an RGN polypeptide or an RGN ribonucleoprotein complex are provided. Such methods include culturing cells containing a nucleotide sequence encoding an RGN polypeptide and, in some embodiments, a nucleotide sequence encoding a guide RNA, under conditions in which the RGN polypeptide (and, in some embodiments, the guide RNA) is expressed. The RGN polypeptide or RGN ribonucleoprotein can then be purified from a lysate of the cultured cells. In embodiments, the nucleotide sequence encoding the RGN polypeptide comprises mRNA (messenger RNA). In some embodiments, the method for assembling an RNP complex comprises combining one or more guide RNAs of the present disclosure with one or more RGN polypeptides of the present disclosure under conditions suitable for the formation of an RNP complex.

[0249] Methods for purifying RGN polypeptides or RGN ribonucleoprotein complexes from lysates of biological samples are known in the art (e.g., size exclusion and / or affinity chromatography, 2D-PAGE, HPLC, reverse-phase chromatography, immunoprecipitation). In certain methods, the RGN polypeptide is recombinantly produced and contains a purification tag to aid in its purification, including, but not limited to, glutathione-S-transferase (GST), chitin-binding protein (CBP), maltose-binding protein, thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tag, myc, AcV5, AU1, AU5, E, ECS, E2, FLAG (e.g., 3X FLAG tag), HA, nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV-G, 6xHis, 10xHis, biotin carboxyl carrier protein (BCCP), and calmodulin. Typically, tagged RGN polypeptides or RGN ribonucleoprotein complexes are purified using immobilized metal affinity chromatography. It will be appreciated that other forms of chromatography or other similar methods known in the art, including for example immunoprecipitation, may also be used alone or in combination.

[0250] An "isolated" or "purified" polypeptide, or biologically active portion thereof, is substantially or essentially free from components that normally accompany or interact with the polypeptide as found in its naturally occurring environment. Thus, an isolated or purified polypeptide may be substantially free of other cellular material or culture medium if produced by recombinant techniques, or substantially free of chemical precursors or other chemicals if chemically synthesized. A protein that is substantially free of cellular material includes preparations of the protein having less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating proteins. When a protein of the invention or a biologically active portion thereof is recombinantly produced, optimally, the culture medium represents less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or non-target protein chemicals. Similarly, an "isolated" polynucleotide or nucleic acid molecule is removed from its naturally occurring environment. An isolated polynucleotide is substantially free of chemical precursors or other chemicals when chemically synthesized or when removed from a gene locus via cleavage of phosphodiester bonds. An isolated polynucleotide can be part of a vector, a composition of matter, or can be contained within a cell, so long as the cell is not the original environment of the polynucleotide.

[0251] Certain methods provided herein for binding and / or cleaving a target sequence of interest involve the use of an in vitro assembled RGN ribonucleoprotein complex. In vitro assembly of the RGN ribonucleoprotein complex can be carried out using any method known in the art for contacting an RGN polypeptide with a guide RNA under conditions that allow the RGN polypeptide to bind to the guide RNA. As used herein, "contacting," "contacting," or "contacted" refers to placing components of a desired reaction together under conditions suitable for the desired reaction to occur. The RGN polypeptide may be purified from a biological sample, cell lysate, or culture medium, produced 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 polypeptide and guide RNA can be contacted in solution (e.g., buffered saline) to allow in vitro assembly of the RGN ribonucleoprotein complex.

[0252] VIII. Methods of Binding to, Cleaving, or Modifying a Target Sequence The present disclosure provides methods for binding to, cleaving, and / or modifying a target nucleotide sequence of interest. The methods include delivering a system comprising at least one guide RNA or a polynucleotide encoding the same and at least one RGN polypeptide or a polynucleotide encoding the same to the target sequence or to a cell, organelle, or embryo containing the target sequence. In some of these embodiments, the RGN comprises any one of the amino acid sequences set forth as SEQ ID NOS: 1-12, or an active variant or fragment thereof. In various embodiments, the guide RNA comprises a CRISPR repeat sequence comprising any one of the nucleotide sequences set forth as SEQ ID NOS: 13-24, or an active variant or fragment thereof. In certain embodiments, the guide RNA comprises a tracrRNA comprising any one of the nucleotide sequences set forth as SEQ ID NOS: 25-36, or an active variant or fragment thereof. The guide RNAs of the system can be single-guide RNAs or dual-guide RNAs.

[0253] The RGN of the system may be a nuclease-inactive RGN, may have nickase activity, or may be a fusion polypeptide. In some embodiments, the fusion polypeptide comprises a base-editing polypeptide, such as a cytosine deaminase or an adenine deaminase. In other embodiments, the RGN fusion protein comprises a reverse transcriptase. In other embodiments, the RGN fusion protein comprises a polypeptide that recruits a member of a functional nucleic acid repair complex, such as a member of the nucleotide excision repair (NER) or transcription-coupled nucleotide excision repair (TC-NER) pathway (Wei et al., 2015, PNAS USA 112(27):E3495-504; Troelstra et al., 1992, Cell 71:939-953; Marnef et al., 2017, J Mol Biol 429(9):1277-1288), which is described in U.S. Provisional Application No. 62 / 966,203, filed January 27, 2020, which is incorporated by reference in its entirety. In some embodiments, the RGN fusion protein comprises CSB, a member of the TC-NER (nucleotide excision repair) pathway that functions in recruiting other members (van den Boom et al., 2004, J Cell Biol 166(1):27-36; van Gool et al., 1997, EMBO J 16(19):5955-65, an example of which is set forth as SEQ ID NO: 324). In further embodiments, the RGN fusion protein comprises an active domain of CSB, such as the acidic domain of CSB comprising amino acid residues 356-394 of SEQ ID NO: 324 (Teng et al., 2018, Nat Commun 9(1):4115).

[0254] In certain embodiments, the RGN and / or guide RNA are heterologous to the cell, organelle, or embryo into which the RGN and / or guide RNA (or polynucleotide(s) encoding at least one of the RGN and guide RNA) are introduced.

[0255] In those embodiments in which the method comprises delivering a polynucleotide encoding a guide RNA and / or an RGN polypeptide, the cell or embryo can then be cultured under conditions in which the guide RNA and / or RGN polypeptide is expressed. In various embodiments, the method comprises contacting the target sequence with an RGN ribonucleoprotein complex. The RGN ribonucleoprotein complex can comprise RGN that is nuclease-inactive or has nickase activity. In some embodiments, the RGN of the ribonucleoprotein complex is a fusion polypeptide comprising a base-editing polypeptide. In certain embodiments, the method comprises introducing the RGN ribonucleoprotein complex into a cell, organelle, or embryo containing the target sequence. The RGN ribonucleoprotein complex can be a complex purified from a biological sample, a complex recombinantly produced and then purified, or a complex assembled in vitro, as described herein. In those embodiments in which the RGN ribonucleoprotein complex contacting the target sequence, organelle, or embryo is assembled in vitro, the method can further comprise in vitro assembly of the complex prior to contacting the target sequence, cell, organelle, or embryo.

[0256] Purified or in vitro assembled RGN ribonucleoprotein complexes can be introduced into cells, organelles, or embryos using any method known in the art, including, but not limited to, electroporation. Alternatively, RGN polypeptides and / or polynucleotides encoding or comprising guide RNAs can be introduced into cells, organelles, or embryos using any method known in the art (e.g., electroporation).

[0257] Upon delivery to or contact with a target sequence or a cell, organelle, or embryo containing the target sequence, the guide RNA guides RGN to bind to the target sequence in a sequence-specific manner. In those embodiments in which RGN has nuclease activity, the RGN polypeptide cleaves the target sequence of interest upon binding. The target sequence may then be modified via endogenous repair mechanisms, such as non-homologous end joining or homology-directed repair using a provided donor polynucleotide.

[0258] Methods for measuring the binding of RGN polypeptides to target sequences are known in the art and include chromatin immunoprecipitation assays, gel mobility shift assays, DNA pull-down assays, reporter assays, and microplate capture and detection assays. Similarly, methods for measuring the cleavage or modification of target sequences are known in the art and include in vitro or in vivo cleavage assays in which cleavage is confirmed using PCR, sequencing, or gel electrophoresis, with or without an appropriate label (e.g., radioisotope, fluorescent substance) attached to the target sequence to facilitate detection of degradation products. Alternatively, a nicking-induced exponential amplification reaction (NTEXPAR) assay can be used (see, e.g., Zhang et al. (2016) Chem. Sci. 7:4951-4957). In vivo cleavage can be assessed using a surveyor assay (Guschin et al. (2010) Methods Mol Biol 649:247-256).

[0259] In some embodiments, the methods involve the use of a single type of RGN complexed with two or more guide RNAs, which can target different regions of a gene or can target multiple genes.

[0260] In those embodiments in which a donor polynucleotide is not provided, the double-strand break introduced by the RGN polypeptide can be repaired by the non-homologous end joining (NHEJ) repair process. Due to the error-prone nature of NHEJ, repair of the double-strand break can result in modification of the target sequence. As used herein, "modification" with respect to a nucleic acid molecule refers to a change in the nucleotide sequence of the nucleic acid molecule, which can be the deletion, insertion, or substitution of one or more nucleotides, or a combination thereof. Modification of the target sequence can result in the expression of an altered protein product or inactivation of the coding sequence.

[0261] In these embodiments in which a donor polynucleotide is present, the donor sequence of the donor polynucleotide can be incorporated into or exchanged with the target nucleotide sequence during repair of the introduced double-strand break, resulting in the introduction of the exogenous donor sequence. Thus, the donor polynucleotide contains the donor sequence desired 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 incorporated donor sequence is not recognized and cleaved by RGN. Donor sequence integration can be enhanced by including flanking sequences, referred to herein as "homology arms," ​​within the donor polynucleotide that share substantial sequence identity with sequences flanking the target nucleotide sequence, enabling the homology-directed repair process. In some embodiments, the homology arms have a length of at least 50 base pairs, at least 100 base pairs, and up to 2000 base pairs, or more, and have at least 90%, at least 95%, or more sequence identity with their corresponding sequences in the target nucleotide sequence.

[0262] In these embodiments in which the RGN polypeptide introduces a double-stranded crossover break, the donor polynucleotide can contain a donor sequence flanked by compatible overhangs, allowing for direct ligation of the donor sequence to the cleaved target nucleotide sequence containing the overhangs by a non-homologous repair process during repair of the double-stranded break.

[0263] In those embodiments in which the method involves the use of an RGN nickase (i.e., capable of cleaving only one strand of a double-stranded polynucleotide), the method can include introducing two RGN nickases that target the same or overlapping target sequences and cleave different strands of the polynucleotide. For example, an RGN nickase that cleaves only the positive (+) strand of a double-stranded polynucleotide can be introduced along with a second RGN nickase that cleaves only the negative (-) strand of the double-stranded polynucleotide.

[0264] In various embodiments, methods are provided for binding to and detecting a target nucleotide sequence, the methods comprising introducing into a cell, organelle, or embryo at least one guide RNA, or a polynucleotide encoding the same, and at least one RGN polypeptide, or a polynucleotide encoding the same; and expressing the guide RNA and / or the RGN polypeptide (if the coding sequences are introduced), wherein the RGN polypeptide is a nuclease-inactive RGN and further comprises a detectable label; and the method further comprises detecting the detectable label. The detectable label may be fused to the RGN as a fusion protein (e.g., a fluorescent protein) or may be a small molecule conjugated to or incorporated within the RGN polypeptide that can be detected visually or by other means.

[0265] Also provided herein are methods for regulating the expression of a target sequence or gene of interest under the control of the target sequence. The methods include introducing at least one guide RNA or a polynucleotide encoding the same and at least one RGN polypeptide or a polynucleotide encoding the same into a cell, organelle, or embryo, and expressing the guide RNA and / or the RGN polypeptide (if a coding sequence is introduced), wherein the RGN polypeptide is a nuclease-inactive RGN. In some of these embodiments, the nuclease-inactive RGN is a fusion protein comprising an expression regulator domain (i.e., an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repression domain) described herein.

[0266] The present disclosure also provides a method for binding to and / or modifying a target nucleotide sequence of interest, the method comprising delivering a system comprising at least one guide RNA or a polynucleotide encoding the same, and at least one fusion polypeptide comprising an RGN and a base-editing polypeptide of the invention, such as a cytosine deaminase or an adenine deaminase, or a polynucleotide encoding the fusion polypeptide, to the target sequence or a cell, organelle, or embryo containing the target sequence.

[0267] Those skilled in the art will understand that any of the methods disclosed herein can be used to target a single target sequence or multiple target sequences. Thus, the methods include the use of a single RGN polypeptide in combination with multiple different guide RNAs, which can target multiple different sequences within a single gene and / or multiple genes. Also encompassed herein are methods in which multiple different guide RNAs are introduced in combination with multiple different RGN polypeptides. These guide RNAs and guide RNA / RGN polypeptide systems can target multiple different sequences within a single gene and / or multiple genes.

[0268] In one aspect, the present invention provides a kit containing any one or more of the elements disclosed in the above methods and compositions. In some embodiments, the kit comprises a vector system and instructions for using the kit. In some embodiments, the vector system comprises: (a) a first regulatory element operably linked to a DNA sequence encoding a crRNA sequence and one or more insertion sites for inserting a guide sequence upstream of the encoded crRNA sequence, where, upon expression, the guide sequence directs sequence-specific binding of an RGN complex to a target sequence in a eukaryotic cell, and the RGN complex comprises an RGN enzyme complexed with a guide RNA polynucleotide; and / or (b) a second regulatory element operably linked to an enzyme encoding the RGN enzyme that comprises a nuclear localization sequence. The elements may be provided individually or in combination and in any suitable container, such as a vial, bottle, or tube.

[0269] In some embodiments, the kit includes instructions in one or more languages. In some embodiments, the kit includes one or more reagents for use in a process utilizing one or more of the elements described herein. The reagents may be provided in any suitable container. For example, the kit may provide one or more reaction or storage buffers. The reagents may be provided in a form that is usable in a particular assay or in a form that requires the addition of one or more other components prior to use (e.g., 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.

[0270] In some embodiments, the kit includes one or more oligonucleotides corresponding to the guide sequence for insertion into a vector so as to operably link the guide sequence and the regulatory element. In some embodiments, the kit includes a homologous recombination template polynucleotide. In one aspect, the present invention provides methods for using one or more elements of an RGN system. The RGN system of the present invention provides an effective means for modifying a target polynucleotide. The RGN system of the present invention has a wide variety of utilities, including modification (e.g., deletion, insertion, translocation, inactivation, activation, base editing) of a target polynucleotide in multiple cell types. As such, the RGN system of the present invention has broad applications in, for example, gene therapy, drug screening, disease diagnosis, and prognosis. An exemplary RGN system, or RGN complex, includes an RGN enzyme complexed with a guide sequence hybridized to a target sequence within a target polynucleotide.

[0271] IX. Target Polynucleotides In one aspect, the present invention provides methods for modifying a target polynucleotide in a eukaryotic cell, which may be in vivo, ex vivo, or in vitro. In some embodiments, the method comprises sampling a cell or population of cells from a human or non-human animal or plant (including microalgae) and modifying the cell or cells. Culturing can occur ex vivo at any stage. The cells may be reintroduced into the non-human animal or plant (including microalgae).

[0272] Using natural variability, plant breeders combine the most useful genes for desirable qualities such as yield, quality, uniformity, durability, and resistance to pests. These desirable qualities also include growth, day-length preference, temperature requirements, the date of onset of flowering or reproductive development, fatty acid content, insect resistance, disease resistance, nematode resistance, fungus resistance, herbicide resistance, and tolerance to various environmental factors, including adverse soil conditions, including drought, heat, humidity, cold, wind, and high salinity. Sources of these useful genes include native or exotic varieties, heirloom varieties, wild plant relatives, and induced mutations, such as treating plant material with mutagens. Using the present invention, plant breeders are provided with new tools for inducing mutations. Thus, those skilled in the art can analyze genomes for sources of useful genes and use the present invention to induce increases in useful genes in varieties with desired characteristics or traits with greater precision than previous mutagens, thus accelerating and improving plant breeding programs.

[0273] The target polynucleotide of the RGN system can be any polynucleotide, endogenous or exogenous to a eukaryotic cell. 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). Without wishing to be bound by theory, the target sequence should associate with a PAM (protospacer adjacent motif), i.e., a short sequence recognized by the RGN system. While the exact sequence and length requirements of the PAM vary depending on the RGN used, the PAM is typically a 2-7 base pair sequence adjacent to the protospacer (i.e., the target sequence).

[0274] Target polynucleotides of the RGN system can include numerous disease-related genes and polynucleotides, as well as signal transduction biochemical pathway-related genes and polynucleotides. Examples of target polynucleotides include sequences associated with signal transduction biochemical pathways, such as signal transduction biochemical pathway-related genes or polynucleotides. 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 at an abnormal level or in an abnormal form in cells derived from disease-affected tissue compared to tissues or cells of non-disease controls. It can be a gene that becomes expressed at an abnormally high level, or it can be a gene that becomes expressed at an abnormally low level, and changes in expression correlate with the onset and / or progression of the disease. A disease-related gene also refers to a gene that possesses mutation(s) or genetic variation that is directly involved in or in linkage disequilibrium with a gene(s) involved in the etiology (e.g., causative mutation) of the disease. The causative mutation may be instability of the transcribed mRNA, altered splicing, or an expanded repeat (present in higher numbers compared to normal individuals) that results in a translation product with reduced activity or instability of the translation product. The transcribed or translated product may be known or unknown, and may be at normal or abnormal levels. In some embodiments, the disease may be an animal disease. In some embodiments, the disease may be an avian disease. In other embodiments, the disease may be a mammalian disease. In further embodiments, the disease may be a human disease. Examples of disease-associated genes and polynucleotides in humans are set forth in Table 6, available on the World Wide Web from the McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and the National Center for Biotechnology Information, National Library of Medicine (Bethesda, Md.).

[0275] While the RGN system is particularly useful due to the relative ease of targeting genomic sequences of interest, questions remain about what RGN can do to address causative mutations. One approach is to produce a fusion protein between RGN (preferably an inactive or nickase variant of RGN) and a base-editing enzyme or the active domain of a base-editing enzyme, such as a cytosine deaminase or adenine deaminase base editor (U.S. Patent No. 9,840,699, incorporated herein by reference). In some embodiments, the method comprises contacting a DNA molecule with (a) a fusion protein comprising an RGN or its nickase variant of the present invention and a base-editing polypeptide, such as a deaminase, and (b) a gRNA that targets the fusion protein of (a) to a target nucleotide sequence in a DNA strand, wherein the DNA molecule is contacted with the fusion protein and the gRNA in an amount and under conditions effective for deamination of nucleobases. In some embodiments, the target DNA sequence comprises a sequence associated with a disease or disorder, and deamination of the nucleobases results in a sequence not associated with the disease or disorder. In some embodiments, the target DNA sequence is present in an allele of a crop plant, and the particular allele of the trait of interest results in a plant with lower agronomic value. Deamination of the nucleobase results in an allele that improves the trait and increases the agronomic value of the plant.

[0276] In some embodiments, the DNA sequence contains a T→C or A→G point mutation associated with a disease or disorder, and deamination of the mutant C or G base results in a sequence that is not associated with the disease or disorder. In some embodiments, deamination corrects the point mutation in the sequence associated with the disease or disorder.

[0277] In some embodiments, the sequence associated with the disease or disorder encodes a protein, and deamination introduces a stop codon into the sequence associated with the disease or disorder, resulting in truncation of the encoded protein. In some embodiments, the contacting is performed in vivo in a subject suspected of having, having, or diagnosed with a disease or disorder. In some embodiments, the disease or disorder is a disease associated with a point mutation or single base mutation in the genome. In some embodiments, the disease is a genetic disease, cancer, metabolic disease, or lysosomal storage disease.

[0278] X. Pharmaceutical Compositions and Methods of Treatment Pharmaceutical compositions are provided comprising the RGN polypeptides of the present disclosure and active variants and fragments thereof, and polynucleotides encoding them, the gRNAs of the present disclosure or polynucleotides encoding them, the systems of the present disclosure, or cells comprising any of the RGN polypeptides or polynucleotides encoding RGN, the gRNAs or polynucleotides encoding gRNAs, or the RGN system, and a pharmaceutically acceptable carrier.

[0279] A pharmaceutical composition is a composition used to prevent, reduce the severity of, cure, or otherwise treat a target condition or disease, comprising an active ingredient (i.e., an RGN polypeptide, an RGN-encoding polynucleotide, a gRNA, a gRNA-encoding polynucleotide, an RGN system, or cells containing any one of these) and a pharmaceutically acceptable carrier.

[0280] As used herein, a "pharmaceutically acceptable carrier" refers to a material that does not cause significant irritation to an organism and does not inhibit the activity and properties of the active ingredient (i.e., an RGN polypeptide, an RGN-encoding polynucleotide, a gRNA, a gRNA-encoding polynucleotide, an RGN system, or a cell containing any of these). The carrier must be of sufficiently high purity and sufficiently low toxicity to be suitable for administration to the subject being treated. The carrier may be inert or may have pharmaceutical benefits. In some embodiments, a pharmaceutically acceptable carrier comprises one or more compatible solid or liquid fillers, diluents, or encapsulating substances that are suitable for administration to humans or other vertebrates. In some embodiments, a pharmaceutically acceptable carrier is not naturally occurring. In some embodiments, the pharmaceutically acceptable carrier and the active ingredient are not found together in nature.

[0281] Pharmaceutical compositions used in the methods of the present disclosure can be formulated with suitable carriers, excipients, and other agents that provide suitable transport, delivery, tolerance, etc. Numerous suitable formulations are known to those skilled in the art. See, for example, Remington, *The Science and Practice of Pharmacy* (21st ed. 2005). Suitable formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (such as LIPOFECTIN vesicles), lipid nanoparticles, DNA complexes, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsion carbowax (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Pharmaceutical compositions for oral or parenteral use can be prepared into unit-dose dosage forms suitable for the dosage of the active ingredient. Such unit-dose dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc.

[0282] In some embodiments in which cells containing or modified with the disclosed RGN, gRNA, RGN system, or polynucleotides encoding same are administered to a subject, the cells are administered as a suspension with a pharmaceutically acceptable carrier. Those skilled in the art will recognize that pharmaceutically acceptable carriers used in cell compositions do not contain amounts of buffers, compounds, cryopreservatives, preservatives, or other agents that substantially interfere with the viability of the cells delivered to a subject. Cell-containing formulations can include, for example, an osmotic buffer that allows the integrity of the cell membrane to be maintained, and optionally, nutrients to maintain cell viability or enhance engraftment upon administration. Such formulations and suspensions are known to those skilled in the art and / or can be adapted for use with the cells described herein using routine experimentation.

[0283] The cell compositions can also be emulsified or presented as liposomal compositions, provided that the emulsification procedure does not adversely affect cell viability. The cells and any other active ingredients can be mixed with excipients that are pharmaceutically acceptable, compatible with the active ingredients, and in amounts suitable for use in the therapeutic methods described herein.

[0284] Additional agents included in the cell composition can include pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the polypeptide) formed with inorganic acids, such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, tartaric acid, mandelic acid, etc. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0285] Physiologically acceptable and pharmaceutically acceptable carriers are well known in the art. An exemplary liquid carrier is a sterile aqueous solution containing no materials in addition to the active ingredient and water, or a buffer solution such as sodium phosphate, physiological saline, or both, e.g., phosphate-buffered saline, at a physiological pH value. Additionally, aqueous carriers can contain two or more buffer salts, as well as salts such as sodium and potassium chloride, dextrose, polyethylene glycol, and other solutes. Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Examples of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of active compound used in a cell composition that will be effective in treating a particular disorder or condition will depend on the nature of the disorder or condition and can be determined by standard clinical techniques.

[0286] The RGN polypeptides, guide RNAs, RGN systems, or polynucleotides encoding them of the present disclosure can be formulated with pharmaceutically acceptable excipients, such as carriers, solvents, stabilizers, adjuvants, and diluents, depending on the particular mode of administration and dosage form. In some embodiments, these pharmaceutical compositions are formulated to achieve a physiologically compatible pH, ranging from about pH 3 to about pH 11, or from about pH 3 to about pH 7, depending on the formulation and route of administration. In some embodiments, the buffer solution has a pH of about pH 5.0 to about pH 8. In some embodiments, the composition can comprise a therapeutically effective amount of at least one compound described herein along with one or more pharmaceutically acceptable excipients. In some embodiments, the composition can comprise a combination of compounds described herein, or a second active ingredient useful for treating or preventing bacterial growth (e.g., but not limited to, an antibacterial or antimicrobial agent), or a combination of reagents of the present disclosure.

[0287] Suitable excipients include carrier molecules comprising large, slowly metabolized macromolecules such as, for example, proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Other exemplary excipients include antioxidants (for example, but not limited to, ascorbic acid), chelating agents (for example, but not limited to, EDTA), carbohydrates (for example, but not limited to, dextrin, hydroxyalkylcellulose, and hydroxyalkylmethylcellulose), stearic acid, liquids (for example, but not limited to, oils, water, saline, glycerol, and ethanol), wetting or emulsifying agents, pH buffering substances, etc.

[0288] In some embodiments, the formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials, and stored under freeze-dried (lyophilized) conditions requiring the addition of a sterile liquid carrier, for example, saline, water for injection, semi-liquid foam, or gel, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets of the kind described above. In some embodiments, the active ingredient is frozen in unit-dose or multi-dose containers and later thawed for injection, or dissolved in a buffer solution that is kept / stabilized under refrigeration until use.

[0289] The therapeutic agent(s) may be contained in a controlled release system. In order to prolong the effect of a drug, it is often desirable to slow the absorption of the drug from subcutaneous, intrathecal, or intramuscular injection. This can be achieved by using a liquid suspension of a crystalline or amorphous material that is poorly water-soluble. The rate of absorption of the drug then depends on its dissolution rate, which may in turn depend on crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is achieved by dissolving or suspending the drug in an oil vehicle. In some embodiments, the use of a long-term sustained-release implant may be particularly suitable for the treatment of chronic diseases. Long-term sustained-release implants are well known to those skilled in the art.

[0290] Provided herein is a method for treating a disease in a subject in need thereof, comprising administering to the subject in need thereof an effective amount of an RGN polypeptide of the present disclosure or an active variant or fragment thereof or a polynucleotide encoding same, a gRNA of the present disclosure or a polynucleotide encoding same, an RGN system of the present disclosure, or a cell modified by or comprising any one of these compositions.

[0291] In some embodiments, the treatment involves in vivo gene editing by administering an RGN polypeptide, gRNA, or RGN system of the present disclosure, or a polynucleotide(s) encoding same. In some embodiments, the treatment involves ex vivo gene editing, in which cells are genetically modified ex vivo using an RGN polypeptide, gRNA, or RGN system of the present disclosure, or a polynucleotide(s) encoding same, and then the modified cells are administered to a subject. In some embodiments, the genetically modified cells are then derived from the subject receiving the modified cells, and the transplanted cells are referred to herein as autologous. In some embodiments, the genetically modified cells are derived from a different subject (i.e., donor) within the same species as the subject receiving the modified cells (i.e., recipient), and the transplanted cells are referred to herein as allogeneic. In some examples described herein, the cells can be expanded in culture before being administered to a subject in need thereof.

[0292] In some embodiments, the disease treated with the composition of the present disclosure is a disease that can be treated with immunotherapy, such as chimeric antigen receptor (CAR) T cells. Such diseases include, but are not limited to, cancer. In some embodiments, the disease treated with the composition of the present disclosure is associated with a causative mutation. As used herein, a "causative mutation" refers to a specific nucleotide, multiple nucleotides, or nucleotide sequence in a genome that contributes to the severity or presence of a disease or disorder in a subject. Correction of the causative mutation leads to amelioration of at least one symptom caused by the disease or disorder. In some embodiments, the causative mutation is adjacent to a PAM site recognized by an RGN disclosed herein. The causative mutation can be corrected with an RGN of the present disclosure or a fusion polypeptide comprising an RGN of the present disclosure and a base-editing polypeptide (i.e., a base editor). Non-limiting examples of diseases associated with causative mutations include cystic fibrosis, Hurler syndrome, Friedreich's ataxia, Huntington's disease, and sickle cell disease. In some embodiments, the disease treated with an RGN of the present disclosure is a disease listed in Table 6. Additional non-limiting examples of disease-associated genes and mutations are available from the McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and the National Center for Biotechnology Information, National Library of Medicine (Bethesda, Md.), and are available on the World Wide Web.

[0293] As used herein, "treatment" or "treating," or "alleviating" or "ameliorating" are used interchangeably. These terms refer to an approach to obtaining a beneficial or desired result, including, but not limited to, therapeutic benefit and / or prophylactic benefit. Therapeutic benefit refers to any therapeutically relevant improvement or effect in one or more diseases, conditions, or symptoms being treated. For prophylactic benefit, the composition may be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more physiological symptoms of a disease, even if the disease, condition, or symptom has not yet manifested. In some embodiments, treatment may be administered after one or more symptoms have developed and / or after a disease has been diagnosed. In certain embodiments, treatment may be administered in the absence of symptoms, e.g., to prevent or delay the onset of symptoms or to inhibit the onset or progression of a disease. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of the condition and / or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example to prevent or delay their recurrence.

[0294] The term "effective amount" or "therapeutically effective amount" refers to an amount of an agent sufficient to produce a beneficial or desired result. A therapeutically effective amount may vary depending on one or more of the subject and disease state being treated, the subject's weight and age, the severity of the disease state, the mode of administration, etc., and these can be readily determined by one of ordinary skill in the art. The specific dose may vary depending on one or more of the particular agent selected, the administration regimen to be followed, whether it is administered in combination with other compounds, the timing of administration, and the delivery system in which it is delivered.

[0295] The term "administering" refers to the placement of an active ingredient in a subject by a method or route that results in at least partial localization of the introduced active ingredient at a desired site, such as a site of injury or repair, so that a desired effect(s) occurs. In some embodiments, the present disclosure provides methods comprising delivering any of the RGN polypeptides, nucleic acid molecules, ribonucleoprotein complexes, vectors, pharmaceutical compositions, and / or gRNAs described herein. In some embodiments, the present disclosure further provides cells produced by such methods, and organisms (such as animals or plants) comprising or produced from such cells. In some embodiments, the RGN polypeptides and / or nucleic acid molecules described herein are delivered to cells in combination with (and optionally complexed with) a guide sequence.

[0296] In those embodiments in which cells are administered, the cells may be administered by any suitable route that results in delivery to a desired location in a subject where at least a portion of the transplanted cells or components of the cells remain viable. The survival period of the cells after administration to a subject may be as short as a few hours (e.g., 24 hours), to several days, to several years, or even the lifespan of the patient (i.e., long-term transplantation). For example, in some aspects described herein, an effective amount of photoreceptor cells or retinal progenitor cells is administered via a systemic route, e.g., intraperitoneal or intravenous.

[0297] In some embodiments, administering comprises administering by viral delivery. Viral vectors containing nucleic acids encoding the RGN polypeptides, ribonucleic acid complexes, or vectors disclosed herein may be administered directly to a patient (i.e., in vivo) or used to treat cells in vitro, and the modified cells may optionally be administered to a patient (i.e., ex vivo). Conventional viral-based systems may include, but are not limited to, retroviral, lentiviral, adenoviral, adeno-associated viral, and herpes simplex viral vectors for gene transfer. Integration into the host genome is possible with retroviral, lentiviral, and adeno-associated viral gene transfer methods, often resulting in long-term expression of the inserted transgene. Lentiviral vectors are retroviral vectors capable of transducing or infecting non-dividing cells and typically produce high viral titers. For applications where transient expression is preferred, adenoviral-based systems may be used. Adenoviral-based vectors are capable of very high transduction efficiency in many cell types and do not require cell division.

[0298] In some embodiments, administering includes administering via other non-viral delivery of nucleic acids. Exemplary non-viral delivery methods include, but are not limited to, RNP complexes, lipofection, nucleofection, microinjection, gene guns, virosomes, liposomes, immunoliposomes, polycations or lipid-nucleic acid complexes, naked DNA, artificial virions, and drug-enhanced uptake of DNA. Lipofection is described, for example, in U.S. Patent Nos. 5,049,386, 4,946,787, and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides include those described by Feigner, WO1991 / 17424, WO1991 / 16024. Delivery can be to cells (eg, in vivo or ex vivo administration) or to a target tissue (eg, in vivo administration).

[0299] In some embodiments, administering comprises administering by a method selected from the group consisting of intravenous, subcutaneous, intramuscular, oral, rectal, by aerosol, parenteral, ophthalmic, pulmonary, transdermal, vaginal, otic, nasal, and topical administration, or any combination thereof. In some embodiments, administration by injection or infusion is used for delivery of the cells.

[0300] As used herein, the term "subject" refers to any individual for whom diagnosis, treatment, or therapy is desired. In some embodiments, the subject is an animal. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0301] The effectiveness of treatment can be determined by a skilled clinician. However, a treatment is considered "effective treatment" if any one or all of the signs or symptoms of a disease or disorder are altered in a beneficial manner (e.g., reduced by at least 10%) or other clinically acceptable symptoms or markers of the disease are improved or ameliorated. Efficacy can also be measured by the individual not getting worse (e.g., the progression of the disease is stopped or at least slowed), as assessed by the need for hospitalization or medical intervention. Methods for measuring these indicators are well known to those skilled in the art. Treatment includes: (1) inhibiting the disease, e.g., preventing or slowing the progression of symptoms, or (2) relieving the disease, e.g., causing regression of symptoms, and (3) preventing or reducing the likelihood of onset of symptoms.

[0302] Pharmaceutical compositions are provided comprising an RGN polypeptide of the present disclosure or a polynucleotide encoding same, a gRNA of the present disclosure or a polynucleotide encoding same, a system of the present disclosure, a cell comprising a ribonucleoprotein complex of the present disclosure or an RGN polypeptide or a polynucleotide encoding RGN, a gRNA or a polynucleotide encoding a gRNA, or the system, and a pharmaceutically acceptable carrier.

[0303] As used herein, "pharmaceutically acceptable carrier" refers to a material that does not cause significant irritation to an organism and does not abolish the activity and properties of an active ingredient (e.g., an RGN polypeptide or a nucleic acid molecule encoding it). The carrier must be of sufficiently high purity and sufficiently low toxicity to be suitable for administration to the subject being treated. The carrier may be inert or may have pharmaceutical benefits. In some embodiments, a pharmaceutically acceptable carrier comprises one or more compatible solid or liquid fillers, diluents, or encapsulating substances that are suitable for administration to humans or other vertebrates. In some embodiments, a pharmaceutical composition comprises a non-naturally occurring pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier and the active ingredient are not found together in nature and are therefore heterologous.

[0304] Pharmaceutical compositions used in the methods of the present disclosure can be formulated with suitable carriers, excipients, and other agents that provide suitable mobility, delivery, tolerability, etc. Many suitable formulations are known to those skilled in the art. See, for example, Remington, *The Science and Practice of Pharmacy* (21st ed. 2005). Non-limiting examples include sterile diluents such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates, and agents for adjusting tonicity such as sodium chloride or dextrose. For intravenous administration, particular carriers are saline or phosphate-buffered saline (PBS). Pharmaceutical compositions for oral or parenteral use can be prepared into a unit-dose dosage form suitable for the dose of the active ingredient. Such dosage forms in unit doses include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. These compositions may also contain adjuvants, including preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of microbial activity can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, etc. It may also be desirable to include isotonic agents, for example, sugars, sodium chloride, etc. Prolonged absorption of injectable pharmaceutical forms can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0305] In some embodiments in which cells containing or modified with the disclosed RGNs, gRNAs, systems, or polynucleotides encoding same are administered to a subject, the cells are administered as a suspension with a pharmaceutically acceptable carrier. Those skilled in the art will recognize that pharmaceutically acceptable carriers used in cell compositions do not contain amounts of buffers, compounds, cryopreservatives, preservatives, or other agents that substantially interfere with the viability of the cells delivered to a subject. Cell-containing formulations can include, for example, an osmotic buffer that allows the integrity of the cell membrane to be maintained, and optionally, nutrients to maintain cell viability or enhance engraftment upon administration. Such formulations and suspensions are known to those skilled in the art and / or can be adapted for use with the cells described herein using routine experimentation.

[0306] The cell compositions can also be emulsified or presented as liposomal compositions, provided that the emulsification procedure does not adversely affect cell viability. The cells and any other active ingredients can be mixed with excipients that are pharmaceutically acceptable, compatible with the active ingredients, and in amounts suitable for use in the therapeutic methods described herein.

[0307] Additional agents included in the cell composition can include pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the polypeptide) formed with inorganic acids, such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, tartaric acid, mandelic acid, etc. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0308] Suitable routes of administration of the pharmaceutical compositions described herein include, but are not limited to, topical, subcutaneous, transdermal, intradermal, intralesional, intraarticular, intraperitoneal, intravesical, transmucosal, gingival, intradental, intracochlear, intratympanic, intravisceral, epidural, intrathecal, intramuscular, intravenous, intravascular, intraosseous, periocular, intratumoral, intracerebral, and intraventricular administration.

[0309] In some embodiments, the pharmaceutical compositions described herein are administered locally to the disease site (e.g., lungs). In some embodiments, the pharmaceutical compositions described herein are administered to a subject by injection, by inhalation (e.g., aerosol) catheter, by suppository, or by implant, where the implant is a porous, non-porous, or gel-like material, including a membrane such as a sialastic membrane or fiber. In some embodiments, the pharmaceutical composition is formulated for delivery to a subject, e.g., for gene editing.

[0310] In some embodiments, pharmaceutical compositions are formulated in accordance with routine procedures as compositions suitable for intravenous or subcutaneous administration to a subject (e.g., a human). In some embodiments, pharmaceutical compositions for administration by injection are solutions in sterile isotonic aqueous buffer. Where necessary, the pharmaceutical may also include a solubilizing agent and a local anesthetic, such as lidocaine, to ease pain at the injection site. Generally, the ingredients are supplied either separately or mixed together, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container, such as an ampoule or sachet, indicating the quantity of active agent. When the pharmaceutical is administered by infusion, it can be dispensed using an infusion bottle containing sterile pharmaceutical-grade water or saline. When the pharmaceutical composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0311] In some embodiments, the pharmaceutical composition may be contained within a lipid particle or vesicle (eg, a liposome or microcrystal that is also suitable for parenteral administration).

[0312] Although the description of pharmaceutical compositions provided herein is primarily directed to pharmaceutical compositions suitable for administration to humans, it will be understood by those skilled in the art that such compositions are generally suitable for administration to any type of animal or organism.

[0313] A. Correcting the causative mutation using base editing An example of a genetic disease that can be corrected using an approach that relies on the RGN base editor fusion protein of the present invention is Hurler syndrome. Hurler syndrome (also known as MPS-1) is the result of a deficiency of α-L-iduronidase (IDUA), resulting in a lysosomal storage disease characterized at the molecular level by the accumulation of dermatan sulfate and heparan sulfate in lysosomes. This disease is generally an inherited genetic disorder caused by mutations in the IDUA gene, which encodes α-L-iduronidase. Common IDUA mutations are W402X and Q70X, both of which are nonsense mutations that result in premature termination of translation. Such mutations are well addressed by precise genome editing (PGE) approaches, as, for example, reversion of a single nucleotide by a base editing approach restores the wild-type coding sequence and results in protein expression controlled by the endogenous regulatory mechanisms of the gene locus. Furthermore, because heterozygotes are known to be asymptomatic, PGE therapy targeting one of these mutations would be useful for the majority of patients with this disease, as only one of the mutated alleles needs to be corrected (Bunge et al. (1994) Hum. Mol. Genet. 3(6):861-866, incorporated herein by reference).

[0314] Current treatments for Hurler syndrome include enzyme replacement therapy and bone marrow transplantation (Vellodi et al. (1997) Arch. Dis. Child. 76(2):92-99; Peters et al. (1998) Blood 91(7):2601-2608, incorporated herein by reference). While enzyme replacement therapy has dramatically impacted the survival and quality of life of patients with Hurler syndrome, this approach requires expensive and time-consuming weekly infusions. Additional approaches include delivery of the IDUA gene on an expression vector or insertion of the gene into a highly expressed locus, such as that of serum albumin (U.S. Patent No. 9,956,247, incorporated herein by reference). However, these approaches do not restore the original IDUA locus to the correct coding sequence. Genome editing strategies would have many advantages, most notably that regulation of gene expression is controlled by natural mechanisms present in healthy individuals. Furthermore, the use of base editing does not require the generation of double-stranded DNA breaks, which can lead to large chromosomal rearrangements, cell death, or cancer inheritance through the disruption of tumor suppressor mechanisms. A general strategy can be directed toward targeting and correcting certain disease-causing mutations in the human genome using the RGN base editor fusion proteins of the present invention, such as those containing LPG10136, LPG10139, LPG10134, or LPG10145. It will be understood that similar approaches can also be pursued to target diseases that can be corrected by base editing. It should further be understood that similar approaches can also be deployed using the RGNs of the present invention to target disease-causing mutations in other species, particularly common household pets or livestock. Common household pets and livestock include dogs, cats, horses, pigs, cows, sheep, chickens, donkeys, snakes, ferrets, and fish, including salmon and shrimp.

[0315] B. Correction of the causative mutation by targeted deletion The RGN of the present invention may also be useful in human therapeutic approaches where the causative mutations are more complex. For example, several diseases, such as Friedreich's ataxia and Huntington's disease, are the result of a significant increase in the repeat of a three-nucleotide motif in specific regions of the gene, which affects the ability of the expressed protein to function or express. Friedreich's ataxia (FRDA) is an autosomal recessive disease that results in progressive degeneration of neural tissue in the spinal cord. Decreased levels of frataxin (FXN) protein in mitochondria cause oxidative damage and iron deficiency at the cellular level. Decreased FXN expression is linked to a GAA triplet expansion within intron 1 of the FXN gene in somatic and germline cells. In FRDA patients, GAA repeats often consist of more than 70, and sometimes more than 1000 (most commonly 600-900) triplets, whereas unaffected individuals have approximately 40 or fewer repeats (Pandolfo et al. (2012) Handbook of Clinical Neurology 103:275-294; Campuzano et al. (1996) Science 271:1423-1427; Pandolfo (2002) Adv. Exp. Med. Biol. 516:99-118, all incorporated herein).

[0316] The expansion of trinucleotide repeat sequences that causes Friedreich's ataxia (FRDA) occurs at a defined locus within the FXN gene, termed the FRDA instability region. RNA-guided nucleases (RGNs) can be used to excise the unstable region in FRDA patient cells. This approach requires 1) an RGN and guide RNA sequence that can be programmed to target an allele within the human genome, and 2) a delivery approach for the RGN and guide sequence. Many nucleases used for genome editing, such as the commonly used Cas9 nuclease (SpCas9) derived from S. pyogenes, are too large to be packaged into adeno-associated virus (AAV) vectors, especially when considering the length of the SpCas9 gene and guide RNA in addition to other genetic elements required for a functional expression cassette. This makes the SpCas9 approach more challenging.

[0317] Certain RNA-guided nucleases of the present invention, such as LPG10134, LPG10145, LPG10136, and LPG10139, are well suited for packaging into AAV vectors together with guide RNAs. The present invention encompasses strategies using the RGNs of the present invention in which regions of genomic instability are removed. Such strategies are applicable to other diseases and disorders with similar genetic underpinnings, such as Huntington's disease. Similar strategies using the RGNs of the present invention may also be applicable to similar diseases and disorders in agriculturally or economically important non-human animals, including dogs, cats, horses, pigs, cows, sheep, chickens, donkeys, snakes, ferrets, and fish, including salmon and shrimp.

[0318] C. Correction of the causative mutation by targeted mutagenesis The RGNs of the present invention can also introduce disruptive mutations that can have beneficial effects. Genetic defects in genes encoding hemoglobin, particularly the beta globin chain (HBB gene), can cause several diseases known as hemoglobinopathies, including sickle cell anemia and thalassemia.

[0319] In adult humans, hemoglobin is a heterotetramer containing two alpha (α)-like globin chains, two beta (β)-like globin chains, and four heme groups. In adults, the α2β2 tetramer is called hemoglobin A (HbA) or adult hemoglobin. Typically, alpha and beta globin chains are synthesized in an approximately 1:1 ratio, which appears to be important in stabilizing hemoglobin and red blood cells (RBCs). In the developing fetus, a different form of hemoglobin, fetal hemoglobin (HbF), is produced, which has a higher oxygen-binding affinity than hemoglobin A, allowing oxygen to be delivered to the infant's system via the mother's bloodstream. Fetal hemoglobin also contains two alpha globin chains, but instead of the adult beta globin chains, it has two fetal gamma (γ) globin chains (i.e., fetal hemoglobin is α2γ2). The regulation of the switch from gamma globin production to beta globin production is highly complex and primarily involves downregulation of gamma globin transcription with concomitant upregulation of beta globin transcription. At approximately 30 weeks of gestation, fetal gamma globin synthesis begins to decline, while beta globin production increases. By approximately 10 months of age, neonatal hemoglobin is almost entirely α2β2, although some HbF persists into adulthood (approximately 1–3% of total hemoglobin). In the majority of patients with hemoglobinopathies, the gene encoding gamma globin is present, but expression is relatively low due to the normal gene silencing that occurs around the time of delivery, as described above.

[0320] Sickle cell disease is caused by the V6E mutation (GAG to GTG at the DNA level) in the beta-globin gene (HbS), and the resulting hemoglobin is called "hemoglobin S" or "HbS." Under hypoxic conditions, HbS molecules aggregate and form fibrous precipitates. These aggregates cause RBC abnormalities or "sickling," resulting in a loss of cellular flexibility. Sickled RBCs can no longer filter into capillary beds, potentially leading to vaso-occlusive crises in sickle cell patients. Additionally, sickled RBCs are more fragile than normal RBCs and more prone to hemolysis, ultimately leading to anemia in the patient.

[0321] Treatment and management of sickle cell patients is a lifelong proposition, involving antibiotic therapy, pain management, and blood transfusions during acute episodes. One approach is the use of hydroxyurea, which exerts its effects in part by increasing gamma globin production. However, the long-term side effects of chronic hydroxyurea therapy remain unknown, and the treatment may result in unwanted side effects and vary in efficacy from patient to patient. Despite the increasing effectiveness of sickle cell treatments, the average life expectancy of patients is still in the mid-to-late 50s, and the associated morbidity of the disease significantly impacts patients' quality of life.

[0322] Thalassemia (alpha and beta thalassemia) is also a hemoglobin-related disorder, typically involving reduced expression of globin chains. This can result from mutations in the regulatory region of the gene or mutations in the globin coding sequence that result in reduced expression or reduced levels or functional globin protein. Treatment for thalassemia typically involves blood transfusions and iron chelation therapy. Bone marrow transplants have also been used to treat people with severe thalassemia if a suitable donor can be identified, although this procedure can carry significant risks.

[0323] One approach proposed for the treatment of both sickle cell disease (SCD) and beta-thalassemia is to increase gamma globin expression so that HbF functionally replaces abnormal adult hemoglobin. As mentioned above, treatment of SCD patients with hydroxyurea is thought to be partially successful due to its effect on increasing gamma globin expression (DeSimone (1982) Proc Nat'l Acad Sci USA 79(14):4428-31; Ley, et al., (1982) N. Engl. J. Medicine, 307:1469-1475; Ley, et al., (1983) Blood 62:370-380; Constantoulakis et al., (1988) Blood 72(6):1961-1967, all of which are incorporated herein by reference). Increasing HbF expression involves identifying the gene whose product plays a role in regulating gamma globin expression. One such gene is BCL11A. BCL11A encodes a zinc finger protein expressed in adult erythroid progenitor cells, and its downregulation leads to increased gamma globin expression (Sankaran et al. (2008) Science 322:1839, incorporated herein by reference). The use of inhibitory RNA targeting the BCL11A gene has been proposed (e.g., US Patent Publication No. 2011 / 0182867, incorporated herein by reference), but this technology has several potential drawbacks, including that complete knockdown may not be achieved, delivery of such RNA may be problematic, and the RNA must be continuously present, requiring multiple treatments throughout life.

[0324] The RGNs of the present invention, such as LPG10134, LPG10145, LPG10136, or LPG10139, may be used to target the BCL11A enhancer region to inhibit BCL11A expression and thereby increase gamma globin expression. This targeted disruption can be achieved by non-homologous end joining (NHEJ), whereby the RGNs of the present invention target specific sequences within the BCL11A enhancer region, create a double-strand break, and the cellular machinery repairs the break, typically simultaneously introducing a deleterious mutation. As with those described for other disease targets, the RGNs of the present invention may have advantages over other known RGNs due to their relatively small size, which allows expression cassettes for the RGN and its guide RNA to be packaged into a single AAV vector for in vivo delivery. Similar strategies using the RGNs of the present invention may also be applicable to similar diseases and disorders in both humans and agriculturally or economically important non-human animals.

[0325] XI. Cells containing genetic modifications Provided herein are cells and organisms comprising a target sequence of interest modified using the RGN, crRNA, and / or tracrRNA-mediated processes described herein. In some of these embodiments, the RGN comprises any one of the amino acid sequences set forth as SEQ ID NOS: 1-12, or an active variant or fragment thereof. In various embodiments, the guide RNA comprises a CRISPR repeat sequence comprising any one of the nucleotide sequences set forth as SEQ ID NOS: 13-24, or an active variant or fragment thereof. In certain embodiments, the guide RNA comprises a tracrRNA comprising any one of the nucleotide sequences set forth as SEQ ID NOS: 25-36, or an active variant or fragment thereof. The guide RNA of the system can be a single guide RNA or a dual guide RNA.

[0326] The modified cells may be eukaryotic (e.g., mammalian, plant, insect, or avian cells) or prokaryotic. Also provided are organelles and embryos comprising at least one nucleotide sequence modified by the processes utilizing RGN, crRNA, and / or tracrRNA described herein. The genetically modified cells, organisms, organelles, and embryos may be heterozygous or homozygous for the modified nucleotide sequence.

[0327] Chromosomal modifications of a cell, organism, organelle, or embryo can result in altered expression (up- or down-regulation), inactivation, or expression of an altered protein product or integrated sequence. In these embodiments, where the chromosomal modification results in either gene inactivation or expression of a non-functional protein product, the genetically modified cell, organism, organelle, or embryo is referred to as a "knockout." The knockout phenotype can be the result of a deletion mutation (i.e., deletion of at least one nucleotide), an insertion mutation (i.e., insertion of at least one nucleotide), or a nonsense mutation (i.e., substitution of at least one nucleotide such that a stop codon is introduced).

[0328] Alternatively, chromosomal modification of a cell, organism, organelle, or embryo can produce a "knock-in" resulting from chromosomal integration of a nucleotide sequence encoding a protein. In some of these embodiments, the coding sequence is integrated into the chromosome such that the chromosomal sequence encoding the wild-type protein is inactivated, but the exogenously introduced protein is expressed.

[0329] In other embodiments, the chromosomal modification results in the production of a variant protein product. The expressed variant protein product can have at least one amino acid substitution and / or at least one amino acid addition or deletion. The variant protein product encoded by the altered chromosomal sequence can exhibit modified properties or activities when compared to the wild-type protein, including, but not limited to, altered enzymatic activity or substrate specificity.

[0330] In yet other embodiments, chromosomal modifications may result in altered expression patterns of proteins. As a non-limiting example, chromosomal changes in regulatory regions controlling expression of a protein product may result in overexpression or downregulation of the protein product, or altered tissue or temporal expression patterns.

[0331] The modified cells can be grown into organisms such as plants according to conventional methods. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants can then be grown and pollinated with either the same modified strain or a different strain, and the resulting hybrids can carry the genetic modification. The present invention provides genetically modified seeds. Progeny, variants, and mutants of the regenerated plants are also included within the scope of the present invention, provided that these portions contain the genetic modification. Further provided are processed plant products or by-products that carry the genetic modification, including, for example, soybean meal.

[0332] The methods provided herein can be used to modify any plant species, including, but not limited to, monocotyledonous or dicotyledonous plants. Examples of plants of interest include, but are not limited to, corn (maize), sorghum, wheat, sunflower, tomato, cruciferous plants, pepper, potato, cotton, rice, soybean, sugar beet, sugarcane, tobacco, barley and rapeseed, Brassica sp., alfalfa, rye, millet, safflower, peanut, sweet potato, cassava, coffee, coconut, pineapple, citrus trees, cocoa, tea, banana, avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond, oat, vegetables, ornamental plants, and conifers.

[0333] Vegetables include, but are not limited to, tomatoes, lettuce, green beans, lima beans, peas, and members of the genus Curcumis, such as cucumbers, cantaloupes, and muskmelons. Ornamental plants include, but are not limited to, azaleas, hydrangeas, hibiscus, roses, tulips, daffodils, petunias, carnations, poinsettias, and chrysanthemums. Preferably, the plants of the present invention are crop plants (e.g., corn, sorghum, wheat, sunflowers, tomatoes, crucifers, peppers, potatoes, cotton, rice, soybeans, sugar beets, sugarcane, tobacco, barley, rapeseed, etc.).

[0334] The methods provided herein can also be used to genetically modify any prokaryotic species, including, but not limited to, archaea and bacteria (e.g., Bacillus sp., Klebsiella sp., Streptomyces sp., Rhizobium sp., Escherichia sp., Pseudomonas sp., Salmonella sp., Shigella sp., Vibrio sp., Yersinia sp., Mycoplasma sp., Agrobacterium, Lactobacillus sp.).

[0335] The methods provided herein can be used to genetically modify any eukaryotic species or cells therefrom, including, but not limited to, animals (e.g., mammals, insects, fish, birds, and reptiles), fungi, amoebae, algae, and yeast. In some embodiments, cells modified by the methods of the present disclosure include cells of hematopoietic origin, such as cells of the 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.

[0336] The modified cells can be introduced into an organism. These cells can be derived from the same organism (e.g., a human) in the case of autologous cell transplantation, where the cells are modified using an ex vivo approach. Alternatively, in the case of allogeneic cell transplantation, the cells are derived from another organism (e.g., another human) within the same species.

[0337] XII. Kit Some aspects of the present disclosure provide kits comprising the RGN polypeptides of the present invention. In addition, in some embodiments, the kits include suitable reagents, buffers, and / or instructions for using the RGN polypeptides, for example, for in vitro or in vivo DNA or RNA editing. In some embodiments, the kits include instructions for designing and using gRNAs suitable for targeted editing of nucleic acid sequences.

[0338] In some embodiments, the pharmaceutical composition may be provided as a pharmaceutical kit, the kit comprising (a) a container containing a composition of the present disclosure in lyophilized form, 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 compound of the present disclosure. Optionally, a notice in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals or biological products may be associated with such container(s), the notice reflecting approval by the agency of manufacture, use, or sale for human administration.

[0339] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "a polypeptide" means one or more polypeptides.

[0340] All publications and patent applications mentioned in this specification are indicative of the level of those skilled in the art to which this disclosure pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0341] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent that certain changes and modifications can be practiced within the scope of the accompanying embodiments.

[0342] Non-limiting embodiments include the following: 1. A nucleic acid molecule comprising a polynucleotide encoding an RNA-guided nuclease (RGN) polypeptide, wherein the polynucleotide comprises a nucleotide sequence encoding an RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1 to 12; A nucleic acid molecule capable of binding to a target DNA sequence in an RNA-guided sequence-specific manner when the RGN polypeptide is bound to a guide RNA (gRNA) that can hybridize to the target DNA sequence.

[0343] 2. The nucleic acid molecule of embodiment 1, wherein said polynucleotide encoding an RGN polypeptide is operably linked to a promoter heterologous to said polynucleotide.

[0344] 3. The nucleic acid molecule of embodiment 1 or 2, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 1-12.

[0345] 4. The nucleic acid molecule of embodiment 1 or 2, wherein the RGN polypeptide comprises an amino acid sequence having 100% sequence identity to any one of SEQ ID NOs: 1-12.

[0346] 5. The nucleic acid molecule of any one of embodiments 1 to 4, wherein said RGN polypeptide is capable of cleaving said target DNA sequence upon binding.

[0347] 6. The nucleic acid molecule of embodiment 5, wherein said RGN polypeptide is capable of generating a double-stranded break.

[0348] 7. The nucleic acid molecule of embodiment 5, wherein the RGN polypeptide is capable of generating a single-strand break.

[0349] 8. The nucleic acid molecule of any one of embodiments 1-4, wherein the RGN polypeptide is nuclease inactive or is a nickase.

[0350] 9. The nucleic acid molecule of any one of embodiments 1-5, 7 and 8, wherein the RGN polypeptide is operably linked to a prime editing polypeptide.

[0351] 10. The nucleic acid molecule of embodiment 9, wherein the prime editing polypeptide comprises a DNA polymerase.

[0352] 11. The nucleic acid molecule of embodiment 9, wherein the prime editing polypeptide comprises a reverse transcriptase.

[0353] 12. The nucleic acid molecule of any one of embodiments 1-8, wherein the RGN polypeptide is operably linked to a base-editing polypeptide.

[0354] 13. The nucleic acid molecule of embodiment 12, wherein the base-editing polypeptide is a deaminase.

[0355] 14. The nucleic acid molecule of embodiment 13, wherein the deaminase is a cytosine deaminase or an adenine deaminase.

[0356] 15. The nucleic acid molecule of embodiment 13, wherein the deaminase has at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 377-448.

[0357] 16. The nucleic acid molecule of embodiment 13, wherein the deaminase has 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 377-448.

[0358] 17. The nucleic acid molecule of any one of embodiments 1-16, wherein the RGN polypeptide comprises one or more nuclear localization signals.

[0359] 18. The nucleic acid molecule of any one of embodiments 1-17, wherein the RGN polypeptide is codon-optimized for expression in a eukaryotic cell.

[0360] 19. The nucleic acid molecule of any one of embodiments 1-18, wherein the target DNA sequence is located adjacent to a protospacer adjacent motif (PAM).

[0361] 20. A vector comprising the nucleic acid molecule of any one of embodiments 1 to 19.

[0362] 21. The vector of embodiment 20, further comprising at least one nucleotide sequence encoding said gRNA capable of hybridizing to said target DNA sequence.

[0363] 22. Guide RNA is a) i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 13; ii) a guide RNA comprising a tracrRNA having at least 90% sequence identity to SEQ ID NO: 25, a guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1; b) i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 14; ii) a guide RNA comprising a tracrRNA having at least 90% sequence identity to SEQ ID NO: 26, a guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 2; c) i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 15; ii) a guide RNA comprising a tracrRNA having at least 90% sequence identity to SEQ ID NO: 27, a guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 3; d) i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 16; ii) a guide RNA comprising a tracrRNA having at least 90% sequence identity to SEQ ID NO: 28, a guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 4; e) i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 17; ii) a guide RNA comprising a tracrRNA having at least 90% sequence identity to SEQ ID NO: 29, a guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 5; f) i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 18; ii) a guide RNA comprising a tracrRNA having at least 90% sequence identity to SEQ ID NO: 30, a guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 6; g) i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 19; ii) a guide RNA comprising a tracrRNA having at least 90% sequence identity to SEQ ID NO: 31, a guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 7; h) i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 20; ii) a guide RNA comprising a tracrRNA having at least 90% sequence identity to SEQ ID NO: 32, a guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 8; i) i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 21; ii) a guide RNA comprising a tracrRNA having at least 90% sequence identity to SEQ ID NO: 33, a guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 9; j) i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 22; ii) a guide RNA comprising a tracrRNA having at least 90% sequence identity to SEQ ID NO: 34, a guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 10; k) i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 23; ii) a guide RNA comprising a tracrRNA having at least 90% sequence identity to SEQ ID NO: 35, a guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 11; and l) i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 24; ii) a guide RNA comprising a tracrRNA having at least 90% sequence identity to SEQ ID NO: 36, 22. The vector of embodiment 21, wherein the RGN polypeptide is selected from the group consisting of: a guide RNA comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 12.

[0364] 23. Guide RNA is a) i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 13; ii) a guide RNA comprising a tracrRNA having at least 95% sequence identity to SEQ ID NO: 25, a guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 1; b) i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 14; ii) a guide RNA comprising a tracrRNA having at least 95% sequence identity to SEQ ID NO: 26, a guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 2; c) i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 15; ii) a guide RNA comprising a tracrRNA having at least 95% sequence identity to SEQ ID NO: 27, a guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 3; d) i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 16; ii) a guide RNA comprising a tracrRNA having at least 95% sequence identity to SEQ ID NO: 28, a guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 4; e) i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 17; ii) a guide RNA comprising a tracrRNA having at least 95% sequence identity to SEQ ID NO: 29, a guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 5; f) i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 18; ii) a guide RNA comprising a tracrRNA having at least 95% sequence identity to SEQ ID NO: 30, a guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:6; g) i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 19; ii) a guide RNA comprising a tracrRNA having at least 95% sequence identity to SEQ ID NO: 31, a guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 7; h) i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 20; ii) a guide RNA comprising a tracrRNA having at least 95% sequence identity to SEQ ID NO: 32, a guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:8; i) i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 21; ii) a guide RNA comprising a tracrRNA having at least 95% sequence identity to SEQ ID NO: 33, a guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 9; j) i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 22; ii) a guide RNA comprising a tracrRNA having at least 95% sequence identity to SEQ ID NO: 34, a guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 10; k) i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 23; ii) a guide RNA comprising a tracrRNA having at least 95% sequence identity to SEQ ID NO: 35, a guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 11; and l) i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 24; ii) a guide RNA comprising a tracrRNA having at least 95% sequence identity to SEQ ID NO: 36, 22. The vector of embodiment 21, wherein the RGN polypeptide is selected from the group consisting of: a guide RNA comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 12.

[0365] 24. Guide RNA is a) i) a CRISPR RNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 13; ii) a guide RNA comprising a tracrRNA having 100% sequence identity to SEQ ID NO: 25, a guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 1; b) i) a CRISPR RNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 14; ii) a guide RNA comprising a tracrRNA having 100% sequence identity to SEQ ID NO: 26, a guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 2; c) i) a CRISPR RNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 15; ii) a guide RNA comprising a tracrRNA having 100% sequence identity to SEQ ID NO: 27, a guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 3; d) i) a CRISPR RNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 16; ii) a guide RNA comprising a tracrRNA having 100% sequence identity to SEQ ID NO: 28, a guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 4; e) i) a CRISPR RNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 17; ii) a guide RNA comprising a tracrRNA having 100% sequence identity to SEQ ID NO: 29, a guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 5; f) i) a CRISPR RNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 18; ii) a guide RNA comprising a tracrRNA having 100% sequence identity to SEQ ID NO: 30, a guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 6; g) i) a CRISPR RNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 19; ii) a guide RNA comprising a tracrRNA having 100% sequence identity to SEQ ID NO: 31, a guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 7; h) i) a CRISPR RNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 20; ii) a guide RNA comprising a tracrRNA having 100% sequence identity to SEQ ID NO: 32, a guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 8; i) i) a CRISPR RNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 21; ii) a guide RNA comprising a tracrRNA having 100% sequence identity to SEQ ID NO: 33, a guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 9; j) i) a CRISPR RNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 22; ii) a guide RNA comprising a tracrRNA having 100% sequence identity to SEQ ID NO: 34, a guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 10; k) i) a CRISPR RNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 23; ii) a guide RNA comprising a tracrRNA having 100% sequence identity to SEQ ID NO: 35, a guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having 100% sequence identity with SEQ ID NO: 11; and l) i) a CRISPR RNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 24; ii) a guide RNA comprising a tracrRNA having 100% sequence identity to SEQ ID NO: 36, 22. The vector of embodiment 21, wherein the RGN polypeptide is selected from the group consisting of: a guide RNA comprising an amino acid sequence having 100% sequence identity to SEQ ID NO: 12.

[0366] 25. The vector of any one of embodiments 21-24, wherein the gRNA is a single guide RNA (sgRNA).

[0367] 26. The vector of embodiment 25, wherein the sgRNA further comprises an extension comprising an editing template for prime editing.

[0368] 27. The vector of any one of embodiments 21-24, wherein the gRNA is a dual guide RNA.

[0369] 28. A cell comprising the nucleic acid molecule of any one of embodiments 1 to 19 or the vector of any one of embodiments 20 to 27.

[0370] 29. The cell of embodiment 28, wherein the cell is a prokaryotic cell.

[0371] 30. The cell of embodiment 28, wherein the cell is a eukaryotic cell.

[0372] 31. The cell of embodiment 30, wherein the eukaryotic cell is a mammalian cell.

[0373] 32. The cell of embodiment 31, wherein the mammalian cell is a human cell.

[0374] 33. The cell of embodiment 32, wherein the human cell is an immune cell.

[0375] 34. The cell of embodiment 33, wherein the immune cell is a stem cell.

[0376] 35. The cell of embodiment 34, wherein the stem cell is an induced pluripotent stem cell.

[0377] 36. The cell of embodiment 30, wherein the eukaryotic cell is an insect or avian cell.

[0378] 37. The cell of embodiment 30, wherein the eukaryotic cell is a fungal cell.

[0379] 38. The cell of embodiment 30, wherein the eukaryotic cell is a plant cell.

[0380] 39. A plant comprising the cell of embodiment 38.

[0381] 40. A seed comprising the cells of embodiment 38.

[0382] 41. A method for producing an RGN polypeptide, comprising culturing a cell of any one of embodiments 28 to 38 under conditions in which the RGN polypeptide is expressed.

[0383] 42. A method for producing an RGN polypeptide, comprising introducing into a cell a heterologous nucleic acid molecule comprising a nucleotide sequence encoding an RNA-guided nuclease (RGN) polypeptide comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-12; introducing the RGN polypeptide, which when bound to a guide RNA (gRNA) capable of hybridizing to a target DNA sequence, is capable of binding to the target DNA sequence in an RNA-guided sequence-specific manner; and culturing the cells under conditions in which the RGN polypeptide is expressed.

[0384] 43. The method of embodiment 42, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 1-12.

[0385] 44. The method of embodiment 42, wherein the RGN polypeptide comprises an amino acid sequence having 100% sequence identity to any one of SEQ ID NOs: 1-12.

[0386] 45. The method of any one of embodiments 41-44, further comprising purifying the RGN polypeptide.

[0387] 46. ​​The method of any one of embodiments 41-44, wherein the cell further expresses one or more guide RNAs capable of binding to the RGN polypeptide to form an RGN ribonucleoprotein complex.

[0388] 47. The method of embodiment 46, further comprising purifying said RGN ribonucleoprotein complex.

[0389] 48. An RNA-guided nuclease (RGN) polypeptide, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-12; An RNA-guided nuclease (RGN) polypeptide capable of binding to a target DNA sequence of a DNA molecule in an RNA-guided sequence-specific manner when the RGN polypeptide is bound to a guide RNA (gRNA) that can hybridize to the target DNA sequence.

[0390] 49. The RGN polypeptide of embodiment 48, wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 1-12.

[0391] 50. The RGN polypeptide of embodiment 48, wherein the RGN polypeptide comprises an amino acid sequence having 100% sequence identity to any one of SEQ ID NOs: 1-12.

[0392] 51. The RGN polypeptide of any one of embodiments 48-50, wherein said RGN polypeptide is an isolated RGN polypeptide.

[0393] 52. The RGN polypeptide of any one of embodiments 48-51, wherein said RGN polypeptide is capable of cleaving said target DNA sequence upon binding.

[0394] 53. The RGN polypeptide of embodiment 52, wherein cleavage by said RGN polypeptide generates a double-stranded break.

[0395] 54. The RGN polypeptide of embodiment 52, wherein cleavage by said RGN polypeptide generates a single-strand break.

[0396] 55. The RGN polypeptide of any one of embodiments 48-51, wherein the RGN polypeptide is nuclease inactive or is a nickase.

[0397] 56. The RGN polypeptide of any one of embodiments 48-52, 54 and 55, wherein the RGN polypeptide is operably linked to a prime editing polypeptide.

[0398] 57. The RGN polypeptide of embodiment 56, wherein the prime editing polypeptide comprises a DNA polymerase.

[0399] 58. The RGN polypeptide of embodiment 56, wherein the prime editing polypeptide comprises a reverse transcriptase.

[0400] 59. The RGN polypeptide of any one of embodiments 48-55, wherein the RGN polypeptide is operably linked to a base-editing polypeptide.

[0401] 60. The RGN polypeptide of embodiment 59, wherein the base-editing polypeptide is a deaminase.

[0402] 61. The RGN polypeptide of embodiment 60, wherein the deaminase is a cytosine deaminase or an adenine deaminase.

[0403] 62. The RGN polypeptide of embodiment 60, wherein the deaminase has at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 377-448.

[0404] 63. The RGN polypeptide of embodiment 60, wherein the deaminase has 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 377-448.

[0405] 64. The RGN polypeptide of any one of embodiments 48-63, wherein the target DNA sequence is located adjacent to a protospacer adjacent motif (PAM).

[0406] 65. The RGN polypeptide of any one of embodiments 48-64, wherein the RGN polypeptide comprises one or more nuclear localization signals.

[0407] 66. A ribonucleoprotein (RNP) complex comprising the RGN polypeptide of any one of embodiments 48 to 65 and a guide RNA (gRNA) bound to the RGN polypeptide.

[0408] 67. The RNP complex of embodiment 66, wherein the gRNA is a single guide RNA (sgRNA).

[0409] 68. The RNP complex of embodiment 67, wherein the gRNA comprises an extension comprising an editing template for prime editing.

[0410] 69. A nucleic acid molecule comprising a CRISPR RNA (crRNA) or a polynucleotide encoding the crRNA, wherein the crRNA comprises a spacer sequence and a CRISPR repeat sequence, and the CRISPR repeat sequence comprises a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 13-24; a) the crRNA; and b) a trans-activating CRISPR RNA (tracrRNA) hybridized to the CRISPR repeat sequence of the crRNA; and a guide RNA comprising A nucleic acid molecule that can hybridize to a target DNA sequence in a sequence-specific manner via the spacer sequence of the crRNA when the guide RNA is bound to an RNA-guided nuclease (RGN) polypeptide.

[0411] 70. The nucleic acid molecule of embodiment 69, wherein said polynucleotide encoding crRNA is operably linked to a promoter heterologous to said polynucleotide.

[0412] 71. The nucleic acid molecule of embodiment 69 or 70, wherein the CRISPR repeat sequence comprises a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 13-24.

[0413] 72. The nucleic acid molecule of embodiment 69 or 70, wherein the CRISPR repeat sequence comprises a nucleotide sequence having 100% sequence identity to any one of SEQ ID NOs: 13-24.

[0414] 73. A vector comprising the nucleic acid molecule of any one of embodiments 69-72, comprising said polynucleotide encoding said crRNA.

[0415] 74. The vector of embodiment 73, wherein the ve...

Claims

1. A nucleic acid molecule comprising a polynucleotide encoding an RNA-guided nuclease (RGN) polypeptide, wherein the polynucleotide comprises a nucleotide sequence encoding an RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-12; A nucleic acid molecule capable of binding to a target DNA sequence in an RNA-guided sequence-specific manner when the RGN polypeptide is bound to a guide RNA (gRNA) capable of hybridizing to the target DNA sequence.

2. The nucleic acid molecule of claim 1, wherein the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 1-12.

3. The nucleic acid molecule of claim 1 or 2, wherein the polynucleotide encoding an RGN polypeptide is operably linked to a promoter heterologous to the polynucleotide.

4. The nucleic acid molecule according to any one of claims 1 to 3, wherein the RGN polypeptide is capable of cleaving the target DNA sequence upon binding.

5. The nucleic acid molecule of claim 4 , wherein the RGN polypeptide is capable of generating a double-stranded break.

6. The nucleic acid molecule of claim 4 , wherein the RGN polypeptide is capable of generating a single-stranded break.

7. The nucleic acid molecule of any one of claims 1 to 3, wherein the RGN polypeptide is nuclease inactive or is a nickase.

8. The nucleic acid molecule of any one of claims 1 to 7, wherein the RGN polypeptide is operably linked to a prime editing polypeptide.

9. The nucleic acid molecule of claim 8 , wherein the prime editing polypeptide comprises a DNA polymerase.

10. The nucleic acid molecule of claim 8 , wherein the prime editing polypeptide comprises a reverse transcriptase.

11. The nucleic acid molecule of any one of claims 1 to 7, wherein the RGN polypeptide is operably linked to a base-editing polypeptide.

12. The nucleic acid molecule of Claim 11, wherein the base-editing polypeptide is a deaminase.

13. The nucleic acid molecule of claim 12, wherein the deaminase is a cytosine deaminase or an adenine deaminase.

14. The nucleic acid molecule of claim 12, wherein the deaminase has at least 90% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 377-448.

15. The nucleic acid molecule of claim 12, wherein the deaminase has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 377-448.

16. The nucleic acid molecule of any one of claims 1 to 15, wherein the RGN polypeptide comprises one or more nuclear localization signals.

17. The nucleic acid molecule of any one of claims 1 to 16, wherein the RGN polypeptide is codon-optimized for expression in a eukaryotic cell.

18. 18. The nucleic acid molecule of any one of claims 1 to 17, wherein the target DNA sequence is located adjacent to a protospacer adjacent motif (PAM).

19. A vector comprising the nucleic acid molecule according to any one of claims 1 to 18.

20. 20. The vector of claim 19, further comprising at least one nucleotide sequence encoding the gRNA capable of hybridizing to the target DNA sequence.

21. The guide RNA is a) i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 13; ii) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 25, A guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:1; b) i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 14; ii) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 26, A guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:2; c) i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 15; ii) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 27, A guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:3; d) i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 16; ii) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 28, A guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:4; e) i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 17; ii) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 29, A guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:5; f) i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 18; ii) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 30, A guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:6; g) i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 19; ii) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 31, A guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:7; h) i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 20; ii) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 32, A guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:8; i) i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 21; ii) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 33, A guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:9; j) i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 22; ii) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 34, A guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 10; k) i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 23; ii) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 35, a guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 11; and l) i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 24; ii) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 36, 21. The vector of claim 20, wherein the RGN polypeptide is selected from the group consisting of a guide RNA comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

12.

22. The guide RNA is a) i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 13; ii) a tracrRNA having 1000% sequence identity to SEQ ID NO: 25, a guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:1; b) i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 14; ii) a tracrRNA having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 26, A guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:2; c) i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 15; ii) a tracrRNA having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 27, A guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:3; d) i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 16; ii) a tracrRNA having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 28, A guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:4; e) i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 17; ii) a tracrRNA having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:29, A guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:5; f) i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 18; ii) a tracrRNA having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 30, A guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:6; g) i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 19; ii) a tracrRNA having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 31, A guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:7; h) i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 20; ii) a tracrRNA having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 32, A guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:8; i) i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:21; ii) a tracrRNA having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 33, A guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:9; j) i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 22; ii) a tracrRNA having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34, A guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 10; k) i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 23; ii) a tracrRNA having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 35, a guide RNA, wherein the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11; and l) i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:24; ii) a tracrRNA having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 36, 21. The vector of claim 20, wherein the RGN polypeptide is selected from the group consisting of a guide RNA comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

12.

23. 22. The vector of claim 20 or 21, wherein the gRNA is a single guide RNA.

24. 24. The vector of embodiment 23, wherein the sgRNA further comprises an extension comprising an editing template for prime editing.

25. 22. The vector of claim 20 or 21, wherein the gRNA is a dual guide RNA.

26. A cell comprising a nucleic acid molecule according to any one of claims 1 to 18 or a vector according to any one of claims 19 to 25.

27. 27. A method for producing an RGN polypeptide, comprising culturing a cell according to claim 26 under conditions in which the RGN polypeptide is expressed.

28. A method for producing an RGN polypeptide, comprising introducing into a cell a heterologous nucleic acid molecule comprising a nucleotide sequence encoding an RNA-guided nuclease (RGN) polypeptide comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-12; Introducing the RGN polypeptide, which is capable of binding to a target DNA sequence in an RNA-guided sequence-specific manner when bound to a guide RNA (gRNA) capable of hybridizing to the target DNA sequence; and culturing the cell under conditions in which the RGN polypeptide is expressed.

29. 29. The method of claim 28, wherein the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 1-12.

30. The method of any one of claims 27 to 29, further comprising purifying the RGN polypeptide.

31. 30. The method of any one of claims 27 to 29, wherein the cell further expresses one or more guide RNAs capable of binding to the RGN polypeptide to form an RGN ribonucleoprotein complex.

32. 32. The method of claim 31 , further comprising purifying the RGN ribonucleoprotein complex.

33. An RNA-guided nuclease (RGN) polypeptide, the RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-12; An RNA-guided nuclease (RGN) polypeptide capable of binding to a target DNA sequence of a DNA molecule in an RNA-guided sequence-specific manner when the RGN polypeptide is bound to a guide RNA (gRNA) capable of hybridizing to the target DNA sequence.

34. The RGN polypeptide of claim 33, wherein the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 1-12.

35. The RGN polypeptide of claim 33 or 34, wherein the RGN polypeptide is an isolated RGN polypeptide.

36. The RGN polypeptide of any one of claims 33 to 35, wherein the RGN polypeptide is capable of cleaving the target DNA sequence upon binding.

37. The RGN polypeptide of claim 36, wherein cleavage by the RGN polypeptide generates a double-stranded break.

38. The RGN polypeptide of claim 36, wherein cleavage by the RGN polypeptide generates a single-stranded break.

39. The RGN polypeptide of any one of claims 33 to 35, wherein the RGN polypeptide is nuclease inactive or is a nickase.

40. The RGN polypeptide of any one of claims 33 to 39, wherein the RGN polypeptide is operably linked to a prime editing polypeptide.

41. The RGN polypeptide of claim 40, wherein the prime editing polypeptide comprises a DNA polymerase.

42. The RGN polypeptide of claim 40, wherein the prime editing polypeptide comprises a reverse transcriptase.

43. The RGN polypeptide of any one of claims 33 to 39, wherein the RGN polypeptide is operably linked to a base-editing polypeptide.

44. The RGN polypeptide of claim 43, wherein the base-editing polypeptide is a deaminase.

45. 45. The RGN polypeptide of claim 44, wherein the deaminase is a cytosine deaminase or an adenine deaminase.

46. 45. The RGN polypeptide of claim 44, wherein the deaminase has at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 377-448.

47. The RGN polypeptide of claim 44, wherein the deaminase has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 377-448.

48. The RGN polypeptide of any one of claims 33 to 47, wherein the target DNA sequence is located adjacent to a protospacer adjacent motif (PAM).

49. The RGN polypeptide of any one of claims 33 to 48, wherein the RGN polypeptide comprises one or more nuclear localization signals.

50. A ribonucleoprotein (RNP) complex comprising an RGN polypeptide according to any one of claims 33 to 49 and a guide RNA bound to the RGN polypeptide.

51. 1. A system for binding to a target DNA sequence of a DNA molecule, said system comprising: a) one or more guide RNAs capable of hybridizing to the target DNA sequence, or one or more polynucleotides comprising one or more nucleotide sequences encoding the one or more guide RNAs (gRNAs); b) an RNA-guided nuclease (RGN) polypeptide comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1 to 12, or a polynucleotide comprising a nucleotide sequence encoding said RGN polypeptide; the one or more guide RNAs are capable of hybridizing to the target DNA sequence; The one or more guide RNAs are capable of forming a complex with the RGN polypeptide to guide the RGN polypeptide to bind to the target DNA sequence of the DNA molecule.

52. 52. The system of claim 51 , wherein at least one of the nucleotide sequences encoding the one or more guide RNAs and the nucleotide sequence encoding the RGN polypeptide is operably linked to a promoter heterologous to the nucleotide sequence.

53. 53. The system of claim 51 or 52, wherein the nucleotide sequence encoding the one or more guide RNAs and the nucleotide sequence encoding the RGN polypeptide are located on one vector.

54. 1. A system for binding to a target DNA sequence of a DNA molecule, said system comprising: a) one or more guide RNAs capable of hybridizing to the target DNA sequence, or one or more polynucleotides comprising one or more nucleotide sequences encoding the one or more guide RNAs (gRNAs); b) an RNA-guided nuclease (RGN) polypeptide comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-12; the one or more guide RNAs are capable of hybridizing to the target DNA sequence; The one or more guide RNAs are capable of forming a complex with the RGN polypeptide to guide the RGN polypeptide to bind to the target DNA sequence of the DNA molecule.

55. 55. The system of any one of claims 51 to 54, wherein the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 1 to 12.

56. 56. The system of any one of claims 51 to 55, wherein at least one of the nucleotide sequences encoding the one or more guide RNAs is operably linked to a promoter heterologous to the nucleotide sequence.

57. The system of any one of claims 51 to 56, wherein the RGN polypeptide and the one or more guide RNAs are not found complexed to each other in nature.

58. 58. A system according to any one of claims 51 to 57, wherein the target DNA sequence is a eukaryotic target DNA sequence.

59. 59. The system of any one of claims 51 to 58, wherein the gRNA is a single guide RNA (sgRNA).

60. 60. The system of Claim 59, wherein the sgRNA further comprises an extension comprising an editing template for prime editing.

61. 59. The system of any one of claims 51 to 58, wherein the gRNA is a dual guide RNA.

62. The gRNA is a) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 13 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 25, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1; b) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 14 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 26, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2; c) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 15 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 27, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 3; d) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 16 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 28, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 4; e) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 17 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 29, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 5; f) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 18 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 30, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 6; g) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 19 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 31, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 7; h) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 20 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 32, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 8; i) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 21 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 33, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 9; j) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 22 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 34, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 10; k) a gRNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 23 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 35, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 11; and 62. The system of any one of claims 51 to 61, wherein the gRNA comprises a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:24 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:36, wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

12.

63. The gRNA is a) a gRNA comprising a CRISPR repeat sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 13 and a tracrRNA having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 25, wherein the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1; b) a gRNA comprising a CRISPR repeat sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 14 and a tracrRNA having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 26, wherein the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2; c) a gRNA comprising a CRISPR repeat sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 15 and a tracrRNA having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 27, wherein the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 3; d) a gRNA comprising a CRISPR repeat sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 16 and a tracrRNA having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 28, wherein the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 4; e) a gRNA comprising a CRISPR repeat sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 17 and a tracrRNA having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 29, wherein the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 5; f) a gRNA comprising a CRISPR repeat sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 18 and a tracrRNA having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 30, wherein the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 6; g) a gRNA comprising a CRISPR repeat sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 19 and a tracrRNA having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 31, wherein the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 7; h) a gRNA comprising a CRISPR repeat sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:20 and a tracrRNA having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:32, wherein the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:8; i) a gRNA comprising a CRISPR repeat sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:21 and a tracrRNA having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:33, wherein the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:9; j) a gRNA comprising a CRISPR repeat sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 22 and a tracrRNA having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34, wherein the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 10; k) a gRNA comprising a CRISPR repeat sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:23 and a tracrRNA having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:35, wherein the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:11; and 62. The system of any one of claims 51 to 61, wherein the gRNA comprises a CRISPR repeat sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:24 and a tracrRNA having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:36, wherein the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

12.

64. 64. The system of any one of claims 51 to 63, wherein the target DNA sequence is located adjacent to a protospacer adjacent motif (PAM).

65. The system of any one of claims 51 to 64, wherein the target DNA sequence is intracellular.

66. 66. The system of any one of claims 51 to 65, wherein the one or more guide RNAs are capable of hybridizing to the target DNA sequence, and the guide RNAs are capable of forming a complex with the RGN polypeptide to guide cleavage of the target DNA sequence.

67. 67. The system of claim 66, wherein the cleavage generates a double-stranded break.

68. 67. The system of claim 66, wherein the cleavage generates a single-stranded break.

69. The system of any one of claims 51 to 65, wherein the RGN polypeptide is nuclease inactive or is a nickase.

70. The system of any one of claims 51 to 69, wherein the RGN polypeptide is operably linked to a prime editing polypeptide.

71. 71. The system of claim 70, wherein the prime editing polypeptide comprises a DNA polymerase.

72. 71. The system of claim 70, wherein the prime editing polypeptide comprises a reverse transcriptase.

73. The system of any one of claims 51 to 69, wherein the RGN polypeptide is operably linked to a base-editing polypeptide.

74. The system of Claim 73, wherein the base-editing polypeptide is a deaminase.

75. 75. The system of claim 74, wherein the deaminase is a cytosine deaminase or an adenine deaminase.

76. 75. The system of claim 74, wherein the deaminase has at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 377-448.

77. 75. The system of claim 74, wherein the deaminase has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence of any one of SEQ ID NOs: 377-448.

78. The system according to any one of claims 51 to 77, wherein the RGN polypeptide comprises one or more nuclear localization signals.

79. The system of any one of claims 51 to 78, wherein the RGN polypeptide is codon-optimized for expression in eukaryotic cells.

80. The system of any one of claims 51 to 79, further comprising one or more donor polynucleotides.

81. A cell comprising the system according to any one of claims 51 to 80.

82. A pharmaceutical composition comprising a nucleic acid molecule according to any one of claims 1 to 18, a vector according to any one of claims 19 to 25, a cell according to claim 26 or 81, an RGN polypeptide according to any one of claims 33 to 49, an RNP complex according to claim 50, or a system according to any one of claims 51 to 80, and a pharma- ceutically acceptable carrier.

83. 81. A method for binding a DNA molecule to a target DNA sequence, comprising delivering a system according to any one of claims 51 to 80 to said target DNA sequence or to a cell containing said target DNA sequence.

84. 84. The method of claim 83, wherein the RGN polypeptide or the guide RNA further comprises a detectable label, thereby allowing detection of the target DNA sequence.

85. 84. The method of claim 83, wherein the guide RNA or the RGN polypeptide further comprises an expression regulator, thereby regulating expression of the target DNA sequence or a gene under transcriptional control by the target DNA sequence.

86. 58. A method for cleaving and / or modifying a target DNA sequence of a DNA molecule, the method comprising delivering a system according to any one of claims 36 to 57 to said target DNA sequence or to a cell comprising said DNA molecule, whereby cleavage or modification of said target DNA sequence occurs.

87. 87. The method of claim 86, wherein the modified target DNA sequence comprises an insertion of heterologous DNA into the target DNA sequence.

88. 87. The method of claim 86, wherein the modified target DNA sequence comprises a deletion of at least one nucleotide from the target DNA sequence.

89. 87. The method of claim 86, wherein the modified target DNA sequence comprises a mutation of at least one nucleotide in the target DNA sequence.

90. 1. A method for binding to a target DNA sequence of a DNA molecule, comprising: a) i) one or more guide RNAs capable of hybridizing to said target DNA sequence; ii) an RGN polypeptide comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1 to 12; assembling an RNA-guided nuclease (RGN) ribonucleotide complex by combining under conditions suitable for the formation of said RGN ribonucleotide complex; b) contacting the target DNA sequence or a cell containing the target DNA sequence with the assembled RGN ribonucleotide complex; The method, wherein the one or more guide RNAs hybridize to the target DNA sequence, thereby directing the RGN polypeptide to bind to the target DNA sequence.

91. 91. The method of claim 90, wherein the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 1-12.

92. 92. The method of claim 90 or 91, wherein the method is carried out in vitro, in vivo, or ex vivo.

93. The method of any one of claims 90 to 92, wherein the RGN polypeptide or the guide RNA further comprises a detectable label, thereby allowing detection of the target DNA sequence.

94. The method according to any one of claims 90 to 92, wherein the guide RNA or the RGN polypeptide further comprises an expression regulator, thereby allowing regulation of the expression of the target DNA sequence.

95. The method of any one of claims 90 to 92, wherein the RGN polypeptide further comprises a prime editing polypeptide, thereby enabling modification of the target DNA sequence.

96. 96. The method of claim 95, wherein the prime editing polypeptide comprises a DNA polymerase.

97. 96. The method of claim 95, wherein the prime editing polypeptide comprises a reverse transcriptase.

98. The method of any one of claims 90 to 92, wherein the RGN polypeptide further comprises a base-editing polypeptide, thereby enabling modification of the target DNA sequence.

99. 99. The method of Claim 98, wherein the base-editing polypeptide comprises a deaminase.

100. 100. The method of claim 99, wherein the deaminase is a cytosine deaminase or an adenine deaminase.

101. The method according to any one of claims 90 to 92, wherein the RGN polypeptide is capable of cleaving the target DNA sequence, thereby allowing cleavage and / or modification of the target DNA sequence.

102. 1. A method for cleaving and / or modifying a target DNA sequence of a DNA molecule, comprising: a) an RNA-guided nuclease (RGN) polypeptide, wherein the RGN comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-12; and b) contacting the DNA molecule with one or more guide RNAs capable of targeting the RGN of (a) to the target DNA sequence; The method, wherein the one or more guide RNAs hybridize to the target DNA sequence, thereby directing the RGN polypeptide to bind to the target DNA sequence, resulting in cleavage and / or modification of the target DNA sequence.

103. The method of claim 102, wherein the RGN polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 1-12.

104. The method of claim 102 or 103, wherein cleavage by the RGN polypeptide generates a double-stranded break.

105. The method of claim 102 or 103, wherein cleavage by the RGN polypeptide generates a single-stranded break.

106. The method of claim 102 or 103, wherein the RGN polypeptide is a nickase and is operably linked to a prime editing polypeptide.

107. 107. The method of Claim 106, wherein the prime editing polypeptide comprises a DNA polymerase.

108. 107. The method of claim 106, wherein the prime editing polypeptide comprises a reverse transcriptase.

109. The method of claim 102 or 103, wherein the RGN polypeptide is nuclease inactive or a nickase and is operably linked to a base-editing polypeptide.

110. The method of Claim 109, wherein the base-editing polypeptide is a deaminase.

111. 111. The method of claim 110, wherein the deaminase is a cytosine deaminase or an adenine deaminase.

112. The method of claim 110, wherein the deaminase has at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 377-448.

113. The method of claim 110, wherein the deaminase has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 377-448.

114. 104. The method of claim 102 or 103, wherein the modified target DNA sequence comprises an insertion of heterologous DNA into the target DNA sequence.

115. 104. The method of claim 102 or 103, wherein the modified target DNA sequence comprises a deletion of at least one nucleotide from the target DNA sequence.

116. 104. The method of claim 102 or 103, wherein the modified target DNA sequence comprises a mutation of at least one nucleotide in the target DNA sequence.

117. 117. The method of any one of claims 83 to 116, wherein the target DNA sequence is located adjacent to a protospacer adjacent motif (PAM).

118. 118. The method of any one of claims 83 to 117, wherein the target DNA sequence is a eukaryotic target DNA sequence.

119. The method of any one of claims 83 to 118, wherein the gRNA is a single guide RNA (sgRNA).

120. 120. The method of Claim 119, wherein the sgRNA further comprises an extension comprising an editing template for prime editing.

121. The method of any one of claims 83 to 118, wherein the gRNA is a dual guide RNA.

122. a) the RGN has at least 90% sequence identity to SEQ ID NO:1, and the guide RNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO:13, and a tracrRNA having at least 90% sequence identity to SEQ ID NO:25; b) the RGN has at least 90% sequence identity to SEQ ID NO:2, and the guide RNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO:14 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:26; c) the RGN has at least 90% sequence identity to SEQ ID NO:3, and the guide RNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO:15, and a tracrRNA having at least 90% sequence identity to SEQ ID NO:27; d) the RGN has at least 90% sequence identity to SEQ ID NO: 4, and the guide RNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO: 16, and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 28; e) the RGN has at least 90% sequence identity to SEQ ID NO:5, and the guide RNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO:17, and a tracrRNA having at least 90% sequence identity to SEQ ID NO:29; f) the RGN has at least 90% sequence identity to SEQ ID NO:6, and the guide RNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO:18, and a tracrRNA having at least 90% sequence identity to SEQ ID NO:30; g) the RGN has at least 90% sequence identity to SEQ ID NO: 7, and the guide RNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO: 19, and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 31; h) the RGN has at least 90% sequence identity to SEQ ID NO:8, and the guide RNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO:20, and a tracrRNA having at least 90% sequence identity to SEQ ID NO:32; i) the RGN has at least 90% sequence identity to SEQ ID NO:9, and the guide RNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO:21 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:33; j) the RGN has at least 90% sequence identity to SEQ ID NO: 10, and the guide RNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO: 22 and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 34; k) the RGN has at least 90% sequence identity to SEQ ID NO: 11, and the guide RNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO: 23, and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 35; or l) The method of any one of claims 83 to 121, wherein the RGN has at least 90% sequence identity to SEQ ID NO: 12 and the guide RNA comprises a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO: 24 and a tracrRNA having at least 90% sequence identity to SEQ ID NO:

36.

123. a) the RGN has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:1, and the guide RNA comprises a crRNA repeat sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:13, and a tracrRNA having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:25; b) the RGN has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:2, and the guide RNA comprises a crRNA repeat sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:14, and a tracrRNA having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:26; c) the RGN has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:3, and the guide RNA comprises a crRNA repeat sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:15, and a tracrRNA having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:27; d) the RGN has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:4, and the guide RNA comprises a crRNA repeat sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:16, and a tracrRNA having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:28; e) the RGN has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:5, and the guide RNA comprises a crRNA repeat sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:17, and a tracrRNA having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:29; f) the RGN has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:6, and the guide RNA comprises a crRNA repeat sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:18, and a tracrRNA having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:30; g) the RGN has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:7, and the guide RNA comprises a crRNA repeat sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:19, and a tracrRNA having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:31; h) the RGN has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:8, and the guide RNA comprises a crRNA repeat sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:20, and a tracrRNA having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:32; i) the RGN has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:9, and the guide RNA comprises a crRNA repeat sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:21, and a tracrRNA having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:33; j) the RGN has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 10, and the guide RNA comprises a crRNA repeat sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 22, and a tracrRNA having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34; k) the RGN has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11, and the guide RNA comprises a crRNA repeat sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 23, and a tracrRNA having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 35, or l) The method of any one of claims 83 to 121, wherein the RGN has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 12, and the guide RNA comprises a crRNA repeat sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 24, and a tracrRNA having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

36.

124. 124. The method of any one of claims 83 to 123, wherein the target DNA sequence is within a cell.

125. The method of claim 124, further comprising culturing the cells under conditions in which the RGN polypeptide is expressed and cleaves and modifies the target DNA sequence to produce a DNA molecule containing the modified target DNA sequence, and selecting cells containing the modified target DNA sequence.

126. A cell comprising a target DNA sequence modified by the method of claim 125.

127. A pharmaceutical composition comprising a cell described in any one of claims 26, 81 and 126 and a pharma- ceutically acceptable carrier.

128. 133. A method of treating a disease, disorder, or condition, the method comprising administering to a subject in need of treatment for the disease, disorder, or condition an effective amount of the pharmaceutical composition of claim 82 or 127.

129. The method of claim 128, wherein the disease is associated with a causative mutation and the effective amount of the pharmaceutical composition corrects the causative mutation.

130. 130. The method of claim 128 or 129, wherein the subject is at risk of developing the disease, disorder, or condition.

131. A nucleic acid molecule comprising a CRISPR RNA (crRNA) or a polynucleotide encoding a crRNA, wherein the crRNA comprises a spacer sequence and a CRISPR repeat sequence, and the CRISPR repeat sequence comprises a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 13-24; a) the crRNA; b) a trans-activating CRISPR RNA (tracrRNA) hybridized to the CRISPR repeat sequence of the crRNA; and A nucleic acid molecule capable of sequence-specifically hybridizing to a target DNA sequence via the spacer sequence of the crRNA when the guide RNA is bound to an RNA-guided nuclease (RGN) polypeptide.

132. The nucleic acid molecule of Claim 131, wherein the polynucleotide encoding the crRNA is operably linked to a promoter heterologous to the polynucleotide.

133. 133. The nucleic acid molecule of claim 131 or 132, wherein the CRISPR repeat sequence comprises a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 13-24.

134. A vector comprising the nucleic acid molecule of any one of claims 131 to 133, comprising the polynucleotide encoding the crRNA.

135. The vector of claim 134, wherein the vector further comprises a polynucleotide encoding the tracrRNA.

136. The tracrRNA is a) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 25, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 13; b) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 26, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 14; c) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 27, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 15; d) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 28, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 16; e) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 29, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 17; f) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 30, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 18; g) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 31, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 19; h) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 32, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 20; i) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 33, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 21; j) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 34, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 22; k) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 35, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 23; and l) a tracrRNA having at least 90% sequence identity to SEQ ID NO: 36, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO:

24. The vector of claim 135,

137. The tracrRNA is a) a tracrRNA having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 25, wherein the CRISPR repeat sequence has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 13; b) a tracrRNA having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 26, wherein the CRISPR repeat sequence has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 14; c) a tracrRNA having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 27, wherein the CRISPR repeat sequence has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 15; d) a tracrRNA having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 28, wherein the CRISPR repeat sequence has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 16; e) a tracrRNA having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 29, wherein the CRISPR repeat sequence has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 17; f) a tracrRNA having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 30, wherein the CRISPR repeat sequence has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 18; g) a tracrRNA having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 31, wherein the CRISPR repeat sequence has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 19; h) a tracrRNA having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 32, wherein the CRISPR repeat sequence has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 20; i) a tracrRNA having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 33, wherein the CRISPR repeat sequence has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 21; j) a tracrRNA having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34, wherein the CRISPR repeat sequence has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 22; k) a tracrRNA having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 35, wherein the CRISPR repeat sequence has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 23; and 136. The vector of claim 135, wherein the vector is selected from the group consisting of: l) a tracrRNA having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 36, wherein the CRISPR repeat sequence has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

24.

138. The vector according to any one of claims 134 to 137, wherein the vector further comprises a polynucleotide encoding the RGN polypeptide.

139. The RGN polypeptide is a) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO:1, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO:13, and the tracrRNA has at least 90% sequence identity to SEQ ID NO:25; b) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO:2, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO:14, and the tracrRNA has at least 90% sequence identity to SEQ ID NO:26; c) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO:3, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO:15, and the tracrRNA has at least 90% sequence identity to SEQ ID NO:27; d) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 4, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 16, and the tracrRNA has at least 90% sequence identity to SEQ ID NO: 28; e) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO:5, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO:17, and the tracrRNA has at least 90% sequence identity to SEQ ID NO:29; f) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO:6, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO:18, and the tracrRNA has at least 90% sequence identity to SEQ ID NO:30; g) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO:7, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO:19, and the tracrRNA has at least 90% sequence identity to SEQ ID NO:31; h) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO:8, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO:20, and the tracrRNA has at least 90% sequence identity to SEQ ID NO:32; i) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO:9, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO:21, and the tracrRNA has at least 90% sequence identity to SEQ ID NO:33; j) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 10, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 22, and the tracrRNA has at least 90% sequence identity to SEQ ID NO: 34; k) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 11, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 23, and the tracrRNA has at least 90% sequence identity to SEQ ID NO: 35; and l) an RGN polypeptide having at least 90% sequence identity to SEQ ID NO: 12, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 24, and the tracrRNA has at least 90% sequence identity to SEQ ID NO:

36. The vector of claim 138, wherein the RGN polypeptide has at least 90% sequence identity to SEQ ID NO: 12, wherein the CRISPR repeat sequence has at least 90% sequence identity to SEQ ID NO: 24, and the tracrRNA has at least 90% sequence identity to SEQ ID NO:

36.

140. The RGN polypeptide is a) an RGN polypeptide having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:1, wherein the CRISPR repeat sequence has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:13, and the tracrRNA has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:25; b) an RGN polypeptide having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:2, wherein the CRISPR repeat sequence has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:14, and the tracrRNA has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:26; c) an RGN polypeptide having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:3, wherein the CRISPR repeat sequence has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:15, and the tracrRNA has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:27; d) an RGN polypeptide having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:4, wherein the CRISPR repeat sequence has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:16, and the tracrRNA has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:28; e) an RGN polypeptide having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:5, wherein the CRISPR repeat sequence has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:17, and the tracrRNA has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:29; f) an RGN polypeptide having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:6, wherein the CRISPR repeat sequence has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:18, and the tracrRNA has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:30; g) an RGN polypeptide having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:7, wherein the CRISPR repeat sequence has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:19, and the tracrRNA has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:31; h) an RGN polypeptide having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:8, wherein the CRISPR repeat sequence has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:20, and the tracrRNA has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:32; i) an RGN polypeptide having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:9, wherein the CRISPR repeat sequence has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:21, and the tracrRNA has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:33; j) an RGN polypeptide having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 10, wherein the CRISPR repeat sequence has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 22, and the tracrRNA has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34; k) an RGN polypeptide having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11, wherein the CRISPR repeat sequence has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 23, and the tracrRNA has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 35; and 1) an RGN polypeptide having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 12, wherein the CRISPR repeat sequence has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 24, and the tracrRNA has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

36.

141. A nucleic acid molecule comprising a trans-activating CRISPR RNA (tracrRNA) or a polynucleotide encoding a tracrRNA comprising a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 25-36, a) the tracrRNA; and b) a crRNA comprising a spacer sequence and a CRISPR repeat sequence, wherein the tracrRNA hybridizes to the CRISPR repeat sequence of the crRNA; and A nucleic acid molecule capable of sequence-specifically hybridizing to a target DNA sequence via the spacer sequence of the crRNA when the guide RNA is bound to an RNA-guided nuclease (RGN) polypeptide.

142. The nucleic acid molecule of Claim 141, wherein the polynucleotide encoding the tracrRNA is operably linked to a promoter heterologous to the polynucleotide.

143. The nucleic acid molecule of claim 141 or 142, wherein the tracrRNA comprises a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 25-36.

144. 144. A vector comprising the nucleic acid molecule of any one of claims 141 to 143, comprising the polynucleotide encoding the tracrRNA.

145. A single guide RNA comprising the nucleic acid molecule of any one of claims 131 to 133 comprising the crRNA and the nucleic acid molecule of any one of claims 141 to 143 comprising the tracrRNA.

146. A dual guide RNA comprising the nucleic acid molecule of any one of claims 131-133 comprising the crRNA and the nucleic acid molecule of any one of claims 141-143 comprising the tracrRNA.

147. A cell comprising a nucleic acid molecule according to any one of claims 131 to 133 and 141 to 143, a vector according to any one of claims 134 to 140 and 144, a single guide RNA according to claim 145, or a dual guide RNA according to claim 146.

Images