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

Abstract

Compositions and methods for binding to a target sequence of interest are provided. The compositions may be used to cleave or modify a target sequence of interest, visualize a target sequence of interest, and modify expression of a target sequence of interest. The composition comprises a RNA (Ribonucleic Acid) guided nuclease (RGN) polypeptide, a CRISPR RNA, a transcriptionally activated CRISPR RNA, a guide RNA and a nucleic acid molecule for coding the RNA guided nuclease (RGN) polypeptide, the CRISPR RNA, the transcriptionally activated CRISPR RNA and the guide RNA. Also provided are host cells and vectors comprising the nucleic acid molecules. Also provided is an RGN system for binding to a target sequence of interest, wherein the RGN system comprises an RNA-guided nuclease polypeptide and one or more guide RNAs.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 302,271 filed on January 24, 2022, and U.S. Provisional Application No. 63 / 386,061 filed on December 5, 2022, each of which is incorporated herein by reference in its entirety.

[0003] Reference to a sequence listing submitted electronically as an XML file

[0004] This application contains the xml format that has been submitted by the USPTO Patent Center 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. Technical Field

[0005] The present invention is related to the fields of molecular biology and gene editing. Background Art

[0006] Targeted genome editing or modification is rapidly becoming an important tool for basic and applied research. The initial method involves engineered nucleases such as meganuclease, zinc finger fusion protein or TALEN, and it is necessary to produce chimeric nucleases with engineered, programmable, sequence-specific DNA binding domains specific to each specific target sequence. RNA-guided nucleases (such as CRISPR-Cas proteins of clustered regularly spaced short palindromic repeats (CRISPR)-associated (Cas) bacterial systems) allow targeting specific sequences by compounding nucleases with guide RNAs (guide RNAs hybridize specifically with specific target sequences). Compared to producing chimeric nucleases for each target sequence, the cost of producing target-specific guide RNAs is lower and more efficient. This RNA-guided nuclease can be used to selectively edit the genome via the introduction of sequence-specific double-strand breaks, which are repaired via error-prone non-homologous end joining (NHEJ) to introduce mutations at specific genomic locations. Alternatively, heterologous DNA can be introduced into genomic sites via homology-directed repair. RNA-guided nucleases (RGNs) can also be used for base editing when fused to deaminases. Summary of the invention

[0007] Compositions and methods for binding to a target sequence of interest are provided. Compositions can be used to cut or modify a target sequence of interest, detect a target sequence of interest, and modify the expression of a sequence of interest. Compositions include RNA-guided nuclease (RGN) polypeptides, CRISPR RNA (crRNA), transcriptionally activated CRISPR RNA (tracrRNA), guide RNA (gRNA), nucleic acid molecules encoding the RGN polypeptides, crRNA, tracrRNA, gRNA, and host cells and vectors including nucleic acid molecules. Also provided is an RGN system for binding to a target sequence of interest, wherein the RGN system includes RNA-guided nuclease polypeptides and one or more guide RNAs. Therefore, the method disclosed herein is described as binding to a target sequence of interest, and in some embodiments, cutting or modifying a target sequence of interest. The target sequence of interest may be modified, for example, due to non-homologous end joining, homology-directed repair of an introduced donor sequence, or base editing.

[0008] 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 the 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; wherein, when bound to a guide RNA (gRNA) capable of hybridizing to a target DNA sequence, the RGN polypeptide is capable of binding to the target DNA sequence in an RNA-guided sequence-specific manner. 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 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.

[0009] In some embodiments of the above aspects, the polynucleotide encoding the RGN polypeptide is operably linked to a promoter that is heterologous to the polynucleotide.

[0010] In some embodiments of the above aspects, the RGN polypeptide is capable of cleaving a target DNA sequence upon binding.

[0011] In some embodiments of the above aspects, the RGN polypeptide is capable of producing a double-stranded break.

[0012] In some embodiments of the above aspects, the RGN polypeptide is capable of producing single-strand breaks.

[0013] In some embodiments of the above aspects, the RGN polypeptide is nuclease-inactive or is a nickase.

[0014] 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 has at least 90% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 377-448. In some embodiments of the above aspects, the deaminase is a cytosine deaminase or an adenine deaminase. 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 with the amino acid sequence of any one of SEQ ID NOs: 377-448.

[0015] In some embodiments of the above aspects, the RGN polypeptide comprises one or more nuclear localization signals.

[0016] In some embodiments of the above aspects, the RGN polypeptide is codon-optimized for expression in eukaryotic cells.

[0017] In some embodiments of the above aspects, the target DNA sequence is positioned adjacent to a protospacer adjacent motif (PAM).

[0018] In another aspect, the present disclosure provides a vector comprising the nucleic acid molecule described above.

[0019] 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.

[0020] In some embodiments of the above aspects, the guide RNA is selected from the group consisting of: a) a guide RNA comprising a CRISPR RNA and a tracrRNA, the CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 13, the 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 guide RNA comprising a CRISPR RNA and a tracrRNA, the CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 14, the 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 guide RNA comprising a CRISPR RNA and a tracrRNA, the CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 15, the 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: 28. NO:3 has an amino acid sequence with at least 90% sequence identity; d) a guide RNA comprising a CRISPR RNA and a tracrRNA, the CRISPR RNA comprising a CRISPR repeat sequence with at least 90% sequence identity to SEQ ID NO:16, the tracrRNA has at least 90% sequence identity to SEQ ID NO:28, wherein the RGN polypeptide comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO:4; e) a guide RNA comprising a CRISPR RNA and a tracrRNA, the CRISPR RNA comprising a CRISPR repeat sequence with at least 90% sequence identity to SEQ ID NO:17, the tracrRNA has at least 90% sequence identity to SEQ ID NO:29, wherein the RGN polypeptide comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO:5; f) a guide RNA comprising a CRISPR RNA and a tracrRNA, the CRISPR RNA comprising a CRISPR repeat sequence with at least 90% sequence identity to SEQ ID NO:18, the tracrRNA has at least 90% sequence identity to SEQ ID NO:30, wherein the RGN polypeptide comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO:4 IDNO:6 has an amino acid sequence with at least 90% sequence identity;g) a guide RNA comprising a CRISPR RNA and a tracrRNA, the CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 19, the 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 and a tracrRNA, the CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 20, the 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 and a tracrRNA, the CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 21, the 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: NO:9 having at least 90% sequence identity; j) a guide RNA comprising a CRISPR RNA and a tracrRNA, the CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:22, the 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; a guide RNA comprising a CRISPR RNA and a tracrRNA, the CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:23, the 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 l) a guide RNA comprising a CRISPR RNA and a tracrRNA, the CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:24, the 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: NO:12 has an amino acid sequence with at least 90% sequence identity. ;

[0021] In some embodiments of the above aspects, the guide RNA is selected from the group consisting of: a) a guide RNA comprising a CRISPR RNA and a tracrRNA, the CRISPR RNA 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, the 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, 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: 1; b) a guide RNA comprising a CRISPR The invention further comprises a CRISPR RNA and a guide RNA for tracrRNA, the CRISPR RNA 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: 14, the 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 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: 2; c) a guide RNA comprising a CRISPR RNA and a tracrRNA, the CRISPR RNA 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: 26 NO:15 has 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, 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: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 guide RNA comprising a CRISPR RNA and a tracrRNA, the CRISPR RNA 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: 16, the 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 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: 4; e) a guide RNA comprising a CRISPR RNA and a tracrRNA, the CRISPR RNA 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: 28 NO:17 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, 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:29, 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 guide RNA comprising a CRISPR RNA and a tracrRNA, the CRISPR RNA 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:29 NO:18 has 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, 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, 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:6;g) a guide RNA comprising a CRISPR RNA and a tracrRNA, the CRISPR RNA 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: 19, the 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 guide RNA comprising a CRISPR RNA and a tracrRNA, the CRISPR RNA 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: 31. NO:20 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, 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, 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 guide RNA comprising a CRISPR RNA and a tracrRNA, the CRISPR RNA comprising NO:21 has 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, 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: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 guide RNA comprising a CRISPR RNA and a tracrRNA, the CRISPR RNA 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:22, the 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 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:10; k) a guide RNA comprising a CRISPR RNA and a tracrRNA, the CRISPR RNA 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:34. NO:23 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, 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: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 1) a guide RNA comprising a CRISPR RNA and a tracrRNA, the CRISPR RNA comprising NO:24 has 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, 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, wherein the RGN polypeptide comprises an amino acid 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:12.;

[0022] In some embodiments of the above aspects, the gRNA is a single guide RNA. In some embodiments, the single guide RNA further comprises an extension comprising an editing template for lead editing.

[0023] In some embodiments of the above aspects, the gRNA is a dual-guide RNA.

[0024] In another aspect, the disclosure provides a cell comprising the nucleic acid molecule described above or the vector described above. In yet another aspect, the disclosure provides a method for producing an RGN polypeptide, comprising: culturing the cell described above under conditions where the RGN polypeptide is expressed.

[0025] In another aspect, the present disclosure provides a method for making an RGN polypeptide, comprising introducing a heterologous nucleic acid molecule into a cell, the heterologous nucleic acid molecule comprising a nucleotide sequence encoding 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; wherein, when combined with a guide RNA (gRNA) capable of hybridizing to a target DNA sequence, the RGN polypeptide is capable of binding to the target DNA sequence in an RNA-guided sequence-specific manner; and culturing the cell under conditions where 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.

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

[0027] In some embodiments of the above aspects, the cell further expresses one or more guide RNAs capable of binding to the RGN polypeptide to form a RGN ribonucleoprotein complex.

[0028] In some embodiments of the above aspects, the method further comprises purifying the RGN ribonucleoprotein complex.

[0029] 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, when bound to a guide RNA (gRNA) capable of hybridizing to a target DNA sequence of a DNA molecule, the RGN polypeptide is capable of binding to the target DNA sequence in an RNA-guided sequence-specific manner. 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.

[0030] In some embodiments of the above aspects, the RGN polypeptide is capable of cleaving a target DNA sequence upon binding.

[0031] In some embodiments of the above aspects, double-stranded breaks are produced by cleavage of the RGN polypeptide.

[0032] In some embodiments of the above aspects, single-strand breaks are produced by cleavage of the RGN polypeptide.

[0033] In some embodiments of the above aspects, the RGN polypeptide is nuclease-inactive or is a nickase.

[0034] In some embodiments of the above aspects, the RGN polypeptide is operably linked to a lead editing polypeptide. In some embodiments, the lead editing polypeptide comprises a DNA polymerase. In some embodiments, the lead editing polypeptide comprises a reverse transcriptase.

[0035] 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 an adenine 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 the amino acid sequence of any one of SEQ ID NOs: 377-448.

[0036] In some embodiments of the above aspects, the target DNA sequence is positioned adjacent to a protospacer adjacent motif (PAM).

[0037] In some embodiments of the above aspects, the RGN polypeptide comprises one or more nuclear localization signals.

[0038] In another aspect, the present disclosure provides a ribonucleoprotein (RNP) complex comprising the RGN polypeptide described above 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, wherein the extension comprises an editing template for lead editing. In yet another aspect, the present disclosure provides a system for binding to a target DNA sequence of a DNA molecule, wherein the system comprises: a) one or more guide RNAs capable of hybridizing to a 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, or a polynucleotide comprising a nucleotide sequence encoding an RGN polypeptide;

[0039] One or more guide RNAs are capable of hybridizing to a target DNA sequence, and one or more guide RNAs are capable of forming a complex with an RGN polypeptide to guide the RGN polypeptide to bind to the target DNA sequence of a DNA molecule.

[0040] 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.

[0041] In some embodiments of the above aspects, at least one of the nucleotide sequence encoding the RGN polypeptide, and the nucleotide sequence encoding the one or more guide RNAs is operably linked to a promoter that is heterologous to the nucleotide sequence.

[0042] 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 one vector.

[0043] In yet another aspect, the present disclosure provides a system for binding a target DNA sequence of a DNA molecule, wherein the system comprises: 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); 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 wherein 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.

[0044] 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.

[0045] 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.

[0046] In some embodiments of the above aspects, at least one nucleotide sequence encoding one or more guide RNAs is operably linked to a promoter that is heterologous to the nucleotide sequence.

[0047] In some embodiments of the above aspects, the RGN polypeptide and the one or more guide RNAs are not found complexed with each other in nature.

[0048] In some embodiments of the above aspects, the target DNA sequence is a eukaryotic target DNA sequence.

[0049] In some embodiments of the above aspects, the gRNA is a single guide RNA (sgRNA). In some embodiments, the sgRNA further comprises an extension comprising an editing template for prime editing.

[0050] In some embodiments of the above aspects, the gRNA is a dual-guide RNA.

[0051] In some embodiments of the above aspects, the gRNA is selected from the group consisting of: 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: 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 NO:16 having at least 90% sequence identity, and a gRNA for 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) comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:17, and a gRNA for 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) comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:18, and a gRNA for 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) comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO:19, and a gRNA for tracrRNA having at least 90% sequence identity to SEQ ID NO: NO:31. a gRNA to a tracrRNA having at least 90% sequence identity, 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 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: NO:35 has a tracrRNA with at least 90% sequence identity, wherein the RGN polypeptide comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO:11; and 1) comprises a CRISPR repeat sequence with at least 90% sequence identity to SEQ ID NO:24, and a gRNA with a tracrRNA with at least 90% sequence identity to SEQ ID NO:36, wherein the RGN polypeptide comprises 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 with at least 90% sequence identity to SEQ ID NO:30. ;

[0052] In some embodiments of the above aspects, the gRNA is selected from the group consisting of: a) a gRNA 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, 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, 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: 1; b) 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: 25. NO:14 has 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, and a gRNA for 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:26, wherein the RGN polypeptide comprises an amino acid 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:2; c) comprises an amino acid 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:2 ID NO:15 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, and a gRNA for 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 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: 16, and a gRNA having 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 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: 4; 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: 28, ID NO:17 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, and a gRNA for 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 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) 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:5; NO:18 having 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, and a gRNA for 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 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;g) 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: 19, and a gRNA having 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 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 NO:20 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, and a gRNA for 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) 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:8; ID NO:21 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, and a gRNA for 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) 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:22, and 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:34, 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:10; k) 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:34 ID NO:23 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, and a gRNA for 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 1) a gRNA 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: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. 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. ;

[0053] In some embodiments of the above aspects, the target DNA sequence is positioned adjacent to a protospacer adjacent motif (PAM).

[0054] In some embodiments of the above aspects, the target DNA sequence is within a cell.

[0055] In some embodiments of the above aspects, the 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 the RGN polypeptide to guide cleavage of the target DNA sequence.

[0056] In some embodiments of the above aspects, the cleavage produces a double-stranded break.

[0057] In some embodiments of the above aspects, the cleavage produces a single strand break.

[0058] In some embodiments of the above aspects, the RGN polypeptide is nuclease-inactive or is a nickase.

[0059] In some embodiments of the above aspects, the RGN polypeptide is operably linked to a lead editing polypeptide. In some embodiments, the lead editing polypeptide comprises a DNA polymerase. In some embodiments, the lead editing polypeptide comprises a reverse transcriptase.

[0060] In some embodiments of the above aspects, the RGN polypeptide is operably linked to a base editing polypeptide.

[0061] In some embodiments of the above aspects, the base editing polypeptide is a deaminase. In some embodiments, the deaminase is a cytosine deaminase or an adenine 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 the amino acid sequence of any one of SEQ ID NOs: 377-448.

[0062] In some embodiments of the above aspects, the RGN polypeptide comprises one or more nuclear localization signals.

[0063] In some embodiments of the above aspects, the RGN polypeptide is codon-optimized for expression in eukaryotic cells.

[0064] In some embodiments of the above aspects, the system further comprises one or more donor polynucleotides.

[0065] In another aspect, the present disclosure provides a cell comprising the system described above.

[0066] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising the nucleic acid molecule, vector, cell, RGN polypeptide, RNP complex, or system described above, and a pharmaceutically acceptable carrier.

[0067] In yet another aspect, the present disclosure provides a method for binding a target DNA sequence of a DNA molecule, comprising delivering the system described above to the target DNA sequence or a cell comprising the target DNA sequence.

[0068] 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.

[0069] In some embodiments of the above aspects, the guide RNA or RGN polypeptide further comprises an expression regulator, thereby regulating the expression of the target DNA sequence or the expression of a gene under the transcriptional control of the target DNA sequence.

[0070] 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 system described above to the target DNA sequence or a cell comprising the DNA molecule, wherein cleavage or modification of the target DNA sequence occurs.

[0071] In some embodiments of the above aspects, the modified target DNA sequence comprises insertion of heterologous DNA into the target DNA sequence.

[0072] In some embodiments of the above aspects, the modified target DNA sequence comprises a deletion of at least one nucleotide in the target DNA sequence.

[0073] 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.

[0074] In another aspect, the present disclosure provides a method for binding a target DNA sequence of a DNA molecule, comprising: a) assembling an RNA-guided nuclease (RGN) ribonucleotide complex by combining one or more guide RNAs capable of hybridizing to a target DNA sequence and 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 forming 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 guiding 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.

[0075] 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.

[0076] In some embodiments of the above aspects, the method is performed in vitro, in vivo, or ex vivo.

[0077] 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.

[0078] In some embodiments of the above aspects, the guide RNA or RGN polypeptide further comprises an expression regulator, thereby allowing regulation of expression of the target DNA sequence.

[0079] In some embodiments of the above aspects, the RGN polypeptide is operably linked to a lead editing polypeptide, thereby allowing modification of a target DNA sequence. In some embodiments, the lead editing polypeptide comprises a DNA polymerase. In some embodiments, the lead editing polypeptide comprises a reverse transcriptase.

[0080] In some embodiments of the above aspects, the RGN polypeptide is operably linked to a base editing polypeptide, thereby allowing modification of a target DNA sequence. In some embodiments, the base editing polypeptide comprises a deaminase. In some embodiments, the deaminase is a cytosine deaminase or an adenine deaminase. In some embodiments of the above aspects, the RGN polypeptide is capable of cleaving a target DNA sequence, thereby allowing cleavage and / or modification of a target DNA sequence.

[0081] In another aspect, the present disclosure provides a method for cleaving and / or modifying a target DNA sequence of a DNA molecule, comprising contacting the DNA molecule with: 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) one or more guide RNAs capable of targeting the RGN of (a) to a target DNA sequence, wherein the one or more guide RNAs hybridize to the target DNA sequence, thereby guiding the RGN polypeptide to bind to the target DNA sequence, and cleavage and / or modification of the target DNA sequence occurs. In some embodiments, the RGN polypeptide has at least 90% sequence identity to SEQ ID NO: 6.

[0082] 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.

[0083] In some embodiments of the above aspects, double-stranded breaks are produced by cleavage of the RGN polypeptide.

[0084] In some embodiments of the above aspects, single-strand breaks are produced by cleavage of the RGN polypeptide.

[0085] In some embodiments of the above aspects, the RGN polypeptide is nuclease-inactive or is a nickase.

[0086] In some embodiments of the above aspects, the RGN polypeptide is operably linked to a lead editing polypeptide, thereby allowing modification of a target DNA sequence. In some embodiments, the lead editing polypeptide comprises a DNA polymerase. In some embodiments, the lead editing polypeptide comprises a reverse transcriptase.

[0087] In some embodiments of the above aspects, the RGN polypeptide is operably linked to a base editing polypeptide.

[0088] In some embodiments of the above aspects, the base editing polypeptide is a deaminase. In some embodiments, the deaminase is a cytosine deaminase or an adenine 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 the amino acid sequence of any one of SEQ ID NOs: 377-448.

[0089] In some embodiments of the above aspects, the modified target DNA sequence comprises insertion of heterologous DNA into the target DNA sequence.

[0090] In some embodiments of the above aspects, the modified target DNA sequence comprises a deletion of at least one nucleotide in the target DNA sequence.

[0091] 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.

[0092] In some embodiments of the above aspects, the target DNA sequence is positioned adjacent to a protospacer adjacent motif (PAM).

[0093] In some embodiments of the above aspects, the target DNA sequence is a eukaryotic target DNA sequence.

[0094] In some embodiments of the above aspects, the gRNA is a single guide RNA (sgRNA). In some embodiments, the sgRNA further comprises an extension comprising an editing template for prime editing.

[0095] In some embodiments of the above aspects, the gRNA is a dual-guide RNA.

[0096] 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) an RGN having at least 90% sequence identity to SEQ ID NO: 3, a guide RNA comprising 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) an RGN having at least 90% sequence identity to SEQ ID NO: 4, a guide RNA comprising a crRNA repeat sequence having at least 90% sequence identity to SEQ ID NO: 28, and a tracrRNA having at least 90% sequence identity to SEQ ID NO: 29; NO:16 has a crRNA repeat sequence with at least 90% sequence identity, and a tracrRNA with at least 90% sequence identity to SEQ ID NO:28; e) an RGN with at least 90% sequence identity to SEQ ID NO:5, a guide RNA comprising a crRNA repeat sequence with at least 90% sequence identity to SEQ ID NO:17, and a tracrRNA with at least 90% sequence identity to SEQ ID NO:29; f) an RGN with at least 90% sequence identity to SEQ ID NO:6, a guide RNA comprising a crRNA repeat sequence with at least 90% sequence identity to SEQ ID NO:18, and a tracrRNA with at least 90% sequence identity to SEQ ID NO:30; g) an RGN with at least 90% sequence identity to SEQ ID NO:7, a guide RNA comprising a crRNA repeat sequence with at least 90% sequence identity to SEQ ID NO:19, and a tracrRNA with at least 90% sequence identity to SEQ ID NO:31; h) an RGN with at least 90% sequence identity to SEQ ID NO:5, a guide RNA comprising a crRNA repeat sequence with at least 90% sequence identity to SEQ ID NO:17, and a tracrRNA with at least 90% sequence identity to SEQ ID NO:29; NO:8 has a RGN with at least 90% sequence identity, a guide RNA comprising a crRNA repeat sequence with at least 90% sequence identity to SEQ ID NO:20, and a tracrRNA with at least 90% sequence identity to SEQ ID NO:32;i) an RGN having at least 90% sequence identity to SEQ ID NO:9, a guide RNA comprising 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) 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: NO:36 has at least 90% sequence identity to tracrRNA; In some embodiments, RGN has at least 90% sequence identity to SEQ ID NO:6, and the guide RNA includes 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. ;

[0097] 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 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, 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: 13, 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) 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: NO:2 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, 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; c) 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:14. NO:3 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, 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, 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;d) 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:4, 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: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; e) 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:4, 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:16 NO:5 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, 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 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; f) 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:17; NO:6 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, 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: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;g) 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: 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; h) 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: 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: NO:8 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, 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 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; i) 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: NO:9 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, 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: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%, at least 99%, or 100% sequence identity to SEQ ID NO:33;j) 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: 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 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; k) 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: 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: NO:11 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, 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: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 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:23. NO:12 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, 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: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;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 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. ;

[0098] In some embodiments of the above aspects, the target DNA sequence is within a cell.

[0099] In some embodiments of the above aspects, the method further comprises: culturing the cells under conditions where 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 cells comprising the modified target DNA sequence.

[0100] In another aspect, the present disclosure provides a cell comprising a modified target DNA sequence according to the method described above.

[0101] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising the cell described above and a pharmaceutically acceptable carrier.

[0102] In yet another aspect, the present disclosure provides a method of treating a disease, disorder or symptom, comprising administering to a subject in need of treatment an effective amount of the pharmaceutical composition described above.

[0103] In some embodiments of the above aspects, the disease is associated with a causal mutation, and the effective amount of the pharmaceutical composition corrects or deletes the causal mutation.

[0104] In some embodiments of the above aspects, the individual is at risk for developing a disease, disorder, or symptom.

[0105] In another aspect, the present disclosure provides 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, wherein the CRISPR repeat sequence comprises a nucleotide sequence having at least 90% sequence identity with any one of SEQ ID NOs: 13-24; wherein, when the guide RNA binds to an RNA-guided nuclease (RGN) polypeptide, the guide RNA (including the crRNA; and a transcriptionally activated CRISPR RNA (tracrRNA) hybridized with the CRISPR repeat sequence of the crRNA) can hybridize with a target DNA sequence in a sequence-specific manner via the spacer sequence of the crRNA. In some embodiments, the CRISPR repeat sequence comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 18.

[0106] In some embodiments of the above aspects, the polynucleotide encoding the crRNA is operably linked to a promoter that is heterologous to the polynucleotide.

[0107] 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 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.

[0108] In another aspect, the present disclosure provides a vector comprising the nucleic acid molecule described above comprising a polynucleotide encoding crRNA.

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

[0110] 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: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:2 NO:30) a tracrRNA having at least 90% sequence identity, 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) a tracrRNA having at least 90% sequence identity to SEQ ID NO:19; h) a tracrRNA having at least 90% sequence identity to SEQ ID NO:32) a tracrRNA having at least 90% sequence identity to SEQ ID NO:20; i) a tracrRNA having at least 90% sequence identity to SEQ ID NO:33) a tracrRNA having at least 90% sequence identity to SEQ ID NO:21; j) a tracrRNA having at least 90% sequence identity to SEQ ID NO:34) a tracrRNA having at least 90% sequence identity to SEQ ID NO:22; k) a tracrRNA having at least 90% sequence identity to SEQ ID NO:35) a tracrRNA having at least 90% sequence identity to SEQ ID NO:36) a tracrRNA having at least 90% sequence identity to SEQ ID NO:37) a tracrRNA having at least 90% sequence identity to SEQ ID NO:38; NO:23 has at least 90% sequence identity; and 1) 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.

[0111] In some embodiments of the above aspects, the tracrRNA is selected from the group consisting of: 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:25, 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:13; b) 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: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: NO:14 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; 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: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: NO:28 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: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: NO: 17 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;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: NO:19 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; 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: NO:33 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:21; j) 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: NO:22 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;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:35, 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 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 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: NO:24 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. In some embodiments, 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, and 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. ;

[0112] In some embodiments of the above aspects, the vector further comprises a polynucleotide encoding a RGN polypeptide.

[0113] 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: 28. NO:28 has at least 90% sequence identity; 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: NO:32 has at least 90% sequence identity; 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. ;

[0114] In some embodiments of the above aspects, the RGN polypeptide is selected from the group consisting of: 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, 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: 13, 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: NO:2 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, 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:27; NO:3 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, 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 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: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 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, 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:28; 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 to SEQ ID NO:28; NO:5 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, 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 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) 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:29; 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: NO:6 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, 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, 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;g) 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:7, 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 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: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: NO:8 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, 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, 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%, at least 99%, or 100% sequence identity to SEQ ID NO:20; NO:9 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:21, 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: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 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, 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: 34; 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 to SEQ ID NO: NO:11 has a 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, 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 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:35;and 1) 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 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.;

[0115] In another aspect, the present disclosure provides a nucleic acid molecule comprising a transcriptionally activated CRISPR RNA (tracrRNA) or a polynucleotide encoding tracrRNA, wherein the tracrRNA comprises a nucleotide sequence having at least 90% sequence identity with any one of SEQ ID NOs: 25-36; wherein, when the guide RNA binds to an RNA-guided nuclease (RGN) polypeptide, the guide RNA (comprising tracrRNA and crRNA, the crRNA comprising a spacer sequence and a CRISPR repeat sequence, wherein the tracrRNA hybridizes with the CRISPR repeat sequence of the crRNA) can hybridize with a target DNA sequence in a sequence-specific manner via the spacer sequence of the crRNA. In some embodiments, the tracrRNA comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 30.

[0116] In some embodiments of the above aspects, the polynucleotide encoding the tracrRNA is operably linked to a promoter that is heterologous to the polynucleotide.

[0117] 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 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.

[0118] In another aspect, the present disclosure provides a vector comprising the nucleic acid molecule comprising a polynucleotide encoding tracrRNA described above.

[0119] In yet another aspect, the present disclosure provides a single guide RNA comprising the above-described nucleic acid molecule comprising crRNA and the above-described nucleic acid molecule comprising tracrRNA.

[0120] In yet another aspect, the present disclosure provides a dual-guide RNA comprising the above-described nucleic acid molecule comprising crRNA and the above-described nucleic acid molecule comprising tracrRNA.

[0121] In another aspect, the present disclosure provides a cell comprising the nucleic acid molecule, the single guide RNA, the dual guide RNA, or the vector described above. BRIEF DESCRIPTION OF THE DRAWINGS

[0122] Figure 1 The editing rates of multiple genes are provided by LPG10134, LPG10136, LPG10138, LPG10139, LPG10141, LPG10145, LPG10146, LPG10147, LPG10150, LPG10155, LPG10159, and LPG10160. Plasmids including expression cassettes for RGN and guide RNA were co-transfected into HEK293T cells.

[0123] Figure 2 Shown: Single guide RNA (sgRNA) SGN002661 together with LPG10136 RNA-guided nuclease (RGN) provided over 75% editing of the T cell receptor alpha (TRAC) gene (measured by flow cytometry) when RGN and sgRNA were delivered as ribonucleoprotein (RNP) complexes in activated human peripheral blood mononuclear cells (PBMCs). B2M: beta-2-microglobulin.

[0124] Figure 3A and 3BIncreasing RNP concentration improves editing. The figure shows the editing efficiency of LPG10145 with a protein:RNA ratio of 1:2 and increasing RNP concentrations of various sgRNAs, measured by flow cytometry, for levels of beta-2-microglobulin (B2M) or T cell receptor alpha (TRAC) in activated human PBMCs. Figure 3B Illustration: Dose-dependency of the increase in editing activity with increasing amounts of RNPs including sgRNAs with two different spacer lengths (20 or 25 nucleotides).

[0125] Figure 4 LPG10145 was shown to edit targets in multiple genes. LPG10145 was delivered to T cells as RNPs via nucleofection. All guides showed a coefficient of variation (CV) of less than 50%. DETAILED DESCRIPTION

[0126] Many modifications and other embodiments of the invention set forth herein will occur to one of ordinary skill in the art, with the benefit of the teachings presented in the foregoing description and the associated drawings. Therefore, it should be understood that the invention is not limited to the specific embodiments disclosed, and modifications and other embodiments are contemplated to be included within the scope of the appended embodiments. Although specific terms are employed herein, these terms are used only in a general and descriptive sense and not for the purpose of limitation.

[0127] I. Overview

[0128] RNA-guided nucleases (RGNs) allow targeted manipulation of specific site(s) within the genome and are useful in therapeutic and research applications in the context of gene targeting. RNA-guided nucleases have been used for genome engineering in various organisms, including mammals, for example, by stimulating nonhomologous end joining and homologous recombination. The compositions and methods described herein are useful for generating single-strand or double-strand breaks in polynucleotides, modifying polynucleotides, detecting specific sites within polynucleotides, or modifying the expression of specific genes.

[0129] RNA-guided nucleases disclosed herein can change gene expression by modifying the target sequence. In a specific embodiment, RNA-guided nucleases are guided to the target sequence by guide RNA (gRNA) as part of a clustered regularly spaced short palindromic repeat (CRISPR) RNA-guided nuclease system. Because guide RNA forms a complex with RNA-guided nuclease to guide RNA-guided nuclease to bind to the target sequence and introduce single-stranded or double-stranded breaks at the target sequence in some embodiments, RGN is considered to be "RNA-guided". After the target sequence has been cut, the break can be repaired so that the DNA sequence of the target sequence is modified during the repair process. Therefore, a method for modifying the target sequence in the DNA of a host cell using RNA-guided nuclease is provided herein. For example, RNA-guided nucleases can be used to modify the target sequence at the genomic locus of a eukaryotic cell or a prokaryotic cell.

[0130] II. RNA-guided nucleases

[0131] Provided herein are RNA-guided nucleases. The term "RNA-guided nuclease (RGN)" refers to a polypeptide that binds to a specific target nucleotide sequence in a sequence-specific manner and is guided to the target nucleotide sequence by a guide RNA molecule that is complexed with the polypeptide and hybridized with the target sequence. Although RNA-guided nucleases can cut the target sequence when combined, the term "RNA-guided nuclease" also includes RNA-guided nucleases that can bind to the target sequence but do not cut the target sequence without nuclease activity. The cutting of the target sequence by RNA-guided nucleases can cause single-stranded or double-stranded breaks. RNA-guided nucleases that can only cut the single strand of a double-stranded nucleic acid molecule are referred to as nickases in this article.

[0132] The RNA-guided nucleases disclosed herein include LPG10134, LPG10136, LPG10138, LPG10139, LPG10141, LPG10145, LPG10146, LPG10147, LPG10150, LPG10155, LPG10159 and LPG10160 RNA-guided nucleases, whose amino acid sequences are shown in SEQ ID NOs: 1-12, respectively; and active fragments or variants thereof, which retain the ability to bind to a target nucleotide sequence in an RNA-guided sequence-specific manner. In some of these embodiments, the active fragments or variants of LPG10134, LPG10136, LPG10138, LPG10139, LPG10141, LPG10145, LPG10146, LPG10147, LPG10150, LPG10155, LPG10159 or LPG10160 RGN are 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 shown in 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 an amino acid sequence as shown in any one of SEQ ID NOs: 1-12. The RNA-guided nucleases provided herein can include at least one nuclease domain (e.g., DNase, RNase domain) and at least one RNA recognition and / or RNA binding domain to interact with the guide RNA.Other domains that can be found in the RNA-guided nucleases provided herein include, but are not limited to, DNA binding domains, helicase domains, protein-protein interaction domains, and dimerization domains. In particular embodiments, the RNA-guided nucleases provided herein may include at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to one or more of the DNA binding domain, the helicase domain, the protein-protein interaction domain, and the dimerization domain.

[0133] The target nucleotide sequence is bound to the RNA-guided nuclease provided herein and hybridized with the guide RNA associated with the RNA-guided nuclease. Then, if the polypeptide has nuclease activity, the target sequence can be subsequently cleaved by the RNA-guided nuclease. The term "cleave" or "cleavage" refers to the hydrolysis of at least one phosphodiester bond within the backbone of the target nucleotide sequence, which can result in a single-strand or double-strand break within the target sequence. The currently disclosed RGN can act as an endonuclease to cleave nucleotides within a polynucleotide, or can be an exonuclease to remove consecutive nucleotides from the ends (5' and / or 3' ends) of a polynucleotide. In other embodiments, the disclosed RGN can cleave nucleotides of a target sequence within any position of a polynucleotide, and thus has the effects of both an endonuclease and an exonuclease. The cleavage of a target polynucleotide by the currently disclosed RGN can result in staggered breaks or blunt ends.

[0134] The RNA-guided nucleases disclosed herein can be wild-type sequences derived from bacterial or archaeal species. Alternatively, the RNA-guided nucleases can be variants or fragments of wild-type polypeptides. For example, wild-type RGNs can be modified to alter nuclease activity or to alter PAM specificity. In some embodiments, the RNA-guided nucleases are not naturally occurring.

[0135] In certain embodiments, the RNA-guided nuclease acts as a nickase, cutting only a single strand of the target nucleotide sequence. Such RNA-guided nucleases have a single functional nuclease domain. In particular embodiments, the nickase can cut either the plus strand or the minus strand. In some of these embodiments, the additional nuclease domain has been mutated such that nuclease activity is reduced or eliminated.

[0136] In other embodiments, RNA-guided nucleases completely lack nuclease activity and are referred to herein as nuclease-dead or nuclease-free. Any method known in the art for introducing mutations into amino acid sequences (e.g., PCR-mediated mutagenesis and site-directed mutagenesis, etc.) can be used to produce RGNs without nickase or nuclease activity. For example, see U.S. Publication No. 2014 / 0068797 and U.S. Publication No. 9,790,490; the entire contents of each of the above patents are incorporated herein by reference. A non-limiting example of a nickase is LPG10145D16A nickase, which is shown in SEQ ID NO: 551 herein. Nickases comprising mutations in the RuvC domain and having a functional HNH domain can be used for base editing, wherein the nickase is fused to a base editing polypeptide (e.g., a deaminase). Another non-limiting example of a nickase is LPG10145 H611A nickase, which is shown herein as SEQ ID NO: 552. Nickases comprising mutations in the HNH domain and having a functional RuvC domain can be used for lead editing, wherein the nickase is fused to a lead editing polypeptide (e.g., a reverse transcriptase). A non-limiting example of a nuclease-free RGN is the LPG10145 D16A H611A sequence shown in SEQ ID NO: 553.

[0137] RNA-guided nucleases lacking nuclease activity can be used to deliver fused polypeptides, polynucleotides, or small molecule payloads to specific genomic locations. In some of these embodiments, the RGN polypeptide or guide RNA can be fused with a detectable label to allow detection of a specific sequence. As a non-limiting example, a RGN without nuclease activity can be fused with a detectable label (e.g., a fluorescent protein) and targeted to a specific sequence associated with a disease to allow detection of a disease-associated sequence.

[0138] Alternatively, RGNs without nuclease activity can be targeted to specific genomic locations to alter the expression of a desired sequence. In some embodiments, the binding of a nuclease-free RNA-guided nuclease to a target sequence results in expression of the target sequence or a reduction in expression of a gene under transcriptional control of the target sequence by interfering with the binding of an RNA polymerase or transcription factor within the targeted genomic region. In other embodiments, the RGN (e.g., an RGN without nuclease activity) or its composite guide RNA further comprises an expression regulator that, when combined with the target sequence, is used to inhibit or activate the expression of a target sequence or a gene under transcriptional control of the target sequence. In some embodiments of these embodiments, the expression regulator regulates the expression of the target sequence or the regulated gene via an epigenetic mechanism.

[0139] In other embodiments, RGNs without nuclease activity or RGNs with nickase activity can be targeted to specific genomic locations to modify the sequence of the target polynucleotide via fusion with a base editing polypeptide (e.g., a deaminase polypeptide, or an active variant or fragment thereof that directly chemically modifies a nucleobase (e.g., deaminase)), resulting in conversion from one nucleobase to another. The base editing polypeptide can be fused to the RGN at the N-terminal or C-terminal end of the RGN. In addition, the base editing polypeptide can be fused to the RGN via a peptide linker. Non-limiting examples of deaminase polypeptides that can be used in such compositions and methods include cytosine deaminase or adenine deaminase (e.g., adenine deaminase base editors described in Gaudelli et al. (2017) Nature 551:464-471, U.S. Publication Nos. 2017 / 0121693 and 2018 / 0073012, and International Publication No. WO / 2018 / 027078, or International Publication No. WO No. 63 / 077,089, filed on September 11, 2020, No. 63 / 146,840, filed on February 8, 2021, and No. 63 / 164,273, filed on March 22, 2021, and International Application No. PCT / US2021 / 049853, filed on September 10, 2021, the entire contents of each of which are incorporated herein by reference). In one embodiment, the deaminase polypeptide useful in such compositions and methods is a cytosine deaminase or an adenine deaminase comprising an amino acid sequence selected from any one of SEQ ID NOs: 377-448. In one embodiment, the deaminase polypeptides useful in such compositions and methods are cytosine deaminases or adenine deaminases 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 greater identity to the amino acid sequence of any one of SEQ ID NOs: 377-448. In some embodiments, the deaminase polypeptides useful in such currently disclosed compositions and methods are deaminases disclosed in Table 17 of International Publication No. WO 2020 / 139873 (the entire contents of which are incorporated herein by reference). In addition, it is known in the art that certain fusion proteins between RGN and base editing enzymes (e.g., cytosine deaminase) may also include at least one uracil stabilizing polypeptide, which increases the mutation rate of cytidine, deoxycytidine or cytosine in nucleic acid molecules to thymidine, deoxythymidine or thymine by the deaminase.Non-limiting examples of uracil stabilizing polypeptides include uracil stabilizing polypeptides disclosed in U.S. Provisional Application No. 63 / 052,175 filed on July 15, 2020, and International Application No. PCT / US2021 / 041809 filed on July 15, 2021 (each of which is incorporated herein by reference in its entirety), including USP2 (SEQ ID NO: 325) and uracil glycosylase inhibitor (UGI) domains (SEQ ID NO: 315), which can increase base editing efficiency. Thus, the fusion protein may include an RGN described herein or a variant thereof, a deaminase, and optionally at least one uracil stabilizing polypeptide (e.g., UGI or USP2). In certain embodiments, the RGN fused to the base editing polypeptide is a nicking enzyme that cuts the DNA strand on which the base editing polypeptide (e.g., deaminase) does not act.

[0140] In some embodiments, RGN can be fused with a lead editing polypeptide. Lead editing is a versatile and precise genome editing method that uses a nucleic acid programmable DNA binding protein associated with a polymerase to write new genetic information directly into a specific DNA site. The lead editing system uses an RGN that is a nickase, and the system is programmed with a lead editing (PE) guide RNA ("PEgRNA"). PEgRNA is a guide RNA that specifies a target sequence and provides a template for polymerization of a replacement chain, which includes an extended edit designed on a guide RNA (e.g., at the 5' end or 3' end, or inside the guide RNA). The RGN nickase / lead editing polypeptide fusion is guided by PEgRNA to the target sequence and to the upstream of the PAM and the upstream of the sequence to be edited. The target chain produces a 3' flap (3'flap) on the target chain. pegRNA includes a primer binding site (PBS) complementary to the 3' flap of the target chain. In some embodiments, the length of 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 certain embodiments, the pegRNA includes a PBS of 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 can include a PBS of at least 8 nucleotides in length. Hybridization of the PBS and the 3' flap of the target strand allows polymerization of the replacement strand containing the editor using the extension of the PEgRNA as a template. The extension of the PEgRNA can be formed by RNA or DNA. In the case of RNA extension, the polymerase of the lead editor can be an RNA-dependent DNA polymerase (e.g., a reverse transcriptase). In the case of DNA extension, the polymerase of the lead editor can be a DNA-dependent DNA polymerase.

[0141] The replacement strand containing the desired edit (e.g., a single nucleobase replacement) shares the same sequence as the target strand of the target sequence to be edited (except that it includes the desired edit). Via 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, because the lead 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 (the editor is set at the position of the corresponding target strand of the target sequence), the lead editor can be considered to be a "search and replace" genome editing technology. Therefore, in some embodiments, the guide RNA of the present disclosure includes an extension, and the extension includes an editing template for the lead editor. In some embodiments, the lead editor polypeptide that can be fused to the RGN includes a DNA polymerase. In certain embodiments, the DNA polymerase is a reverse transcriptase. In certain embodiments, the RGN is a nickase.

[0142] RNA-guided nucleases fused to a polypeptide or domain can be connected or separated by a joint. The term "joint" used herein refers to a chemical group or molecule that connects two molecules or parts (e.g., a binding domain and a cleavage domain of a nuclease). In some embodiments, the joint connects the gRNA binding domain of an RNA-guided nuclease with a base editing polypeptide such as a deaminase. In some embodiments, the joint connects an RGN with a deaminase without nuclease activity. Typically, the joint is located between or on both sides of two groups, molecules or other parts and is connected to each group, molecule or other part via a covalent bond, thereby connecting the two. In some embodiments, the joint is an amino acid or multiple amino acids (e.g., a peptide or protein). In some embodiments, the joint is an organic molecule, a group, a polymer or a chemical part. In some embodiments, the length of the linker is 5-100 amino acids, 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. Longer or shorter linkers are also contemplated.

[0143] The presently disclosed RNA-guided nucleases may include at least one nuclear localization signal (NLS) to enhance delivery of the RGN to the nucleus of a cell. Nuclear localization signals are known in the art and typically include a stretch of basic amino acids (e.g., see Lange et al., J. Biol. Chem. (2007) 282:5101-5105). In some embodiments, the RGN includes 2, 3, 4, 5, 6 or more nuclear localization signals. The (one or more) nuclear localization signals may be heterologous NLSs. Non-limiting examples of nuclear localization signals for the presently disclosed RGNs are the nuclear localization signals of SV40 large T antigen, nucleoplasmin, and c-Myc (e.g., see Ray et al. (2015) Bioconjug Chem 26(6):1004-7). In specific embodiments, the RGN includes an NLS sequence as shown in SEQ ID NO:316 or 317. The RGN may include one or more NLS sequences at its N-terminus, C-terminus, or both the N-terminus and the C-terminus. For example, an RGN may include two NLS sequences at the N-terminal region and four NLS sequences at the C-terminal region.

[0144] Other localization signal sequences known in the art that localize polypeptides to specific subcellular location(s) can also be used to target RGNs, including but not limited to plastid localization sequences, mitochondrial localization sequences, and dual targeting signal sequences that target both plastids and mitochondria (e.g., see 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 55:219-225). Biol 5:529-535; Carrie and Small (2013) Biochimica et Biophysica Acta 1833:253-259; Carrie et al. (2009) FEBS J 276:1187-1195; Silva-Filho (2003) Current Opinion in Plant Biol 6:589-595; Peeters and Small (2001) Biochimica et Biophysica 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).

[0145] In certain embodiments, the presently disclosed RNA-guided nucleases include at least one cell-penetrating domain that facilitates cellular uptake of the RGN. Cell-penetrating domains are known in the art and typically include stretches of positively charged amino acid residues (i.e., polycationic cell-penetrating domains), alternating polar and nonpolar amino acid residues (i.e., amphiphilic cell-penetrating domains), or hydrophobic amino acid residues (i.e., hydrophobic cell-penetrating domains) (e.g., see Milletti F. (2012) Drug Discov Today 17:850-860). A non-limiting example of a cell-penetrating domain is the transcriptional activator (TAT) from human immunodeficiency virus 1.

[0146] A nuclear localization signal, a plastid localization signal, a mitochondrial localization signal, a dual targeting localization signal, and / or a cell penetrating domain can be located at the amino terminus (N-terminus), the carboxyl terminus (C-terminus), or an internal location of the RNA-guided nuclease.

[0147] The currently disclosed RGNs can be fused directly or indirectly to effector domains such as cleavage domains, deaminase domains, or expression regulator domains via a linker peptide. Such domains can be located at the N-terminus, C-terminus, or internal locations of the RNA-guided nuclease. In some of these embodiments, the RGN composition of the fusion protein is a nuclease-inactive RGN or a nickase.

[0148] In some embodiments, the RGN fusion protein includes a cleavage domain that is any domain capable of cleaving a polynucleotide (i.e., RNA, DNA, or RNA / DNA hybrid), and includes, but is not limited to, restriction endonucleases and homing endonucleases, such as Type IIS endonucleases (e.g., FokI) (e.g., see Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388; Linn et al. (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993).

[0149] In other embodiments, the RGN fusion protein includes a deaminase domain that deaminates a nucleobase resulting in conversion from one nucleobase to another, including but not limited to a cytosine deaminase or an adenine deaminase (e.g., see Gaudelli et al. (2017) Nature 551:464-471, U.S. Publication Nos. 2017 / 0121693 and 2018 / 0073012, U.S. Patent No. 9,840,699 and International Publication No. WO / 2018 / 027078, or Any of the deaminases disclosed in International Patent Publication No. WO2020 / 139873, U.S. Provisional Application No. 63 / 077,089 filed on September 11, 2020, U.S. Provisional Application No. 63 / 146,840 filed on February 8, 2021, and U.S. Provisional Application No. 63 / 164,273 filed on March 22, 2021, and International Application No. PCT / US2021 / 049853 filed on September 10, 2021, the entire contents of each of which are incorporated herein by reference).

[0150] In some embodiments, the effector domain of the RGN fusion protein can be an expression regulator domain, which is a domain used to positively or negatively regulate transcription. The expression regulator domain can be an epigenetic modification domain, a transcription repressor domain, or a transcription activation domain.

[0151] In some embodiments of these embodiments, the expression regulator of the RGN fusion protein includes an epigenetic modification domain, which covalently modifies DNA or histones to change histone structure and / or chromosome structure without changing the DNA sequence, resulting in changes in gene expression (i.e., positive regulation or negative regulation). Non-limiting examples of epigenetic modifications include acetylation or methylation of lysine residues in DNA, arginine methylation, serine and threonine phosphorylation, and lysine ubiquitination and sumoylation of histones, and methylation and hydroxymethylation of cytosine residues. 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.

[0152] In other embodiments, the expression regulator of the fusion protein includes a transcriptional repressor domain that interacts with transcriptional control elements and / or transcriptional regulatory proteins such as 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 repressors, IκB, and Krüppel-associated box (KRAB) domains.

[0153] In still other embodiments, the expression regulator of the fusion protein includes a transcriptional activation domain, which interacts with transcriptional control elements and / or transcriptional regulatory proteins such as RNA polymerase and transcription factors to increase or activate the transcription of at least one gene. The transcriptional activation domain is known in the art and includes but is not limited to herpes simplex virus VP16 activation domain and NFAT activation domain.

[0154] The currently disclosed RGN polypeptides may include a detectable label or purification tag. The detectable label or purification tag may be located directly or indirectly via a linker peptide at the N-terminus, C-terminus, or internal position of the RNA-guided nuclease. In some of these embodiments, the RGN composition of the fusion protein is a RGN without nuclease activity. In other embodiments, the RGN composition of the fusion protein is a RGN with nickase activity.

[0155] A detectable label is a molecule that can be seen or otherwise observed. A detectable label can be fused to an RGN as a fusion protein (e.g., a fluorescent protein), or a small molecule that can be conjugated to an RGN polypeptide that can be detected visually or otherwise. Detectable labels that can be fused to the presently disclosed RGNs as a fusion protein include any detectable protein domain, including but not limited to a protein domain detectable with a specific antibody or a fluorescent protein. 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 radioactive labels, such as, 3 H and 35 S.

[0156] RGN polypeptides may also include a purification tag, which is any molecule that can be used to separate 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.

[0157] III. Guide RNA

[0158] The present disclosure provides guide RNA and polynucleotides encoding guide RNA. 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 guide the associated RNA-guided nuclease to specifically bind to the target nucleotide sequence. Therefore, the guide RNA of each RGN is one or more RNA molecules (usually one or two), which can bind to the RGN and guide the RGN to bind to a specific target nucleotide sequence, and in those embodiments where the RGN has nickase or nuclease activity, the target nucleotide sequence is also cut. In general, guide RNA includes CRISPR RNA (crRNA) and transcriptionally activated CRISPR RNA (tracrRNA). The natural guide RNA including both crRNA and tracrRNA generally includes two separate RNA molecules, which hybridize to each other via the repetitive sequence of crRNA and the anti-repetitive sequence of tracrRNA.

[0159] The length of the natural direct repeat sequence in the CRISPR array is generally in the range of 28 to 37 base pairs, but the length may vary between about 23bp and about 55bp. The length of the spacer sequence in the CRISPR array is generally in the range of about 32 to about 38bp, but the length may be between about 21bp and about 72bp. Each CRISPR array generally includes less than 50 units of CRISPR repeat-spacer sequences. CRISPR is transcribed as a part of a long transcript called a primary CRISPR transcript, which includes a large part of the CRISPR array. The primary CRISPR transcript is cut by the Cas protein to produce crRNA, or in some cases, a precursor crRNA (pre-crRNA) is produced, which is further processed into mature crRNA by other Cas proteins. Mature crRNA includes spacer sequences and CRISPR repeats. In some embodiments where the precursor crRNA is processed into mature (or processed) crRNA, maturation involves removing about 1 to about 6 or more 5', 3', or 5' and 3' nucleotides. These nucleotides that are removed during maturation of the pre-crRNA molecule are not necessary for the generation or design of guide RNA for the purpose of genome editing or targeting a specific target nucleotide sequence of interest.

[0160] CRISPR RNA (crRNA) includes spacer sequences and CRISPR repeat sequences. A "spacer sequence" is a nucleotide sequence that directly hybridizes with a target nucleotide sequence of interest. The spacer sequence is engineered to be completely or partially complementary to the target sequence of interest. In various embodiments, the spacer sequence may include about 8 nucleotides to about 30 nucleotides or more nucleotides. For example, the length of the spacer sequence may be about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30 or more nucleotides. In some embodiments, the length of 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 some embodiments, the length of the spacer sequence is about 10 to about 26 nucleotides, or about 12 to about 30 nucleotides. In some embodiments, the length of the spacer sequence is 19-27 nucleotides. In certain embodiments, particularly when the RGN has an amino acid sequence as shown in SEQ ID NO:6 or an active variant or fragment thereof, the length of the spacer sequence is 21-24 nucleotides. In a specific embodiment, the length of the spacer sequence is about 30 nucleotides. In some embodiments, the length of the spacer sequence is 30 nucleotides. In some embodiments, when optimally aligned using a suitable alignment algorithm, the degree of complementarity between the spacer sequence and its corresponding target sequence is between 50% and 99% or higher, including but not limited to about or greater than about 50%, about 60%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more. In certain embodiments, the degree of complementarity between the spacer sequence and its corresponding target sequence is 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more when optimally aligned using a suitable alignment algorithm.In particular embodiments, the spacer sequence contains no secondary structure that can be predicted using any suitable polynucleotide folding algorithm known in the art, including but not limited to mFold (e.g., see Zuker and Stiegler (1981) Nucleic Acids Res 9:133-148) and RNAfold (e.g., see Gruber et al. (2008) Cell 106(1):23-24).

[0161] The CRISPR RNA repeat sequence includes a nucleotide sequence that forms a structure that can be recognized by the RGN molecule, which is formed by the nucleotide sequence alone or with the hybridized tracrRNA. In various embodiments, the CRISPR RNA repeat sequence may include about 8 nucleotides to about 30 nucleotides or more nucleotides. For example, the length of the CRISPR repeat sequence may be about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30 or more nucleotides. In specific embodiments, the length of the CRISPR repeat sequence is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides. In some embodiments, the degree of complementarity between a CRISPR repeat sequence and its corresponding tracrRNA sequence is about or greater than about 50%, about 60%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more when optimally aligned using a suitable alignment algorithm. In certain embodiments, the degree of complementarity between a CRISPR repeat sequence and its corresponding tracrRNA sequence is 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more when optimally aligned using a suitable alignment algorithm.

[0162] In specific embodiments, the CRISPR repeat sequence comprises a nucleotide sequence of any one of SEQ ID NOs: 13-24, or an active variant or fragment thereof, which, when included in 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 sequence 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 a nucleotide sequence as shown in any one of SEQ ID NOs: 13-24. In certain embodiments, the active CRISPR repeat sequence fragment of the wild-type sequence includes at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 13-24.

[0163] In certain embodiments, crRNA is not naturally occurring. In some embodiments of these embodiments, a specific CRISPR repeat sequence is not essentially connected to an engineered spacer sequence, and the CRISPR repeat sequence is considered to be heterologous to the spacer sequence. In certain embodiments, the spacer sequence is a non-naturally occurring engineered sequence.

[0164] The transcriptionally activated CRISPR RNA or tracrRNA molecule includes a nucleotide sequence, and the nucleotide sequence includes a region having sufficient complementarity to hybridize with the CRISPR repeat sequence of crRNA, which is referred to as an anti-repeat region in this article. In some embodiments, the tracrRNA molecule further includes a region with a secondary structure (e.g., a stem loop) or forms a secondary structure when hybridizing with the crRNA corresponding thereto. In a specific embodiment, the region of the tracrRNA that is completely or partially complementary to the CRISPR repeat sequence is at the 5' end of the molecule, and the 3' end of the tracrRNA includes a secondary structure. This secondary structure region generally includes several hairpin structures, including a connection (nexus) hairpin that is found to be adjacent to the anti-repeat sequence. The connection forms an interactive core between the guide RNA and the RGN and is at the intersection between the guide RNA, the RGN and the target DNA. The connection hairpin generally has a retained nucleotide sequence in the base of the hairpin stem, and the motif UNANNC (SEQ ID NO: 318) is present in many connection hairpins in tracrRNA. Interestingly, several RGNs of the invention use tracrRNAs that include atypical sequences in the bases of their hairpin stems that connect to the hairpin, including UNANNA, UNANNG, and CNANNC (SEQ ID NOs: 319, 321, and 322, respectively). There is often a terminal hairpin at the 3' end of the tracrRNA, which can vary in structure and number, but typically includes a GC-rich Rho-independent transcription terminator hairpin followed by a run of Us at the 3' end. See, for example, 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, the entire contents of each of which are incorporated herein by reference.

[0165] In various embodiments, the anti-repeat region of the tracrRNA that is fully or partially complementary to the CRISPR repeat sequence comprises about 8 nucleotides to about 30 nucleotides or more nucleotides. For example, the length of the base pairing 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 particular embodiments, the base-paired region between the tracrRNA anti-repeat sequence and the CRISPR repeat sequence can be 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 is about or greater than about 50%, about 60%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more when optimally aligned using a suitable alignment algorithm. In certain embodiments, the degree of complementarity between a CRISPR repeat sequence and its corresponding tracrRNA anti-repeat sequence is about or greater than 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 when optimally aligned using a suitable alignment algorithm.

[0166] In various embodiments, the entire tracrRNA can include about 60 nucleotides to more than about 210 nucleotides. For example, the length of the tracrRNA can be about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210 or more nucleotides. In a specific embodiment, the length of 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 particular 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 specific 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 in length.

[0167] In certain embodiments, the tracrRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 25-36, or an active variant or fragment thereof, which, when included in 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 tracrRNA sequence 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 a nucleotide sequence as shown in any one of SEQ ID NOs: 25-36. In certain embodiments, the active tracrRNA sequence fragment of the wild-type sequence includes at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 or more consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 25-36.

[0168] Two polynucleotide sequences are considered to be substantially complementary when the two sequences hybridize to each other under stringent conditions. Similarly, if the guide RNA bound to the RGN binds to the target sequence under stringent conditions, then the RGN is considered to bind to a specific target sequence in a sequence-specific manner. "Stringent conditions" or "stringent hybridization conditions" means that under the conditions, the detectable degree of hybridization of two polynucleotide sequences to each other is greater than the detectable degree of hybridization to other sequences (e.g., at least 2 times higher than background). Stringent conditions are sequence-dependent and will be different in different environments. Typically, stringent conditions will be those conditions in which: the salt concentration is less than about 1.5M Na ions (typically about 0.01 to 1.0M Na ion concentration (or other salts)) 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., greater than 50 nucleotides). Stringent conditions can also be achieved by adding destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization in a buffer solution of 30 to 35% formamide, 1M NaCl, 1% SDS (sodium dodecyl sulfate) at 37°C, and washing in 1X to 2X SSC (20X SSC=3.0M NaCl / 0.3M trisodium citrate) at 50 to 55°C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0M NaCl, 1% SDS at 37°C, and washing in 0.5X to 1X SSC at 55 to 60°C. Exemplary high stringency conditions include hybridization in 50% formamide, 1M NaCl, 1% SDS at 37°C, and washing in 0.1X SSC at 60 to 65°C. Optionally, the wash buffer may include about 0.1% to about 1% SDS. The duration of hybridization is generally less than about 24 hours, typically about 4 to about 12 hours. The duration of the wash time will be at least the length of time sufficient to reach equilibrium.

[0169] The Tm is the temperature (under defined ionic strength and pH) at which 50% of the complementary target sequence hybridizes to the perfectly matched sequence. For DNA-DNA hybrids, the Tm can be roughly estimated from 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 length of the hybrid in base pairs. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) of the specific sequence and its complement at a defined ionic strength and pH. However, extremely stringent conditions can be carried out at 1, 2, 3 or 4°C below the thermal melting point (Tm) for hybridization and / or washing; moderately stringent conditions can be carried out at 6, 7, 8, 9 or 10°C below the thermal melting point (Tm); and low stringency conditions can be carried out at 11, 12, 13, 14, 15 or 20°C below the thermal melting point (Tm). One of ordinary skill in the art will appreciate that using the equation, hybridization and wash compositions, and desired Tm, variations in the stringency of hybridization and / or wash solutions are inherently described. An extensive guide to nucleic acid hybridization is available 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 (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, New York).

[0170] The term "sequence specificity" may also refer to binding to a target sequence at a higher frequency than binding to a randomized background sequence.

[0171] The guide RNA can be a single guide RNA (sgRNA) or a dual guide RNA system. The single guide RNA includes crRNA and tracrRNA on a single RNA molecule, and the dual guide RNA system includes crRNA and tracrRNA present in two different RNA molecules that hybridize to each other via at least a portion of the CRISPR repeat sequence of crRNA and at least a portion of tracrRNA, and at least a portion of tracrRNA can be completely or partially complementary to the CRISPR repeat sequence of crRNA. In some embodiments in which the guide RNA is a single guide RNA, crRNA and tracrRNA are separated by a joint nucleotide sequence. In general, to avoid the formation of secondary structures in the nucleotides of the joint nucleotide sequence or to avoid the formation of secondary structures including nucleotides, the joint nucleotide sequence is a nucleotide sequence that does not include complementary bases. In some embodiments, the length of the joint nucleotide sequence between crRNA and tracrRNA is at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12 or more nucleotides. In a specific embodiment, the length of the joint nucleotide sequence of a single guide RNA is at least 4 nucleotides. In certain embodiments, the linker nucleotide sequence is the nucleotide sequence shown in SEQ ID NO:323.

[0172] Single guide RNA or dual guide RNA can be synthesized chemically or via in vitro transcription. Assays for determining sequence-specific binding between RGN and guide RNA are known in the art and include, but are not limited to, in vitro binding assays between expressed RGN and guide RNA, which can be labeled with a detectable label (e.g., biotin) and used in pull-down detection assays, wherein the guide RNA:RGN complex is captured via a detectable label (e.g., using streptavidin magnetic beads). Control guide RNAs with sequences or structures unrelated to the guide RNAs can serve as negative controls for non-specific binding of RGN to RNA. In certain embodiments, the guide RNA is any one of SEQ ID NOs: 37-48, wherein the spacer sequence can be any sequence and is represented by a poly-N sequence.

[0173] In certain embodiments, guide RNA can be introduced into target cell, organelle or embryo as RNA molecule.Guide RNA can be transcribed in vitro or chemically synthesized.In other embodiments, the nucleotide sequence encoding guide RNA is introduced into cell, organelle or embryo.In some embodiments of these embodiments, the nucleotide sequence encoding guide RNA is operably connected to promoter (for example, RNA polymerase III promoter).Promoter can be a natural promoter or heterologous to the nucleotide sequence encoding guide RNA.

[0174] In various embodiments, as described herein, a guide RNA can be introduced into a target cell, organelle, or embryo as a ribonucleoprotein complex in which the guide RNA is associated with an RNA-guided nuclease polypeptide.

[0175] Via hybridization of the guide RNA with the target nucleotide sequence, the guide RNA guides the associated RNA-guided nuclease to the specific target nucleotide sequence of interest. The target nucleotide sequence may include DNA, RNA, or a combination of the two, 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, short interfering RNA). The target nucleotide sequence may be bound (and in some embodiments cut) by an RNA-guided nuclease in vitro or in a cell. The chromosomal sequence targeted by the RGN may be a chromosomal sequence of a nucleus, plastid, or mitochondria. In some embodiments, the target nucleotide sequence is unique in the target genome.

[0176] The target nucleotide sequence is adjacent to a pre-spacer adjacent motif (PAM). The pre-spacer adjacent motif is generally within about 1 to about 10 nucleotides from 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 specific embodiments, the PAM is within 1 to 10 nucleotides from the target nucleotide sequence, including 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from 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 of the currently disclosed RGN. Typically, the PAM is a consensus sequence of about 3-4 nucleotides, but in specific embodiments, the length of the PAM can be 2, 3, 4, 5, 6, 7, 8, 9, or more nucleotides. In various embodiments, the PAM sequence recognized by the currently disclosed RGN includes a consensus sequence as shown in any one of SEQ ID NOs: 73-84.

[0177] 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 as shown in any one of SEQ ID NOs: 73-84. In some embodiments, the RGN binds to a guide sequence comprising a CRISPR repeat sequence as shown in any one of SEQ ID NOs: 13-24, or an active variant or fragment thereof, and a tracrRNA sequence as shown in any one of SEQ ID NOs: 25-36, or an active variant or fragment thereof, respectively. The RGN system is further described in Examples 1-3 and Tables 1 and 2 of the specification.

[0178] In some embodiments, when bound to a guide RNA comprising a CRISPR repeat sequence as shown in SEQ ID NO:13, or an active variant or fragment thereof, and a tracrRNA sequence as shown in SEQ ID NO:25, or an active variant or fragment thereof, the RNA-guided nuclease having SEQ ID NO:1 or an active variant or fragment thereof binds to a target nucleotide sequence adjacent to a PAM sequence as shown in SEQ ID NO:73.

[0179] In some embodiments, when bound to a guide RNA comprising a CRISPR repeat sequence as shown in SEQ ID NO:14, or an active variant or fragment thereof, and a tracrRNA sequence as shown in SEQ ID NO:26, or an active variant or fragment thereof, an RNA-guided nuclease having SEQ ID NO:2, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to a PAM sequence as shown in SEQ ID NO:74.

[0180] In some embodiments, when bound to a guide RNA comprising a CRISPR repeat sequence as shown in SEQ ID NO:15, or an active variant or fragment thereof, and a tracrRNA sequence as shown in SEQ ID NO:27, or an active variant or fragment thereof, an RNA-guided nuclease having SEQ ID NO:3, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to a PAM sequence as shown in SEQ ID NO:75.

[0181] In some embodiments, when bound to a guide RNA comprising a CRISPR repeat sequence as shown in SEQ ID NO:16, or an active variant or fragment thereof, and a tracrRNA sequence as shown in SEQ ID NO:28, or an active variant or fragment thereof, an RNA-guided nuclease having SEQ ID NO:4, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to a PAM sequence as shown in SEQ ID NO:76.

[0182] In some embodiments, an RNA-guided nuclease having SEQ ID NO:5 or an active variant or fragment thereof binds to a target nucleotide sequence adjacent to a PAM sequence as shown in SEQ ID NO:77 when bound to a guide RNA comprising a CRISPR repeat sequence as shown in SEQ ID NO:17, or an active variant or fragment thereof, and a tracrRNA sequence as shown in SEQ ID NO:29, or an active variant or fragment thereof.

[0183] In some embodiments, when bound to a guide RNA comprising a CRISPR repeat sequence as shown in SEQ ID NO:18, or an active variant or fragment thereof, and a tracrRNA sequence as shown in SEQ ID NO:30, or an active variant or fragment thereof, an RNA-guided nuclease having SEQ ID NO:6, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to a PAM sequence as shown in SEQ ID NO:78.

[0184] In some embodiments, when bound to a guide RNA comprising a CRISPR repeat sequence as shown in SEQ ID NO:19, or an active variant or fragment thereof, and a tracrRNA sequence as shown in SEQ ID NO:31, or an active variant or fragment thereof, an RNA-guided nuclease having SEQ ID NO:7, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to a PAM sequence as shown in SEQ ID NO:79.

[0185] In some embodiments, when bound to a guide RNA comprising a CRISPR repeat sequence as shown in SEQ ID NO:20, or an active variant or fragment thereof, and a tracrRNA sequence as shown in SEQ ID NO:32, or an active variant or fragment thereof, an RNA-guided nuclease having SEQ ID NO:8, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to a PAM sequence as shown in SEQ ID NO:80.

[0186] In some embodiments, when bound to a guide RNA comprising a CRISPR repeat sequence as shown in SEQ ID NO:21, or an active variant or fragment thereof, and a tracrRNA sequence as shown in SEQ ID NO:33, or an active variant or fragment thereof, an RNA-guided nuclease having SEQ ID NO:9, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to a PAM sequence as shown in SEQ ID NO:81.

[0187] In some embodiments, when bound to a guide RNA comprising a CRISPR repeat sequence as shown in SEQ ID NO:22, or an active variant or fragment thereof, and a tracrRNA sequence as shown in SEQ ID NO:34, or an active variant or fragment thereof, the RNA-guided nuclease having SEQ ID NO:10, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to a PAM sequence as shown in SEQ ID NO:82.

[0188] In some embodiments, when bound to a guide RNA comprising a CRISPR repeat sequence as shown in SEQ ID NO:23, or an active variant or fragment thereof, and a tracrRNA sequence as shown in SEQ ID NO:35, or an active variant or fragment thereof, an RNA-guided nuclease having SEQ ID NO:11, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to a PAM sequence as shown in SEQ ID NO:83.

[0189] In some embodiments, when bound to a guide RNA comprising a CRISPR repeat sequence as shown in SEQ ID NO:24, or an active variant or fragment thereof, and a tracrRNA sequence as shown in SEQ ID NO:36, or an active variant or fragment thereof, an RNA-guided nuclease having SEQ ID NO:12, or an active variant or fragment thereof, binds to a target nucleotide sequence adjacent to a PAM sequence as shown in SEQ ID NO:84.

[0190] It is well known in the art that PAM sequence specificity for a given nuclease enzyme is affected by enzyme concentration (e.g., see Karvelis et al. (2015) Genome Biol 16:253), which can be modified by changing the promoter used to express the RGN or the amount of ribonucleoprotein complex delivered to the cell, organelle, or embryo.

[0191] Upon recognizing the PAM sequence corresponding thereto, the RGN can cleave the target nucleotide sequence at a specific cleavage site. As used herein, the cleavage site is composed of two specific nucleotides within the target nucleotide sequence, and the nucleotide sequence between the two specific nucleotides is cleaved by the RGN. The cleavage site may include the 1st and 2nd, 2nd and 3rd, 3rd and 4th, 4th and 5th, 5th and 6th, 7th and 8th, or 8th and 9th nucleotides from the 5' or 3' direction of the PAM. In some embodiments, the cleavage site may be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or more than 20 nucleotides from the 5' or 3' direction of the PAM. In some embodiments, particularly those wherein the RGN has an amino acid sequence as shown in SEQ ID NO: 6 or an active variant or fragment thereof, the cleavage site of the RGN is between the 3rd and 4th nucleotides of the 5' of the PAM, producing a blunt end. Because RGNs can cleave target nucleotide sequences, resulting in staggered ends, in some embodiments, the cleavage site is defined based on the distance of two nucleotides on the positive (+) strand of the polynucleotide from the PAM and the distance of two nucleotides on the negative (-) strand of the polynucleotide from the PAM.

[0192] IV. Nucleotides encoding RNA-guided nucleases, CRISPR RNAs and / or tracrRNAs

[0193] The present disclosure provides polynucleotides including the currently disclosed CRISPR RNA, tracrRNA and / or sgRNA and polynucleotides including nucleotide sequences encoding the currently disclosed RNA-guided nucleases, CRISPR RNA, tracrRNA and / or sgRNA. The currently disclosed polynucleotides include those including or encoding CRISPR repeat sequences, the CRISPR repeat sequences including nucleotide sequences as shown in any one of SEQ ID NO: 13-24 or active variants or fragments thereof, which when included in the guide RNA can guide the sequence-specific binding of the associated RNA-guided nuclease to the target sequence of interest. Also disclosed are polynucleotides including or encoding tracrRNA, tracrRNA including nucleotide sequences as shown in any one of SEQ ID NO: 25-36 or active variants or fragments thereof, which when included in the guide RNA can direct the sequence-specific binding of the associated RNA-guided nuclease to the target sequence of interest. Also provided is a polynucleotide encoding an RNA-guided nuclease, the RNA-guided nuclease comprising an amino acid sequence as shown in any one of SEQ ID NOs: 1-12 and an active fragment or variant thereof, which retains the ability to bind to a target nucleotide sequence in an RNA-guided sequence-specific manner.

[0194] The use of the term "polynucleotide" or "nucleic acid molecule" is not intended to limit the present disclosure to polynucleotides including DNA. One of ordinary skill 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 (PNA), PNA-DNA chimeras, locked nucleic acids (LNA), and sequences linked by phosphorothioate. The polynucleotides disclosed herein also include all forms of sequences, including but not limited to single-stranded forms, double-stranded forms, DNA-RNA hybrids, triplex structures, stem-loop structures, and the like.

[0195] In some embodiments, the polynucleotide encoding the presently disclosed RGN 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 composition of an mRNA molecule includes at least one coding region, a 5'UTR, a 3'UTR, a 5'cap, and a poly-A tail. In embodiments, the mRNA encoding the RGN useful in the presently disclosed methods and compositions may include one or more structural and / or chemical modifications or changes that confer useful properties to the polynucleotide. For example, useful properties of mRNA include not substantially inducing an innate immune response of a cell into which the mRNA is introduced. A "structural" feature or modification is the insertion, deletion, duplication, inversion, or randomization of two or more linked nucleotides in an mRNA without significant chemical modification of the nucleotides themselves. Because chemical bonds will necessarily be broken and reformed to produce structural modifications, structural modifications are chemical in nature and are therefore chemical modifications. However, structural modifications will result in different nucleotide sequences. Chemical modifications to the mRNA can involve including 5-methylcytosine, N1-methyl-pseudouridine, pseudouridine, 2-thiouridine, 4-thiouridine, 5-methoxyuridine, 2'fluoroguanosine, 2'fluorouridine, 5-bromouridine, 5-(2-methoxycarbonylvinyl)uridine, 5-[3(1-E-propenylamino)]uridine, α-thiocytidine, N6-methyladenosine, 5-methylcytidine, N4-acetylcytidine, 5-formylcytidine, or a combination thereof in the mRNA.

[0196] Nucleic acid molecules encoding RGNs can be codon optimized for expression in an organism of interest. A "codon optimized" coding sequence is a polynucleotide coding sequence whose codon usage frequency is designed to mimic the preferred codon usage frequency or transcription conditions of a particular host cell. Expression in a particular host cell or organism is enhanced due to the alteration of 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. Other references and codon tables 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 discussion of plant-preferred codon usage). Methods are available in the art for synthesizing plant-preferred genes or mammalian (e.g., human) codon-optimized coding sequences. See, for example, US 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.

[0197] The polynucleotides encoding the RGN, crRNA, tracrRNA and / or sgRNA provided herein can be provided in an expression cassette for in vitro expression or expression in a cell, organelle, embryo or organism of interest. The cassette will include 5' and 3' regulatory sequences, which are operably connected to the polynucleotides encoding the RGN, crRNA, tracrRNA and / or sgRNA provided herein that allow the polynucleotides to be expressed. The cassette may additionally contain at least one additional gene or genetic element to be co-transformed in an organism. In the case of including additional genes or elements, these compositions are operably connected. The term "operably connected" is intended to describe the functional connection between two or more elements. For example, the operably connected between a promoter and a coding region of interest (e.g., a region encoding RGN, crRNA, tracrRNA and / or sgRNA) is a functional connection that allows the coding region of interest to be expressed. The operably connected elements may be continuous or non-continuous. When used to refer to the connection of two protein coding regions, by operably connected means that the coding region is in the same reading frame. Alternatively, (one or more) additional genes or elements may be provided on multiple expression cassettes. For example, a nucleotide sequence encoding a currently disclosed RGN may be present on one expression cassette, while a nucleotide sequence encoding crRNA, tracrRNA or a complete guide RNA may be on a separate expression cassette. Such an expression cassette is provided with multiple restriction sites and / or recombination sites for transcriptional regulation of the insertion regulated region of the polynucleotide. The expression cassette may additionally contain a screening marker gene.

[0198] The expression cassette will include a transcription (and in some embodiments, translation) initiation region (i.e., a promoter), a polynucleotide encoded by RGN-, crRNA-, tracrRNA- and / or sgRNA- of the present invention, and a transcription (and in some embodiments, translation) termination region (i.e., termination region) that functions in the organism of interest in the 5'-3' direction of transcription. The promoter of the present invention is capable of directing or driving the expression of the coding sequence in a host cell. The regulatory region (e.g., promoter, transcription regulatory region, and translation termination region) may be endogenous or heterologous to the host cell, or endogenous or heterologous to each other. As used herein, "heterologous" with respect to a sequence is a sequence derived from an alien species, or, if from the same species, a sequence that is substantially modified in composition and / or genomic loci from its native form by deliberate human intervention. As used herein, a chimeric gene includes a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence.

[0199] Suitable termination regions can be obtained from the Ti plasmid of Agrobacterium (A. tumefaciens), such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet. 262: 141-14; 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 Research 17: 7891-7903; and Joshi et al. (1987) Nucleic Acids Research 15: 9627-9639.

[0200] Additional regulatory signals include, but are not limited to, transcription initiation start sites, operators, activators, enhancers, other regulatory elements, ribosome binding sites, initiation codons, termination signals, etc. See, e.g., U.S. Pat. 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 Laboratory, New York, hereinafter "Sambrook 11"; Davis et al., eds. (1980) Advanced Bacterial Genetics (Cold Spring Harbor Laboratory Press), Cold Spring Harbor Laboratory, New York, and references cited therein.

[0201] In preparing the expression cassette, the various DNA fragments may be manipulated to provide the DNA sequence in the appropriate orientation and, where appropriate, in the appropriate reading frame. To this end, adapters or linkers may be used to connect the DNA fragments, or other manipulations may be involved to provide appropriate restriction sites, remove excess DNA, remove restriction sites, etc. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, re-substitution (e.g., transition and transversion) may be involved.

[0202] Many promoters can be used to implement the present invention. Promoters can be selected based on the desired results. Nucleic acids can be combined with persistent, inducible, growth stage specific, cell type specific, tissue-preferred, tissue-specific or other promoters to express in the organism of interest. See, e.g., promoters as shown in WO 99 / 43838 and in U.S. Pat. Nos. 8,575,425; 7,790,846; 8,147,856; 8,586832; 7,772,369; 7,534,939; 6,072,050; 5,659,026; 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611; which are incorporated herein by reference.

[0203] For expression in plants, constitutive promoters also 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) EMBO J. 3:2723-2730).

[0204] Examples of inducible promoters are the Adh1 promoter, which can be induced by hypoxia or cold stress, the Hsp70 promoter, which can be induced by heat stress, the PPDK promoter, which can be induced by light, and the pepcarboxylase promoter. Chemically induced promoters are also useful, such as the protectant-induced In2-2 promoter (U.S. Patent No. 5,364,780), the auxin-induced Axig1 promoter, which is specific to the tapetum but also active in callus (PCT US01 / 22169), steroid-responsive promoters (e.g., see Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88: 10421-10425 and McNellis et al. (1998) Plant Journal (Plant J.) 14(2):247-257), and tetracycline-inducible and tetracycline-repressible promoters (e.g., see Gatz et al. (1991) Mol. Gen. Genet. 227:229-237 and U.S. Pat. Nos. 5,814,618 and 5,789,156), which are incorporated herein by reference.

[0205] Tissue-specific or tissue-preferred promoters can be used for the expression of expression constructs in targeted specific tissues. In certain embodiments, tissue-specific or tissue-preferred promoters are active in plant tissues. Examples of promoters that are developmentally controlled in plants include promoters that preferentially initiate transcription in certain tissues such as leaves, roots, fruits, seeds, or flowers. "Tissue-specific" promoters are promoters that initiate transcription only in certain tissues. Different from the persistent expression of genes, tissue-specific expression is the result of several interaction levels of gene regulation. In this way, promoters from homologous or closely related plant species can be used preferentially in specific tissues to achieve effective and reliable expression of transgenics. In certain embodiments, expression includes promoters of tissue preference. "Tissue-preferred" promoters are promoters that preferentially initiate transcription in certain tissues, but not necessarily completely or only in certain tissues.

[0206] In some embodiments, the nucleic acid molecules encoding RGN, crRNA and / or tracrRNA include cell type specific promoters. A "cell type specific" promoter is a promoter that drives expression mainly in certain cell types in one or more organs. Cell type specific promoters that work in plants can be some examples of plant cells that are mainly active, including, for example, BETL cells, roots, vascular cells in leaves, stalk cells, and stem cells. Nucleic acid molecules may also include promoters that are cell type preferred. "Cell type preferred" promoters are promoters that mainly drive expression in most, but not necessarily completely or only in certain cell types in one or more organs. Some examples of plant cells that are preferentially active include, for example, BETL cells, roots, vascular cells in leaves, stalk cells, and stem cells.

[0207] The nucleic acid sequence encoding the RGN, crRNA, tracrRNA and / or sgRNA can be operably linked to a promoter sequence that is recognized by, for example, a bacteriophage RNA polymerase 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 methods of genome modification described herein.

[0208] In certain embodiments, the polynucleotides encoding the RGN, crRNA, tracrRNA and / or sgRNA may also be linked to a polyadenylation signal (e.g., SV40 polyA signal and other signals that function in plants) and / or at least one transcription termination sequence. In addition, the sequence encoding the RGN may also be linked to a sequence encoding at least one nuclear localization signal, at least one cell penetration domain, and / or at least one signal peptide capable of transporting the protein to a specific subcellular location, as described elsewhere herein.

[0209] The polynucleotides encoding RGN, crRNA, tracrRNA and / or sgRNA may be present in one or more vectors. "Vector" refers to a polynucleotide composition for transferring, delivering or introducing 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). The vector may include additional expression control sequences (e.g., enhancer sequences, Kozak sequences, polyadenylation sequences, transcription termination sequences), screening marker sequences (e.g., antibiotic resistance genes), replication origins, etc. Other information can be found in "Molecular Biology Laboratory Guide" Ausubel et al., John Wiley & Sons, New York, 2003 or "Molecular Cloning: Laboratory Manual" Sambrook and Russell, Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory, New York, 3rd edition, 2001.

[0210] The vector may also include a selectable marker gene for selecting transformed cells. Screenable marker genes are used for the selection of transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, for example, genes encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT); and genes that confer resistance to herbicidal compounds such as glufosinate, bromoxynil, imidazolinone, and 2,4-dichlorophenoxyacetate (2,4-D).

[0211] In some embodiments, the expression cassette or vector comprising a sequence encoding an RGN polypeptide may further include a sequence encoding crRNA and / or tracrRNA, or a combination of crRNA and tracrRNA to produce sgRNA. The (one or more) sequences encoding crRNA and / or tracrRNA may be operably connected to at least one transcription control sequence for expressing crRNA and / or tracrRNA in an organism or host cell of interest. For example, a polynucleotide encoding crRNA and / or tracrRNA may be operably connected 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, for example, human U6 promoters as shown in SEQ ID NO: 329, and promoters disclosed in U.S. Provisional Application No. 63 / 209,660 filed on June 11, 2021 (the entire contents of which are incorporated herein by reference), including those shown in SEQ ID NO: 449-458 herein.

[0212] As noted, expression constructs comprising nucleotide sequences encoding RGN, crRNA, tracrRNA and / or sgRNA can be used to transform an organism of interest. The method for transformation involves introducing a nucleotide construct into an organism of interest. "Introduction" is intended to introduce a nucleotide construct into a host cell in such a way that the construct can enter the interior of the host cell. The method of the present invention does not require a specific method for introducing a nucleotide construct into a host organism, only that the nucleotide construct can enter the interior of at least one cell of the host organism. The host cell may be a eukaryotic cell or a prokaryotic cell. In a particular embodiment, 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 or expressing a currently disclosed RGN or modified by a currently disclosed RGN is a human cell. In some embodiments, the eukaryotic cells comprising or expressing the presently disclosed RGNs or modified by the presently disclosed RGNs are cells of hematopoietic origin, such as immune cells (i.e., cells of the innate or acquired immune system), including but not limited to B cells, T cells, natural killer (NK) cells, enriched stem cells, induced enriched stem cells, chimeric antigen receptor T (CAR-T) cells, monocytes, macrophages, and dendritic cells. In some embodiments, the eukaryotic cells comprising or expressing the presently disclosed RGNs or modified by the presently disclosed RGNs are eye cells, muscle cells (e.g., skeletal muscle cells), epithelial cells (e.g., lung epithelial cells), diseased cells (e.g., tumor cells).

[0213] Methods for introducing nucleotide constructs into plants 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.

[0214] The method obtains a transformed organism, such as a plant, including whole plants and plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, propagules, embryos and their progeny. The plant cell can be differentiated or undifferentiated (e.g., callus, suspension culture cells, protoplasts, leaf cells, root cells, phloem cells, pollen).

[0215] "Transgenic organism" or "transformed organism" or "stably transformed" organism or cell or tissue refers to an organism that has incorporated or integrated a polynucleotide encoding the RGN, crRNA and / or tracrRNA of the present invention. It should be recognized that other exogenous or endogenous nucleic acid sequences or DNA fragments can also be incorporated into the host cell. Agrobacterium and gene gun-mediated transformation remain the two main methods used for plant cell transformation. However, transformation of host cells can be carried out by infection, transfection, microinjection, electroporation, microinjection, gene gun or particle bombardment, electroporation, silica / carbon fiber, ultrasound mediation, PEG mediation, calcium phosphate coprecipitation, polycation DMSO technology, DEAE dextran procedure, and virus mediation, liposome mediation, etc. Virus-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 cauliflower mosaic virus, geminivirus and RNA plant viruses.

[0216] Transformation protocols and protocols for introducing polypeptide or polynucleotide sequences into plants can vary depending on the type of host cell targeted for transformation (e.g., monocot or dicot plant cells). Methods for transformation are known in the art and include those described in U.S. Pat. Nos. 8,575,425, 7,692,068, 8,802,934, 7,541,517, each of which is incorporated herein by reference. See 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 Journal) 2:275-281; ​​Christou, P. (1995) Euphytica 85:13-27; Tzfira 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) Cultivation Methods 1:5.

[0217] Transformation can result in stable or transient incorporation of nucleic acids into cells. "Stable transformation" means that the nucleotide construct introduced into a host cell is integrated into the host cell's genome and can be inherited by its progeny. "Transient transformation" means that a polynucleotide is introduced into a host cell and is not integrated into the host cell's genome.

[0218] Methods for chloroplast transformation are known in the art, see, e.g., Svab et al. (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530; Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Svab and Maliga (1993) EMBO J. 12:601-606. Such methods rely on particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome via homologous recombination. In addition, plastid transformation can be achieved by transcriptionally activating silent plastid-borne transgenes through tissue-preferred expression of a nuclear-encoded and plastid-directed RNA polymerase. Such a system has been reported by McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91:7301-7305.

[0219] According to the traditional method, the transformed cells can be grown into transgenic organisms, for example, plants. For example, see McCormick et al. (1986) Plant Cell Reports 5: 81-84. Then, these plants can be grown and pollinated with the same transformed strain or different strains, and the resulting hybrids with the persistent expression of the desired expression type characteristics are identified. Two or more generations can be grown to ensure stable maintenance and inheritance of the expression of the desired expression type characteristics, and then seeds are harvested to ensure that the expression of the desired expression type characteristics has been achieved. In this way, the present invention provides transformed seeds (also referred to as "transgenic seeds") having the nucleotide construct of the present invention (e.g., expression cassette of the present invention) stably incorporated into its genome.

[0220] Alternatively, transformed cells may be introduced into an organism. These cells may be derived from said organism, wherein the cells were transformed in an ex vivo process.

[0221] The sequences provided herein can be used to transform any plant species, including but not limited to monocots and dicots. Examples of plants of concern include but are not limited to maize (corn), sorghum, wheat, sunflower, tomato, cruciferous plants, pepper, potato, cotton, rice, soybean, beet, sugarcane, tobacco, barley and rape, Brassica sp., alfalfa, rye, millet, safflower, peanut, sweet potato, cassava, coffee, coconut, pineapple, citrus, cocoa, tea, banana, avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond, oat, vegetables, ornamental plants and conifers.

[0222] Vegetables include, but are not limited to, tomatoes, lettuces, green beans, beans, peas, and members of the genus Curcumis such as cucumbers, cantaloupes, and cantaloupes. Ornamental plants include, but are not limited to, azaleas, hydrangeas, hibiscus, roses, tulips, narcissus, petunias, carnations, Christmas reds, and chrysanthemums. Preferably, the plants of the present invention are crops (e.g., corn, sorghum, wheat, sunflowers, tomatoes, cruciferous plants, peppers, potatoes, cotton, rice, soybeans, sugar beets, sugar cane, tobacco, barley, rapeseed, etc.).

[0223] As used herein, the term "plant" includes plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and intact plant cells in plants or parts of plants (e.g., embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, kernels, spikes, cobs, shells, stems, roots, root tips, anthers, etc.). Cereals refer to mature seeds produced by commercial growers for purposes other than growing or propagating species. Progeny, variants, and mutants of regenerated plants are also included within the scope of the present invention, provided that these parts include the introduced polynucleotides. Processed plant products or by-products retaining sequences disclosed herein are also provided, including, for example, soybean meal.

[0224] Polynucleotides encoding RGN, crRNA and / or tracrRNA, or comprising crRNA and / or tracrRNA, can also be used to transform any prokaryotic species, including but not limited to Archaea and bacteria (e.g., Bacillus, Klebsiella, Streptomyces, Rhizobium, Escherichia sp., Pseudomonas, Salmonella, Shigella, Vibrio, Yersinia, Mycoplasma, Agrobacterium, Lactobacillus).

[0225] Polynucleotides encoding RGN, crRNA and / or tracrRNA, or comprising 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, amoeba, algae, and yeast.

[0226] Conventional viral and non-viral gene transfer methods can be used to introduce nucleic acids into mammalian, insect or avian cells or target tissues. Such methods can be used to administer nucleic acids encoding RGN system components to cells in culture or cells in a host organism. Non-viral vector delivery systems include DNA plasmids, RNA (e.g., transcripts of vectors described herein), naked nucleic acids, and nucleic acids complexed with delivery vehicles such as liposomes. Viral vector delivery systems include DNA and RNA viruses that have episomal or integrated genomes after delivery to cells. For a review of gene therapy procedures, see Anderson, Science 256:808-813 (1992); Nabel and Feigner, TIBTECH 11:211-217 (1993); Mitani and 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 and Perricaudet, British Medical Bulletin 11:164-166 (1993); Bulletin 51(1):31-44 (1995); Haddada et al., Current Topics in Microbiology and Immunology, Doerfler and Bohm (eds.) (1995); and Yu et al., Gene Therapy 1:13-26 (1994).

[0227] Non-viral delivery methods for nucleic acids include lipofection, nucleofection, microinjection, gene guns, virosomes, liposomes, immunoliposomes, polycations or lipid:nucleic acid conjugates, naked DNA, artificial virus particles, and pharmacologically enhanced uptake of DNA. For example, lipofection is described in U.S. Pat. Nos. 5,049,386, 4,946,787, and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam TM Lipofectin TM). Cationic and neutral lipids suitable for efficient receptor recognition liposome transfection of polynucleotides include those of Feigner, WO 91 / 17424; WO 91 / 16024. Delivery can be to cells (eg, in vitro or ex vivo administration) or to target tissues (eg, in vivo administration). The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to those of ordinary skill in the art (see, 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 Ther. 2:710-722 (1995); Ahmad et al., Cancer Res. 2:710-722 (1995); Res.) 52:4817-4820 (1992); U.S. Patents 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).

[0228] The use of RNA or DNA virus-based systems to deliver nucleic acids utilizes highly evolved processes to target specific cells in the virus body and transport the viral load to the nucleus. Viral vectors can be directly applied to patients (in vivo) or can be used for in vitro treatment of cells, and modified cells can be optionally applied to patients (ex vivo). Traditional viral-based systems can include retroviruses, slow viruses, adenoviruses, adeno-associated and herpes simplex virus vectors for gene transfer. Integration in the host genome with retrovirus, slow virus and adeno-associated virus gene transfer methods is possible, often resulting in long-term expression of the inserted transgene. In addition, high transduction efficiency has been observed in many different cell types and target tissues.

[0229] The tropism of retroviruses can be altered by incorporating foreign envelope proteins, thereby expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and generally produce high viral titers. Therefore, the choice of retroviral gene transfer system will depend on the target tissue. Retroviral vectors are composed of cis-acting long terminal repeats, which have the ability to package foreign sequences up to 6-10 kb. The minimal cis-acting LTR is sufficient to replicate and package the vector, which is then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression. Widely used retroviral vectors include those based on mouse leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (e.g., see Buchscher et al., J. Viral. 66:2731-2739 (1992); Johann et al., J. Viral. 66:1635-1640 (1992); Sommnerfelt et al., J. 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).

[0230] In applications where transient expression is preferred, adenovirus-based systems can be used. Adenovirus-based vectors can have very high transduction efficiencies in many cell types and do not require cell division. Using such vectors, high titers and high expression levels have been achieved. This vector can be produced in large quantities in a relatively simple system. Adeno-associated virus ("AAV") vectors can also be used to transduce cells with target nucleic acids, such as in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (e.g., see 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, Journal of Clinical Research (I. Clin. Invest.) 94:1351 (1994)). The construction of recombinant AAV vectors is described in many publications, including U.S. Patent No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat and Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., Journal of Virology 63:03822-3828 (1989). Packaging cells are generally used to form viral particles capable of infecting host cells. Such cells include 293 cells for packaging adenoviruses and ψJ2 cells or PA317 cells for packaging retroviruses.

[0231] The viral vectors used in gene therapy are usually produced by producing a cell line that packages the nucleic acid vector in a viral particle. The vector usually contains the minimum viral sequence required for packaging and subsequent integration into the host, and other viral sequences replaced by the expression cassette of the polynucleotide to be expressed. The missing viral function is usually provided in trans by the packaging cell line. For example, the AAV vector used in gene therapy usually has only the ITR sequence from the AAV genome required for packaging and integration into the host genome. The viral DNA is packaged in a cell line that contains a helper plasmid encoding other AAV genes (i.e., rep and cap) but lacks ITR sequences.

[0232] Adenovirus can also be used as a helper to infect cell lines. The helper virus promotes the replication of the AAV vector and the expression of the AAV genes from the helper plasmid. Due to the lack of ITR sequences, the helper plasmid is not packaged in large quantities. Adenovirus contamination can be reduced by, for example, heat treatment, and adenovirus is more sensitive to heat treatment than AAV. Other methods for delivering nucleic acids to cells are known to those of ordinary skill in the art. For example, see US20030087817, which is incorporated herein by reference.

[0233] In some embodiments, host cells are transiently or non-transiently transfected with one or more vectors described herein. In some embodiments, cells are transfected when they are naturally present in an individual. In some embodiments, the transfected cells are taken from an individual. In some embodiments, the cells are derived from cells taken from an individual, such as a cell line. In some embodiments, the cell line can be a mammalian, insect, or avian cell. A variety of cell lines for tissue culture are known in the art. Examples of cell lines include, but are not limited to, C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLaS3, Huhl, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panel, PC-3, TF1, CTLL-2, CIR, Rat6, CVI, RPTE, A10, T24, 182, A375, ARH-77, Calul, SW480, SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB55, lurkat, 145.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A, BS-C-1 monkey kidney epithelial cells, BALB / 3T3 mouse embryonic fibroblasts, 3T3 Swiss, 3T3-Ll, 132-d5 human fetal fibroblasts; 10.1 mouse fibroblasts, 293-T, 3T3, 721, 9L, A2780, A2780ADR, A2780cis, A172, A20, A253, A431, A-549, ALC, B16, B35, BCP-I cells, BEAS-2B, bEnd.3, BHK-21, BR 293, BxPC3, C3H-10Tl / 2, C6 / 36, Cal-27, CHO, CHO-7, CHO-IR, CHO-Kl, CHO-K2, CHO-T, CHO Dhfr- / -, COR-L23, COR-L23 / CPR, COR-L235010, CORL23 / R23, COS-7, COV-434, CML Tl, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3, EMT6 / AR1, EMT6 / AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa, Hepalclc7, HL-60, HMEC, HT-29, lurkat, LY cells, K562 cells, Ku812, KCL22, KGl, KYOl, LNCap, Ma-Mel 1-48, MC-38, MCF-7, MCF-10A, MDA-MB-231, MDA-MB-468, MDA-MB-435, MDCKII, MDCKII, MOR / 0.2R, MONO-MAC 6, MTD-1A, MyEnd, NCI-H69 / CPR, NCI-H69 / LX10, NCI-H69 / LX20, NCI-H69 / LX4, NIH-3T3, NALM-1, NW-145, OPCN / OPCT cell lines, Peer, PNT-1A / PNT 2, RenCa, RIN-5F, RMA / RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell lines, U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1, YAR, and transgenic variants thereof. Cell lines can be obtained from a variety of sources known to those of ordinary skill in the art (e.g., see American Type Culture Collection (ATCC) (Manassas, Virginia)). .

[0234] In some embodiments, cells transfected with one or more vectors described herein are used to establish new cell lines comprising one or more vector-derived sequences. In some embodiments, cells transiently transfected with a composition of an RGN system described herein (e.g., by transient transfection of one or more vectors, or transfection with RNA) and modified via the activity of the RGN system are used to establish new cell lines comprising cells containing the modification 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 to evaluate one or more test compounds.

[0235] In some embodiments, one or more vectors described herein are used to generate non-human transgenic animals or transgenic plants. In some embodiments, the transgenic animal is a mammal, e.g., a mouse, rat, hamster, rabbit, cow, or pig. In some embodiments, the transgenic animal is a bird, e.g., a chicken or duck. In some embodiments, the transgenic animal is an insect, e.g., a mosquito or a tick.

[0236] V. Variants and fragments of polypeptides and polynucleotides

[0237] The present disclosure provides active variants and fragments of naturally occurring (i.e., wild-type) RNA-guided nucleases, whose amino acid sequences are shown in any one of SEQ ID NOs: 1-12; and active variants and fragments of naturally occurring CRISPR repeat sequences, for example, a sequence as shown in any one of SEQ ID NOs: 13-24; and active variants and fragments of naturally occurring tracrRNAs, for example, a sequence as shown in any one of SEQ ID NOs: 25-36; and polynucleotides encoding the sequences.

[0238] Although the activity of a variant or fragment may be altered compared to the polynucleotide or polypeptide of interest, the variant and fragment should retain the functionality of the polynucleotide or polypeptide of interest. For example, a variant or fragment may have increased activity, decreased activity, a different activity spectrum, or any other change in activity when compared to the polynucleotide or polypeptide of interest.

[0239] Fragments and variants of naturally occurring RGN polypeptides, such as those disclosed herein, will retain sequence-specific RNA-guided DNA binding activity. In specific embodiments, fragments and variants of naturally occurring RGN polypeptides, such as those disclosed herein, will retain nuclease activity (single-stranded or double-stranded).

[0240] Fragments and variants of naturally occurring CRISPR repeat sequences, such as those disclosed herein, when incorporated as part of a guide RNA, including tracrRNA, retain the ability to bind to and guide an RNA-guided nuclease (complexed with the guide RNA) to a target nucleotide sequence in a sequence-specific manner.

[0241] Fragments and variants of naturally occurring tracrRNA, such as those disclosed herein, when incorporated as 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.

[0242] The term "fragment" refers to a portion of a polynucleotide or polypeptide sequence of the invention. "Fragments" or "biologically active portions" include polynucleotides that include a sufficient number of contiguous nucleotides to retain biological activity (i.e., when included in a guide RNA, bind the RNA to and guide the RGN to a target nucleotide sequence in a sequence-specific manner). "Fragments" or "biologically active portions" include polypeptides that include a sufficient number of contiguous amino acid residues to retain biological activity (i.e., bind to a target nucleotide sequence in a sequence-specific manner when complexed with a guide RNA). Fragments of RGN proteins include those fragments that are 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 a polypeptide comprising, for example, 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 specificity, RNA-guided DNA binding activity. A biologically active fragment of a CRISPR repeat sequence can include at least 8 consecutive amino acids of any one of SEQ ID NOs: 13-24. The biologically active portion of the CRISPR repeat sequence can be a polynucleotide comprising, e.g., 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. The biologically active portion of the tracrRNA can be a polynucleotide comprising, e.g., 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.

[0243] In general, "variant" is intended to mean substantially similar sequences. For polynucleotides, variants include the deletion and / or addition of one or more nucleotides at one or more internal sites in natural polynucleotides, and / or the substitution of one or more nucleotides at one or more sites in natural polynucleotides. As used herein, "natural" or "wild-type" polynucleotides or polypeptides include naturally occurring nucleotide sequences or amino acid sequences, respectively. For polynucleotides, conservative variants include those sequences that encode the native amino acid sequence of the gene of interest because of the degeneracy of the genetic code. For example, well-known molecular biology techniques can be used to identify naturally occurring allelic variants (e.g., using polymerase chain reaction (PCR) and hybridization techniques as outlined below). Variant polynucleotides also include synthetically derived polynucleotides, such as those produced, for example, by using site-directed mutagenesis, but they still encode polypeptides or polynucleotides of interest. Generally speaking, 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 that particular polynucleotide as determined by the sequence alignment programs and parameters described elsewhere herein.

[0244] The variants of specific polynucleotides disclosed herein (i.e., reference polynucleotides) can also be evaluated by comparing the sequence identity percentage between the polypeptide encoded by the variant polynucleotide and the polypeptide encoded by the reference polynucleotide. The sequence alignment program and parameters described elsewhere herein can be used to calculate the sequence identity percentage between any two polypeptides. When evaluating any given polynucleotide pair disclosed herein by comparing the sequence identity percentage shared by two polypeptides encoded by any given polynucleotide disclosed herein, the sequence identity percentage between the two encoded polypeptides is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher consistency.

[0245] In certain embodiments, the polynucleotides disclosed herein encode RNA-guided nuclease polypeptides 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 the amino acid sequence shown in any one of SEQ ID NOs: 1-12.

[0246] Biologically active variants of the RGN polypeptides of the invention may differ by as few as about 1-15 amino acid residues, as few as about 1-10 (e.g., about 6-10), as few as 5, as few as 4, as few as 3, as few as 2, or as few as 1 amino acid residue. , 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700 or more amino acids from the N-terminus or C-terminus of the polypeptide.

[0247] In certain embodiments, the polynucleotides disclosed herein comprise or encode a CRISPR repeat sequence 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 the nucleotide sequence shown in any one of SEQ ID NOs: 13-24.

[0248] The polynucleotides disclosed in the present invention include or encode tracrRNA, and the tracrRNA includes 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 the nucleotide sequence shown in any one of SEQ ID NOs: 25-36.

[0249] Biologically active variants of the CRISPR repeat sequences or tracrRNA of the invention may differ by as few as about 1-15 nucleotides, as few as about 1-10 (e.g., about 6-10), as few as 5, as few as 4, as few as 3, as few as 2, or as few as 1 nucleotide. In specific embodiments, the polynucleotide may include a 5' or 3' truncation, which may include a deletion of at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 95, 100, 105, 110 or more nucleotides from the 5' or 3' end of the polynucleotide.

[0250] It is recognized that the RGN polypeptides, CRISPR repeat sequences and tracrRNA provided herein can be modified to produce variant proteins and polynucleotides. Artificially designed changes can be introduced through the application of site-directed mutagenesis techniques. Alternatively, natural, unknown or unidentified polynucleotides and / or polypeptides that are structurally and / or functionally related to the sequences disclosed herein can also be considered to fall 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 that improve RGN activity can be made.

[0251] Variant polynucleotides and proteins also include sequences and proteins derived from, for example, mutagenesis and recombination procedures (e.g., DNA shuffling). Using such procedures, one or more of the different RGN proteins disclosed herein (e.g., SEQ ID NOs: 1-12) are manipulated to generate new RGN proteins with desired properties. In this manner, a library of recombinant polynucleotides is generated from a population of related sequence polynucleotides that include sequence regions that have substantial sequence identity and can be homologously recombined 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 obtain proteins with improved properties of interest (e.g., in the case of an enzyme, increased K m) of a new gene encoding a protein of the same or similar origin. Strategies for such DNA shuffling are known in the art. For example, see Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotechnol. 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. Patents Nos. 5,605,793 and 5,837,458. A "shuffled" nucleic acid is a nucleic acid produced by a shuffling procedure, such as any of the shuffling procedures described herein. The nucleic acid of reorganization is produced by, for example, reorganizing two or more nucleic acids (or strings) in an artificial and optionally recursive manner (physically or virtually). In general, one or more screening steps are used in the reorganization process to identify the nucleic acid of interest; this screening step can be performed before or after any reorganization step. In some (but not all) reorganization embodiments, it is desirable to perform multiple rounds of reorganization before selection to increase the diversity of the pool to be screened. The entire process of reorganization and selection is optionally repeated recursively. Depending on the context, reorganization can refer to the entire process of reorganization and selection, or alternatively, can refer only to the reorganization part of the entire process.

[0252] As used herein, "sequence identity" or "identity" used in the context of two polynucleotides or polypeptide sequences refers to residues in two sequences that are identical when aligned over a specified comparison window for maximum correspondence. When using percentages of sequence identity associated with proteins, it should be recognized that different residue positions are typically different due to retention of amino acid substitutions, where the amino acid residues are replaced by other amino acid residues with similar chemical properties (e.g., charged or hydrophobic), and therefore do not change the functional properties of the molecule. When the sequences differ in retention of substitutions, the percentage of sequence identity can be adjusted upward to correct the retention of the substitutions. Sequences that differ by such retention of substitutions are referred to as having "sequence similarity" or "similarity". Means for making such adjustments are well known to those of ordinary skill in the art. Typically, this involves counting retention substitutions as partial mismatches rather than all mismatches, thereby increasing the percentage of sequence identity. Thus, for example, where the score for the same amino acid is 1 and the score for the non-retention of substitutions is 0, the score for the retention of substitutions is a score between 0 and 1. The score of conserved substitutions is calculated, for example, as implemented in the program PC / GENE (Intelligenetics, Mountain View, California).

[0253] As used herein, "sequence identity percentage" refers to a value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may include additions or deletions (i.e., gaps) compared to a reference sequence (excluding 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 appears in the two sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the sequence identity percentage.

[0254] Unless otherwise indicated, the sequence identity / similarity values ​​provided herein refer to the values ​​obtained using GAP version 10 using the following parameters: % identity and % similarity for nucleotide sequences using GAP weight 50 and length weight 3 and the nwsgapdna.cmp scoring matrix; % identity and % similarity for amino acid sequences using GAP weight 8 and length weight 2 and the BLOSUM62 scoring matrix; or any equivalent programs thereof. "Equivalent program" refers to any sequence comparison program that, when compared to the corresponding alignment generated by GAP version 10, produces an alignment with identical nucleotide or amino acid residue matches and identical sequence identity percentages for any two sequences involved.

[0255] Two sequences are "optimally aligned" when the similarity scores of two sequences are aligned using a defined amino acid substitution matrix (e.g., BLOSUM62), a gap existence penalty, and a gap extension penalty to achieve the highest score possible for the pair of sequences. Amino acid substitution matrices and their use in quantifying the 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 Supplement 3 (Edited by 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 workflows. The gap existence penalty is implemented for introducing a single amino acid gap in one of the alignment sequences, and the gap extension penalty is implemented for each other empty amino acid position inserted into the gap that has been opened. By comparing the amino acid position of each sequence at the beginning and end of the comparison, and optionally defining the comparison by inserting one or more gaps in one or two sequences, to reach the highest possible scoring. Although the best comparison and scoring can be completed manually, the process can be promoted by using the comparison algorithm implemented by a computer (for example, Altschul et al. (1997) Nucleic Acids Research 25: described in 3389-3402, and gapped BLAST 2.0 open to the public on the website (www.ncbi.nlm.nih.gov) of the National Center for Biotechnology Information (National Center for Biotechnology Information). For example, available via www.ncbi.nlm.nih.gov, and PSI-BLAST described in Altschul et al. (1997) Nucleic Acids Research 25: 3389-3402 prepares the best comparison including multiple comparisons.

[0256] With respect to an amino acid sequence that is optimally aligned with a reference sequence, an amino acid residue "corresponds to" the position in the reference sequence being aligned where the residue is paired with it. The "position" is represented by a number that identifies each amino acid in the reference sequence in order based on its position relative to the N-terminus. Due to deletions, insertions, truncations, fusions, etc. that must be considered when determining the optimal alignment, the number of amino acid residues in the test sequence, which is usually determined by simply counting from the N-terminus, does not have to be the same as the number of their corresponding positions in the reference sequence. For example, in the case of a deletion in the aligned test sequence, there will be no amino acid corresponding to the position at the deletion site in the reference sequence. In the case of an insertion in the aligned reference sequence, the insertion will not correspond to any amino acid position in the reference sequence. In the case of a truncation or fusion, there may be an amino acid stretch (stretch) in the reference sequence or the aligned sequence that does not correspond to any amino acid in the corresponding sequence.

[0257] VI. Antibodies

[0258] Also included are antibodies against the RGN polypeptides of the invention or ribonucleoproteins comprising the RGN polypeptides, including those having an amino acid sequence as set forth in any one of SEQ ID NOs: 1-12 or active variants or fragments thereof. Methods for producing antibodies are well known in the art (e.g., see Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York; and U.S. Patent No. 4,196,265). These antibodies can be used in kits for the detection and isolation of RGN polypeptides or ribonucleoproteins. Thus, the present disclosure provides kits comprising antibodies that specifically bind to the polypeptides or ribonucleoproteins described herein, including, for example, polypeptides having an amino acid sequence as set forth in any one of SEQ ID NOs: 1-12.

[0259] VII. Systems and ribonucleoprotein complexes for binding target sequences of interest and methods for making the same

[0260] The present disclosure provides a system for binding to a target sequence of interest, wherein the system includes at least one guide RNA or a nucleotide sequence encoding at least one guide RNA, and at least one RNA-guided nuclease or a nucleotide sequence encoding at least one RNA-guided nuclease. The guide RNA hybridizes with the target sequence of interest and also forms a complex with the RGN polypeptide, thereby guiding the RGN polypeptide to bind to the target sequence. In some of these embodiments, the RGN includes an amino acid sequence as shown in any one of SEQ ID NO: 1-12, or an active variant or fragment thereof. In various embodiments, the guide RNA includes a CRISPR repeat sequence, and the CRISPR repeat sequence includes a nucleotide sequence as shown in any one of SEQ ID NO: 13-24, or an active variant or fragment thereof. In a specific embodiment, the guide RNA includes a tracrRNA, and the tracrRNA includes a nucleotide sequence as shown in any one of SEQ ID NO: 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. In a specific embodiment, the system includes an RNA-guided nuclease heterologous to the guide RNA, wherein the RGN and the guide RNA are not complexed with each other (i.e., bound to each other) in nature.

[0261] The system provided herein for binding a target sequence of interest can be a ribonucleoprotein complex, which is at least one molecule of RNA bound to at least one protein. The ribonucleoprotein complex provided herein includes at least one guide RNA as an RNA composition and an RNA-guided nuclease as a protein composition. Such ribonucleoprotein complexes can be purified from cells or organisms that naturally express RGN polypeptides, and such ribonucleoprotein complexes have been engineered to express specific guide RNAs that are specific to the target sequence of interest. Alternatively, the ribonucleoprotein complex can be purified from cells or organisms that have been transformed with polynucleotides encoding RGN polypeptides and guide RNAs and cultured under conditions that allow the expression of RGN polypeptides and guide RNAs. Therefore, a method for making an RGN polypeptide or an RGN ribonucleoprotein complex is provided. Such methods include: culturing cells comprising a nucleotide sequence encoding an RGN polypeptide under conditions where the RGN polypeptide (and in some embodiments, guide RNA) is expressed, and in some embodiments, culturing cells comprising a nucleotide sequence encoding the guide RNA. Then, the RGN polypeptide or RGN ribonucleoprotein can be purified from the 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 the RNP complex comprises: combining one or more guide RNAs disclosed herein and one or more RGN polypeptides disclosed herein in a manner suitable for forming the RNP complex.

[0262] Methods for purifying RGN polypeptides or RGN ribonucleoprotein complexes from lysates of biological samples are known in the art (e.g., size analysis and / or affinity chromatography, 2D-PAGE, HPLC, reverse phase chromatography, immunoprecipitation). In certain methods, the RGN polypeptide is recombinantly produced and includes a purification tag that facilitates 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. Generally, labeled RGN polypeptides or RGN ribonucleoprotein complexes are purified using immobilized metal affinity chromatography. It will be appreciated that other similar methods known in the art, including other forms of chromatography or, for example, immunoprecipitation, may be used alone or in combination.

[0263] An "isolated" or "purified" polypeptide or biologically active portion thereof is substantially or essentially free of components found in its naturally occurring environment that normally accompany or interact with the polypeptide. Thus, when produced by recombinant techniques, an isolated or purified polypeptide is substantially free of other cellular material or culture medium, or when chemically synthesized, an isolated or purified polypeptide is substantially free of chemical precursors or other chemicals. Proteins substantially free of cellular material include protein preparations having less than about 30%, 20%, 10%, 5% or 1% (by dry weight) of contaminating proteins. When recombinantly producing the protein of the invention or its biologically active portion, the optimal culture medium presents less than about 30%, 20%, 10%, 5% or 1% (by dry weight) of chemical precursors or non-protein chemicals of interest. 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 has been removed from the locus of the genome via the cleavage of a phosphodiester bond. An isolated polynucleotide may be part of a vector, a composition of matter, or may be contained within a cell (so long as the cell is not the native environment of the polynucleotide).

[0264] The specific methods provided herein for binding and / or cleaving the target sequence of interest involve the use of RGN ribonucleoprotein complexes assembled in vitro. The in vitro assembly of the RGN ribonucleoprotein complex can be performed using any method known in the art, wherein the RGN polypeptide is contacted with the guide RNA under conditions that allow the RGN polypeptide to bind to the guide RNA. As used herein, "contact" and "contacted" refer to putting together the components of the desired reaction under conditions suitable for the desired reaction. The RGN polypeptide can be purified from a biological sample, cell lysate, or culture medium, produced via in vitro translation, or chemically synthesized. The guide RNA can be purified from a biological sample, cell lysate, or culture medium, transcribed in vitro, or chemically synthesized. The RGN polypeptide and the guide RNA can be contacted in a solution (e.g., a buffered saline solution) to allow the in vitro assembly of the RGN ribonucleoprotein complex.

[0265] VIII. Methods of Binding, Cleavage or Modification of Target Sequences

[0266] The present disclosure provides methods for binding, cutting and / or modifying a target nucleotide sequence of interest. The method includes delivering a system to a target sequence or a cell, organelle or embryo comprising a target sequence, the system including at least one guide RNA or a polynucleotide encoding at least one guide RNA, and at least one RGN polypeptide or a polynucleotide encoding at least one RGN polypeptide. In some of these embodiments, the RGN includes an amino acid sequence as shown in any one of SEQ ID NO: 1-12, or an active variant or fragment thereof. In various embodiments, the guide RNA includes a CRISPR repeat sequence, and the CRISPR repeat sequence includes a nucleotide sequence as shown in any one of SEQ ID NO: 13-24, or an active variant or fragment thereof. In a specific embodiment, the guide RNA includes a tracrRNA, and the tracrRNA includes a nucleotide sequence as shown in any one of SEQ ID NO: 25-36, or an active variant or fragment thereof. The guide RNA of the system may be a single guide RNA or a dual guide RNA.

[0267] The RGN of the system may be a nuclease-free RGN, have nickase activity, or may be a fusion polypeptide. In some embodiments, the fusion polypeptide includes a base editing polypeptide, for example, a cytosine deaminase or an adenine deaminase. In other embodiments, the RGN fusion protein includes a reverse transcriptase. In other embodiments, the RGN fusion protein includes a polypeptide that recruits members of a functional nucleic acid repair complex, for example, members of a 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, Journal of Molecular Biology 429 (9): 1277-1288), as described in U.S. Provisional Patent Application No. 62 / 966,203 filed on January 27, 2020, and the entire contents of which are incorporated herein by reference. In some embodiments, the RGN fusion protein includes CSB (van den Boom et al., 2004, J. Mol. Biol. 166(1):27-36; van Gool et al., 1997, EMBO J 16(19):5955-65; an example thereof is shown in SEQ ID NO:324), which is a member of the TC-NER (nucleotide excision repair) pathway and functions in the recruitment of other members. In further embodiments, the RGN fusion protein includes the active domain of CSB, for example, the acidic domain of CSB including amino acid residues 356-394 of SEQ ID NO:324 (Teng et al., 2018, Nat Commun. 9(1):4115).

[0268] In particular embodiments, the RGN and / or guide RNA are heterologous to the cell, organelle, or embryo into which the RGN and / or guide RNA (or a polynucleotide encoding at least one of the RGN and guide RNA) is introduced.

[0269] In those embodiments where the method includes delivering a polynucleotide encoding a guide RNA and / or an RGN polypeptide, the cell or embryo can then be cultured under conditions where the guide RNA and / or the RGN polypeptide is expressed. In various embodiments, the method includes contacting the target sequence with an RGN ribonucleoprotein complex. The RGN ribonucleoprotein complex may include an RGN without nuclease activity or with nickase activity. In some embodiments, the RGN of the ribonucleoprotein complex is a fusion polypeptide including a base editing polypeptide. In certain embodiments, the method includes introducing the RGN ribonucleoprotein complex into a cell, organelle, or embryo including the target sequence. The RGN ribonucleoprotein complex may be a complex that has been purified from a biological sample, recombinantly produced and subsequently purified, or assembled in vitro as described herein. In those embodiments where the RGN ribonucleoprotein complex in contact with the target sequence or cell organelle or embryo has been assembled in vitro, the method may further include assembling the complex in vitro before contacting the target sequence, cell, organelle, or embryo.

[0270] 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, polynucleotides encoding or comprising guide RNAs and / or RGN polypeptides can be introduced into cells, organelles, or embryos using any method known in the art, such as electroporation.

[0271] When delivered to or contacted with a target sequence or a cell, organelle, or embryo comprising a target sequence, the guide RNA guides the RGN to bind to the target sequence in a sequence-specific manner. In those embodiments where the RGN has nuclease activity, the RGN polypeptide cleaves the target sequence of interest upon binding. Subsequently, the target sequence can be modified via endogenous repair mechanisms such as non-homologous end joining or homology-mediated repair using a provided donor polynucleotide.

[0272] Methods for measuring binding of RGN polypeptides to target sequences are known in the art and include chromatin immunoprecipitation assays, gel shift assays, DNA pull-down assays, reporter assays, microplate capture and detection assays. Likewise, methods for measuring cleavage or modification of target sequences are known in the art and include in vitro or in vivo cleavage assays, where 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 nick-triggered exponential amplification reaction (NTEXPAR) assay can be used (e.g., see Zhang et al. (2016) Chem. Sci. 7:4951-4957). In vivo cleavage can be assessed using the Surveyor assay (Guschin et al. (2010) Methods Mol Biol 649:247-256).

[0273] In some embodiments, the methods involve the use of a single type of RGN complexed with more than one guide RNA. The more than one guide RNA can target different regions of a single gene, or can target multiple genes.

[0274] In those embodiments where a donor polynucleotide is not provided, double-strand breaks introduced by the RGN polypeptide can be repaired by a non-homologous end joining (NHEJ) repair process. Due to the error-prone nature of NHEJ, repair of double-strand breaks 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 a nucleic acid molecule, which can be a deletion, insertion or substitution of one or more nucleotides, or a combination thereof. Modification of the target sequence can result in expression of an altered protein product or inactivation of the coding sequence.

[0275] In those embodiments in which a donor polynucleotide is present, the donor sequence in the donor polynucleotide can be integrated into the target nucleotide sequence or exchanged with the target nucleotide sequence during the process of repairing the introduced double-strand break, resulting in the introduction of an exogenous donor sequence. Thus, the donor polynucleotide includes a donor sequence that is 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 integrated donor sequence will not be recognized and cut by the RGN. The integration of the donor sequence can be enhanced by including adjacent sequences in the donor polynucleotide, which are referred to herein as "homology arms" that have substantial sequence identity to the sequence adjacent to the target nucleotide sequence to allow for a homology-guided 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 homology with their corresponding sequences within the target nucleotide sequence.

[0276] In those embodiments in which the RGN polypeptide introduces double-stranded staggered breaks, the donor polynucleotide can include a donor sequence adjacent to a compatible overhang to allow for direct ligation of the donor sequence to the cleaved target nucleotide sequence including the overhang by a non-homologous repair process during repair of the double-stranded break.

[0277] In those embodiments where the method involves the use of an RGN that is a nickase (i.e., capable of cleaving only a single 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 plus (+) strand of a double-stranded polynucleotide can be introduced together with a second RGN nickase that cleaves only the minus (-) strand of a double-stranded polynucleotide.

[0278] In various embodiments, a method for binding to a target nucleotide sequence and detecting the target sequence is provided, wherein the method comprises introducing at least one guide RNA or a polynucleotide encoding at least one guide RNA, and at least one RGN polypeptide or a polynucleotide encoding at least one RGN polypeptide into a cell, organelle, or embryo; expressing the guide RNA and / or RGN polypeptide (if the coding sequence is 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 can be fused to the RGN as a fusion protein (e.g., a fluorescent protein), or can be a small molecule conjugated to or incorporated into the RGN polypeptide that can be detected visually or by other means.

[0279] Also provided herein are methods for regulating the expression of a target sequence or gene of interest under the control of a target sequence. The method comprises introducing at least one guide RNA or a polynucleotide encoding at least one guide RNA, and at least one RGN polypeptide or a polynucleotide encoding at least one RGN polypeptide into a cell, an organelle, or an embryo; expressing the guide RNA and / or the RGN polypeptide (if the 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 repressor domain) as described herein.

[0280] The present disclosure also provides a method for binding and / or modifying a target nucleotide sequence of interest. The method includes delivering a system to a target sequence or a cell, organelle, or embryo comprising a target sequence, the system comprising at least one guide RNA or a polynucleotide encoding at least one guide RNA, and at least one fusion polypeptide, at least one fusion polypeptide comprising an RGN of the present invention and a base editing polypeptide (e.g., cytosine deaminase or adenine deaminase) or a polynucleotide encoding a fusion polypeptide.

[0281] One of ordinary skill in the art will appreciate that any of the methods disclosed herein can be used to target a single target sequence or multiple target sequences. Thus, these methods include using 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 included 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.

[0282] In one aspect, the present invention provides a kit comprising any one or more elements disclosed in the above methods and compositions. In some embodiments, the kit includes a vector system and instructions for using the kit. In some embodiments, the vector system includes (a) a first regulatory element, the first regulatory element is operably connected 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, wherein, when expressed, the guide sequence guides the sequence-specific binding of the RGN complex to a target sequence in a eukaryotic cell, wherein the RGN complex includes an RGN enzyme complexed with a guide RNA polynucleotide; and / or (b) a second regulatory element, the second regulatory element is operably connected to an enzyme coding sequence, and the enzyme coding sequence encodes the RGN enzyme including a nuclear localization sequence. Elements can be provided individually or in combination and can be provided in any suitable container, for example, a vial, a bottle or a tube.

[0283] In certain embodiments, the kit includes instructions in one or more languages. In certain embodiments, the kit includes one or more reagents used in the method utilizing one or more elements as described herein. Reagent can be provided in any suitable container. For example, the kit can provide one or more reaction or storage buffers. Reagent can be provided in a form that can be used for special determination or in a form (for example, in concentrated or lyophilized form) that needs to add one or more other compositions before use. Buffer can 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 certain embodiments, buffer is alkaline. In certain embodiments, buffer has a pH of about 7 to about 10.

[0284] In some embodiments, the kit includes one or more oligonucleotides corresponding to the guide sequence for insertion into the vector, so as to operably connect the guide sequence to the regulatory element. In some embodiments, the kit includes a homologous recombination template polynucleotide. In one aspect, the present invention provides a method for using one or more elements of the 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 variety of uses, including modifying (e.g., deletion, insertion, displacement, inactivation, activation, base editing) target polynucleotides in a variety of cell types. In this way, the RGN system of the present invention has a wide range of 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, and the guide sequence hybridizes with a target sequence in a target polynucleotide.

[0285] IX. Target Polynucleotide

[0286] In one aspect, the invention provides methods for modifying a target polynucleotide in a eukaryotic cell, which can be in vivo, ex vivo or in vitro. In some embodiments, the method comprises sampling a cell or cell population from a human or non-human animal or plant (including microalgae), and modifying the cell or cells. Cultivation can occur at any stage in vitro. The cell or cells can even be reintroduced into a non-human animal or plant (including microalgae).

[0287] Using natural variability, plant breeders combine most useful genes to obtain desired qualities, such as yield, quality, consistency, tolerance, and resistance to pests. These desired qualities also include growth, day length preference, temperature requirements, start date of flower or reproductive development, fatty acid content, insect resistance, disease resistance, nematode resistance, fungal resistance, herbicide resistance, tolerance to various environmental factors (including drought, heat, humidity, cold, wind, and unfavorable soil conditions including high salinity). The sources of these useful genes include natural or exotic varieties, native species (heirloom variety), wild plant relatives, and induced mutations such as treatment of plant materials with mutagens. Using the present invention, a new tool for inducing mutations is provided for plant breeders. Therefore, a person of ordinary skill in the art can analyze the genome to find the source of useful genes, and adopt the present invention in varieties with desired characteristics or traits to induce the increase of useful genes more accurately than previous mutagens, and thus accelerate and improve plant breeding programs.

[0288] The target polynucleotide of the RGN system can be any polynucleotide that is 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 be associated with a PAM (pre-spacer adjacent motif); that is, a short sequence recognized by the RGN system. The exact sequence and length requirements of the PAM vary depending on the RGN used, but the PAM is typically a 2-7 base pair sequence adjacent to the pre-spacer sequence (i.e., the target sequence).

[0289] The target polynucleotides of the RGN system can include many disease-associated genes and polynucleotides and genes and polynucleotides associated with signal transduction biochemical pathways. Examples of target polynucleotides include sequences associated with signal transduction biochemical pathways, for example, genes or polynucleotides associated with signal transduction biochemical pathways. Examples of target polynucleotides include genes or polynucleotides associated with diseases. "Disease-associated" genes or polynucleotides refer to any gene or polynucleotide that produces transcription or translation products at abnormal levels or in abnormal forms in cells taken from diseased tissues compared to non-disease-controlled tissues or cells. It can be a gene that becomes expressed at an abnormally high level; it can be a gene that becomes expressed at an abnormally low level, wherein the altered expression is associated with the occurrence and / or progression of the disease. Genes associated with diseases also refer to genes with mutations or gene variations, genes with mutations or gene variations that directly cause the disease (e.g., causal mutations), or are in linkage disequilibrium with genes that cause the disease (e.g., causal mutations). The causal mutation can be an extended repetition (more than the number of normal individuals), resulting in instability of the transcribed mRNA, splicing of the change or instability of the translation product or reduced translation product activity. The product of transcription or translation can be known or unknown, and can also be at a normal or abnormal level. In some embodiments, the disease can be an animal disease. In some embodiments, the disease can be a bird disease. In other embodiments, the disease can be a mammalian disease. In a further embodiment, the disease can be a human disease. Examples of human disease-associated genes and polynucleotides are provided in Table 6, and can also be obtained from the Johns Hopkins University (Baltimore, Maryland) McKusick-Nathans Institute of Genetic Medicine and the National Library of Medicine (Bethesda, Md.) National Center for Biotechnology Information available on the World Wide Web.

[0290] While the RGN system is particularly useful because of its relative ease of targeting genomic sequences of interest, there remains the question of what RGNs can do to address causal mutations. One approach is to produce a fusion protein between an RGN (preferably an inactive or nickase variant of an RGN) and an active domain of a base editing enzyme or base editing enzyme (e.g., 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 of the invention or a nickase variant thereof and a base editing polypeptide such as a deaminase; and contacting with (b) a gRNA that targets the fusion protein of (a) to a target nucleotide sequence of a DNA chain; wherein the DNA molecule is contacted with the fusion protein and gRNA in an effective amount and under conditions suitable for deamination of a nucleobase. In some embodiments, the target DNA sequence comprises a sequence associated with a disease or disorder, and wherein deamination of the nucleobase results in a sequence that is not associated with the disease or disorder. In some embodiments, the target DNA sequence is located in an allele of a crop plant, wherein a particular allele of a trait of interest results in a plant of lower agronomic value. Deamination of the nucleobase results in an allele that improves the trait of the plant and increases the agronomic value of the plant.

[0291] In some embodiments, the DNA sequence comprises a T→C or A→G point mutation associated with a disease or condition, and wherein deamination of the mutant C or G base results in a sequence not associated with the disease or condition. In some embodiments, deamination corrects a point mutation in a sequence associated with a disease or condition.

[0292] In some embodiments, the sequence associated with the disease or condition encodes a protein, and wherein deamination introduces a stop codon into the sequence associated with the disease or condition, resulting in truncation of the encoded protein. In some embodiments, contacting is performed in an individual susceptible to, suffering from, or diagnosed with a disease or condition. In some embodiments, the disease or condition is a disease associated with a point mutation or a single base mutation in the genome. In some embodiments, the disease is a genetic disease, cancer, metabolic disease, or lysosomal storage disease.

[0293] X. Pharmaceutical compositions and methods of treatment

[0294] Provided are pharmaceutical compositions comprising RGN polypeptides disclosed herein, active variants and fragments thereof, and polynucleotides encoding RGN polypeptides and active variants and fragments thereof, gRNAs disclosed herein or polynucleotides encoding gRNAs, systems disclosed herein, or cells comprising any one of RGN polypeptides or RGN encoding polynucleotides, gRNAs or gRNA encoding polynucleotides, or RGN systems, and pharmaceutically acceptable carriers.

[0295] A pharmaceutical composition is a composition used to prevent, reduce the intensity, cure or treat a target condition or disease, comprising an active ingredient (i.e., an RGN polypeptide, a polynucleotide encoding an RGN, a gRNA, a polynucleotide encoding a gRNA, an RGN system, or a cell comprising any of these) and a pharmaceutically acceptable carrier.

[0296] As used herein, a "pharmaceutically acceptable carrier" refers to a material that does not cause significant irritation to an organism and does not abrogate the activity and properties of the active ingredient (i.e., RGN polypeptide, RGN encoded polynucleotide, gRNA, gRNA encoded polynucleotide, RGN system, or a cell comprising any of these). The carrier must be of sufficiently high purity and sufficiently low toxicity to make it suitable for administration to the individual being treated. The carrier may be inert, or it may have a pharmaceutical benefit. In some embodiments, a pharmaceutically acceptable carrier comprises one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to humans or other vertebrates. In some embodiments, the pharmaceutically acceptable carrier is not naturally occurring. In some embodiments, the pharmaceutically acceptable carrier is not found in nature together with the active ingredient.

[0297] The pharmaceutical composition used in the method disclosed in the present invention can be formulated with suitable carriers, excipients and other medicaments that provide suitable transfer, delivery, tolerance, etc. Many suitable formulations are known to those of ordinary skill in the art. For example, see Remington: The Science and Practice of Pharmacy (21st edition, 2005). For example, suitable formulations include powders, pastes, ointments, jellies, waxes, oils, lipids, vesicles containing lipids (cations or anions) (e.g., LIPOFECTIN vesicles), lipid nanoparticles, DNA conjugates, anhydrous absorption pastes, water-oil emulsions and water-oil emulsions, emulsions carbowax (polyethylene glycol of various molecular weights), semi-solid gels and semi-solid mixtures containing carbowax. The pharmaceutical composition for oral or parenteral use can be prepared as a dosage form suitable for adapting to the dosage of the active ingredient in a unit dose. For example, these dosage forms of unit doses include tablets, pills, capsules, injections (ampoules), suppositories, etc.

[0298] In some embodiments in which cells comprising or containing RGNs, gRNAs, RGN systems, or polynucleotides encoding RGNs, gRNAs, RGN systems disclosed herein are administered to an individual, the cells are administered as a suspension along with a pharmaceutically acceptable carrier. One of ordinary skill in the art will recognize that pharmaceutically acceptable carriers used in cell compositions will not include buffers, compounds, cryopreservatives, preservatives, or other formulations in an amount that substantially interferes with the viability of the cells to be delivered to the individual. Formulations comprising cells may include, for example, osmotic pressure buffers that allow the cell membrane to maintain integrity, and optionally include nutrients that maintain cell viability or enhance implantation upon administration. Such formulations and suspensions are known to those of ordinary skill in the art, and / or can be adapted for use with the cells disclosed herein using routine experimentation.

[0299] The cell composition may also be emulsified or presented as a ribosomal composition, provided that the emulsification procedure does not adversely affect cell viability.The cells and any other active ingredients may be mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredients, and in amounts suitable for use in the therapeutic methods disclosed herein.

[0300] The additives included in the cell composition may include pharmaceutically acceptable salts of the compositions therein. For example, pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of polypeptides) formed with inorganic acids such as hydrochloric acid or phosphoric acid, or with organic acids such as acetic acid, tartaric acid, mandelic acid, etc. For example, salts formed with free carboxyl groups may also be derived from inorganic bases such as hydroxides of sodium, potassium, ammonium, calcium or iron, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0301] Physiologically tolerable and pharmaceutically acceptable carriers are known in the art. Exemplary liquid carriers are sterile aqueous solutions that contain no other materials except active ingredients and water, or contain, for example, sodium phosphate, saline or a buffer of both at physiological pH (e.g., phosphate buffered saline). In addition, aqueous carriers may contain more than one buffer salt and salts such as sodium chloride and potassium chloride, glucose, polyethylene glycol and other solutes. Liquid compositions may also contain a liquid phase other than water and excluding water. Examples of such added liquid phases are glycerol, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of active compound used in an effective cell composition for the treatment of a particular disorder or condition may depend on the nature of the disorder or condition and may be determined by standard clinical techniques.

[0302] The RGN polypeptides, guide RNAs, RGN systems, or polynucleotides encoding RGN polypeptides, guide RNAs, RGN systems disclosed herein can be formulated with pharmaceutically acceptable excipients such as carriers, solvents, stabilizers, adjuvants, diluents, etc., depending on the particular mode of administration and dosage form. In some embodiments, these pharmaceutical compositions are formulated to achieve a physiologically compatible pH, and range from about pH 3 to about pH 11, about pH 3 to about pH 7, depending on the formulation and route of administration. In some embodiments, the pH can be adjusted to a range of about pH 5.0 to about pH 8. In some embodiments, the composition can include a therapeutically effective amount of at least one compound described herein, together with one or more pharmaceutically acceptable excipients. In some embodiments, the composition includes a combination of the compounds described herein, or includes a second active ingredient useful in treating or preventing bacterial growth (for example, without limitation, an antibacterial or antimicrobial agent), or includes a combination of agents disclosed herein.

[0303] For example, suitable excipients include carrier molecules, which include large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymerized amino acids, amino acid copolymers, and inactive viral particles. Other exemplary excipients may include antioxidants (such as, but not limited to, ascorbic acid), chelating agents (such as, but not limited to, EDTA), carbohydrates (such as, but not limited to, dextrin, hydroxyalkyl cellulose, and hydroxyalkyl methyl cellulose), stearic acid, liquids (such as, but not limited to, oil, water, saline, glycerol, and ethanol), wetting or emulsifiers, pH buffer substances, and the like.

[0304] In some embodiments, the formulations are provided in unit dose or multi-dose containers (e.g., sealed ampoules and vials) and can be stored in freeze-dried (lyophilized) conditions, requiring the addition of a sterile liquid carrier (e.g., saline, water for injection, semi-liquid foam or gel) immediately prior to use. Immediate injection solutions and suspensions can be prepared from sterile powders, granules and tablets of the aforementioned types. In some embodiments, the active ingredient is dissolved in a buffered liquid solution, which is frozen in a unit dose or multi-dose container and then thawed for injection or refrigerated for storage / stabilization until use.

[0305] One or more therapeutic agents can be included in the controlled release system. In order to prolong the effect of the drug, it is often desired to slow down the absorption of the drug from subcutaneous, intrathecal cavity, or intramuscular injection. This can be accomplished by using a liquid suspension of a crystalline or non-crystalline material with poor water solubility. Then, the absorption rate of the drug depends on its dissolution rate, and its dissolution rate can depend on crystal size and crystalline form. Alternatively, the delayed absorption of non-oral drug forms is accomplished by dissolving the drug or being suspended in an oil medium. In certain embodiments, the use of the implant of long-term sustained release is particularly suitable for the treatment of chronic conditions. The implant of long-term sustained release is known to those of ordinary skill in the art.

[0306] Provided herein are methods for treating a disease in an individual in need thereof. The method comprises administering to an individual in need thereof an effective amount of an RGN polypeptide disclosed herein or an active variant or fragment thereof, or a polynucleotide encoding an RGN polypeptide or an active variant or fragment thereof, a gRNA disclosed herein or a polynucleotide encoding a gRNA, an RGN system disclosed herein, or a cell modified by or comprising any of these compositions.

[0307] In some embodiments, treatment includes in vivo gene editing by administering RGN polypeptides, gRNAs, or RGN systems disclosed herein, or (one or more) polynucleotides encoding RGN polypeptides, gRNAs, or RGN systems. In some embodiments, treatment includes in vitro gene editing, wherein cells are in vitro genetically modified with RGN polypeptides, gRNAs, or RGN systems disclosed herein, or (one or more) polynucleotides encoding RGN polypeptides, gRNAs, or RGN systems, and then the modified cells are administered to individuals. In some embodiments, genetically modified cells are derived from individuals to whom modified cells are subsequently administered, and transplanted cells are referred to herein as autologous. In some embodiments, genetically modified cells are derived from different individuals (i.e., donors) within the same species as the individuals (i.e., recipients) to whom modified cells are administered, and transplanted cells are referred to herein as allogeneic. In some examples described herein, cells may be expanded in culture before being administered to individuals in need.

[0308] In some embodiments, the disease to be treated with the composition disclosed in the present invention is a disease that can be treated with immunotherapy (e.g., with chimeric antigen receptor (CAR) T cells). Such diseases include, but are not limited to, cancer. In some embodiments, the disease to be treated with the composition disclosed in the present invention is associated with a causal mutation. As used herein, a "causal mutation" refers to a specific nucleotide, multiple nucleotides, or nucleotide sequence in the genome that contributes to the severity or presence of a disease or disorder in an individual. Correction of a causal mutation results in improvement of at least one symptom caused by a disease or disorder. In some embodiments, the causal mutation is adjacent to the PAM site identified by the RGN disclosed herein. The causal mutation can be corrected with an RGN disclosed in the present invention or a fusion polypeptide comprising an RGN disclosed in the present invention and a base-edited polypeptide (i.e., a base editor). Non-limiting examples of diseases associated with causal mutations include cystic fibrosis, Hurler syndrome, Friedreich's Ataxia, Huntington's Disease, and sickle cell disease. In some embodiments, the disease to be treated with the RGN disclosed in the present invention is a disease listed in Table 6. Additional non-limiting examples of disease-associated genes and mutations are available from the McCosker-Nathans Institute for Genetic Medicine at Johns Hopkins University (Baltimore, MD) and the National Center for Biotechnology Information at the National Library of Medicine (Bethesda, MD) available on the World Wide Web.

[0309] As used herein, "treatment" or "treatment", or "mitigation" or "improvement" can be used interchangeably. These terms refer to methods for obtaining beneficial or desired results, including but not limited to therapeutic benefits and / or preventive benefits. Therapeutic benefit means any treatment-related improvement in one or more diseases, disorders or symptoms in treatment or the effect on one or more diseases, disorders or symptoms in treatment. For preventive benefit, the composition can be applied to individuals at risk of development of a specific disease, disorder or symptom, or to individuals reporting one or more physiological symptoms of the disease, even if the disease, disorder or symptom may not yet present signs. In some embodiments, treatment can be performed after one or more symptoms appear and / or the disease is diagnosed. In specific embodiments, treatment can be performed in the absence of symptoms, for example, to prevent or delay the appearance of symptoms or to inhibit the appearance or progression of the disease. For example, susceptible individuals can be treated before symptoms appear (for example, according to symptom history and / or according to genetic or other sensitive factors). For example, treatment can also be continued after the symptoms resolve and subside, to prevent or delay its prevention or recurrence.

[0310] The term "effective amount" or "therapeutically effective amount" refers to an amount of a drug sufficient to achieve a beneficial or desired result. The therapeutically effective amount may vary depending on one or more of the following: the individual and disease condition being treated, the weight and age of the individual, the severity of the disease condition, the mode of administration, etc., and can be easily determined by one of ordinary skill in the art. The specific dose may vary depending on one or more of the following: the specific agent selected, the dosing regimen to be followed, whether it is administered in combination with other compounds, the time of administration, and the delivery system in which it is carried.

[0311] The term "administering" refers to placing an active ingredient in an individual by a method or approach that causes the introduced active ingredient to be at least partially localized at a desired site (e.g., a site of injury or repair) so that (one or more) desired effects are produced. In some embodiments, the disclosure provides methods comprising delivering any of the RGN polypeptides, nucleic acid molecules, ribonucleoprotein complexes, vectors, pharmaceutical compositions, and / or gRNA disclosed herein. In some embodiments, the disclosure further provides cells produced by such methods, and organisms (e.g., animals or plants) comprising or produced by such cells. In some embodiments, RGN polypeptides and / or nucleic acid molecules combined (and optionally complexed) with guide sequences as described herein are delivered to cells.

[0312] In those embodiments in which cells are administered, the cells can be administered by any suitable route resulting in delivery to a desired location in an individual, where at least a portion of the implanted cells or composition of cells remain viable. The survival period of the cells after administration to an individual can be as short as a few hours (e.g., twenty-four hours), to a few days, to as long as several years, or even the life span of the patient, i.e., long-term implantation. For example, in some aspects described herein, an effective amount of photoreceptor cells or retinal progenitor cells is administered via a systemic route of administration (e.g., intraperitoneal or intravenous route).

[0313] In some embodiments, administration comprises administration by viral delivery. Viral vectors comprising nucleic acids encoding RGN polypeptides disclosed herein, ribonucleoprotein complexes or vectors can be administered directly to a patient (i.e., in vivo) or can be used to treat cells in vitro, and the modified cells can optionally be administered to a patient (i.e., ex vivo). Conventional viral-based systems can include, but are not limited to, retroviral, lentiviral, adenoviral, adeno-associated and herpes simplex viral vectors for gene transfer. Integration in the host genome is possible using retroviral, lentiviral and adeno-associated viral gene transfer methods, often resulting in long-term expression of the inserted transgene. Lentiviral vectors are retroviral vectors that are capable of transducing or infecting non-dividing cells and generally produce high viral titers. In applications where transient expression is preferred, adenoviral-based systems can be used. Adenoviral-based vectors can have very high transduction efficiencies in many cell types and do not require cell division.

[0314] In some embodiments, administration includes administration by other non-viral delivery of nucleic acid. In the absence of limitation, exemplary non-viral delivery methods include RNP complexes, lipofection, nuclear transfection, microinjection, gene guns, virosomes, liposomes, immunoliposomes, polycationic or lipid nucleic acid conjugates, naked DNA, artificial virus particles, and drug-enhanced uptake of DNA. For example, lipofection is described in U.S. Patents 5,049,386, 4,946,787, and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam TM Lipofectin TM ). Cationic and neutral lipids suitable for efficient receptor recognition liposome transfection of polynucleotides include those of Feigner WO1991 / 17424; WO1991 / 16024. Delivery can be to cells (eg, in vitro or ex vivo administration) or to target tissues (eg, in vivo administration).

[0315] In some embodiments, administration comprises administration by a method selected from the group consisting of: intravenously, subcutaneously, intramuscularly, orally, rectally, by aerosol, parenterally, ocularly, pulmonary, transdermally, vaginally, aurally, nasally, and by external administration, or any combination thereof. In some embodiments, for delivery of cells, administration by injection or perfusion is used.

[0316] As used herein, the term "subject" refers to any individual for whom diagnosis, treatment, or therapy is desired. In some embodiments, the individual is an animal. In some embodiments, the individual is a mammal. In some embodiments, the individual is a human.

[0317] The efficacy of a treatment can be determined by a skilled clinician. However, 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 accepted symptoms or markers of a disease are ameliorated or improved, then the treatment is considered "effective treatment". Efficacy can also be measured by the individual not experiencing a deterioration as assessed by hospitalization, or not requiring medical intervention (e.g., the progression of the disease is stopped or at least slowed). Methods for measuring these indicators are known to those of ordinary skill in the art. Treatment includes: (1) inhibiting the disease, e.g., arresting or slowing the progression of symptoms; or (2) mitigating the disease, e.g., causing regression of symptoms; and (3) preventing or reducing the likelihood of symptom development.

[0318] Provided are pharmaceutical compositions comprising an RGN polypeptide or a polynucleotide encoding an RGN polypeptide disclosed herein, a gRNA or a polynucleotide encoding a gRNA disclosed herein, a system disclosed herein, a ribonucleoprotein complex or cell comprising any of an RGN polypeptide or a polynucleotide encoding an RGN polypeptide, a gRNA or a polynucleotide encoding a gRNA, or a system disclosed herein, and a pharmaceutically acceptable carrier.

[0319] As used herein, "pharmaceutically acceptable carrier" refers to a material that does not cause significant irritation to an organism and does not abrogate the activity and properties of the active ingredient (e.g., RGN polypeptide or nucleic acid molecule encoding RGN polypeptide). The carrier must be of sufficiently high purity and sufficiently low toxicity to make it suitable for administration to the individual being treated. The carrier may be inert, or may have a pharmaceutical benefit. In some embodiments, a pharmaceutically acceptable carrier comprises one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to humans or other vertebrates. In some embodiments, a pharmaceutical composition comprises a pharmaceutically acceptable carrier that is not naturally occurring. In some embodiments, the pharmaceutically acceptable carrier is not found in nature with the active ingredient and is therefore heterologous.

[0320] The pharmaceutical compositions used in the methods disclosed herein can be formulated with suitable carriers, excipients and other agents that provide suitable transfer, delivery, tolerance, etc. Numerous suitable formulations are known to those of ordinary skill in the art. For example, see Remington: Pharmaceutical Science and Practice (21st Edition, 2005). Non-limiting examples include sterile diluents, such as water for injection, saline solution, non-volatile oils, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methyl paraben; 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 tension, such as sodium chloride or glucose. Specific carriers for intravenous administration are physiological saline or phosphate buffered saline (PBS). Pharmaceutical compositions for oral or parenteral use can be prepared in unit dosage forms suitable for adapting to the dosage of the active ingredient. For example, these unit dosage forms include tablets, pills, capsules, injections (ampoules), suppositories, etc. These compositions may also contain adjuvants, including preservatives, wetting agents, emulsifiers and dispersants. Protection against the action of microorganisms may be ensured by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, etc.). It may also be desirable to include isotonic agents, such as sugars, sodium chloride, etc. The absorption of injectable drug forms may be prolonged by the use of agents that delay absorption (e.g., aluminum monostearate and gelatin).

[0321] In some embodiments in which cells comprising or with RGNs, gRNAs, systems, or polynucleotides encoding RGNs, gRNAs, systems disclosed herein are administered to an individual, these cells are administered as a suspension along with a pharmaceutically acceptable carrier. One of ordinary skill in the art will recognize that pharmaceutically acceptable carriers used in cell compositions will not include buffers, compounds, cryopreservatives, preservatives, or other formulations that substantially interfere with the viability of the cells to be delivered to the individual. Formulations comprising cells may include, for example, osmotic pressure buffers that allow the cell membrane to maintain integrity, and optionally include nutrients that maintain cell viability or enhance implantation upon administration. Such formulations and suspensions are known to those of ordinary skill in the art, and / or can be adapted for use with the cells disclosed herein using routine experiments.

[0322] The cell composition may also be emulsified or presented as a ribosomal composition, provided that the emulsification procedure does not adversely affect cell viability.The cells and any other active ingredients may be mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredients, and in amounts suitable for use in the therapeutic methods disclosed herein.

[0323] The additives included in the cell composition may include pharmaceutically acceptable salts of the compositions therein. For example, pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of polypeptides) formed with inorganic acids such as hydrochloric acid or phosphoric acid, or with organic acids such as acetic acid, tartaric acid, mandelic acid, etc. For example, salts formed with free carboxyl groups may also be derived from inorganic bases such as hydroxides of sodium, potassium, ammonium, calcium or iron, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0324] 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, transtympanic, intraorgan, epidural, intrathecal, intramuscular, intravenous, intravascular, intraosseous, periocular, intratumoral, intracerebral, and intraventricular administration.

[0325] In some embodiments, the pharmaceutical compositions described herein are topically applied to a diseased site (e.g., lung). In some embodiments, the pharmaceutical compositions described herein are administered to an individual by injection, inhalation (e.g., aerosol), by catheter means, by suppository means, or by implant means, and the implant is a porous, non-porous, or gel-like material including a membrane (e.g., a silicone rubber membrane or fiber). In some embodiments, the pharmaceutical composition is formulated for delivery to an individual, such as for gene editing.

[0326] In some embodiments, the pharmaceutical composition is formulated according to conventional procedures as a composition suitable for intravenous or subcutaneous administration to an individual (e.g., human). In some embodiments, the pharmaceutical composition for injection is a solution in a sterile isotonic aqueous buffer. Where necessary, the medicine may also include a cosolvent and a local anesthetic (e.g., lidocaine) to relieve pain at the injection site. In general, for example, the composition is provided as a dry frozen powder or anhydrous concentrate in a sealed container (e.g., an ampoule or a pouch) indicating the amount of the active agent, either alone or in combination. In the case where the medicine is to be administered by perfusion, it may be distributed with a perfusion bottle containing sterile pharmaceutical grade water or saline. In the case where the pharmaceutical composition is administered by injection, an ampoule containing sterile water for injection or saline may be provided to mix the composition before administration.

[0327] In some embodiments, the pharmaceutical composition may be contained in lipid particles or vesicles, such as liposomes or microcrystals, which are also suitable for parenteral administration.

[0328] Although the descriptions of pharmaceutical compositions provided herein are primarily directed to pharmaceutical compositions suitable for administration to humans, the skilled artisan will appreciate that such compositions are generally suitable for administration to animals or organisms of all kinds.

[0329] A. Modification of causal mutations using base editing

[0330] An example of a genetic disease that can be corrected using a method depending on the RGN base editor fusion protein of the present invention is Hurler's disease. Hurler's disease (also known as MPS-1) is the result of a lack of α-L-iduronidase (IDUA), resulting in a lysosomal storage disease characterized by the accumulation of heparan sulfate and dermatan sulfate in lysosomes at the molecular level. This disease is usually a hereditary genetic disorder caused by a mutation in the IDUA gene encoding α-L-iduronidase. Common IDUA mutations are W402X and Q70X, both of which are nonsense mutations that cause premature termination of translation. This mutation is well addressed by precise genome editing (PGE) methods, since the recovery of a single nucleotide (e.g., by base editing methods) will restore the wild-type coding sequence and result in protein expression controlled by the endogenous regulatory mechanism of the genetic locus. Additionally, since heterozygotes are known to be asymptomatic, PGE therapy targeting one of these mutations is useful for most patients with the disease because 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).

[0331] 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). Although enzyme replacement therapy has a significant impact on the survival and quality of life of patients with Hurler syndrome, this approach requires expensive and time-consuming perfusions every week. Other methods include delivering the IDUA gene on an expression vector or inserting the gene into a highly expressed locus, for example, the locus of serum albumin (U.S. Patent No. 9,956,247, incorporated herein by reference). However, these methods cannot restore the original IDUA locus to the correct coding sequence. Genome editing strategies can have many advantages, the most notable of which is that the regulation of gene expression will be controlled by natural mechanisms present in healthy individuals. In addition, the use of base editing does not necessarily cause double-stranded DNA breaks, which may lead to large-scale chromosomal rearrangements, cell death or carcinogenesis due to the destruction of tumor suppression mechanisms. A general strategy can be directed to using the RGN base editor fusion proteins of the invention (e.g., those RGN base editor fusion proteins including LPG10136, LPG10139, LPG10134, or LPG10145) to target and correct certain pathogenic mutations in the human genome. It should be understood that similar methods can also be sought to target diseases that can be corrected by base editing. It should be further understood that similar methods can also be used to target pathogenic mutations in other species (particularly general household pets or livestock) using the RGNs of the invention. General household pets and livestock include dogs, cats, horses, pigs, cattle, sheep, chickens, donkeys, snakes, ferrets, fish (including salmon), and shrimp.

[0332] B. Modification of causal mutations by targeted deletion

[0333] The RGNs of the invention are also useful in human therapeutic approaches where the causal mutation is more complex. For example, some diseases such as Friedrich's ataxia and Huntington's disease are the result of a significant increase in the repeat of a three nucleotide motif in a specific region of the gene, which affects the ability of the expressed protein to function or be expressed. Friedrich's ataxia (FRDA) is an autosomal recessive disease that causes progressive degeneration of spinal cord neural tissue. Reduced levels of frataxin (FXN) protein in mitochondria cause oxidative damage and iron deficiency at the cellular level. Reduced FXN expression has been associated with a GAA triplet expansion within intron 1 of the somatic and germline FXN gene. In FRDA patients, GAA repeats typically 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 by reference).

[0334] The expansion of trinucleotide repeat sequences that cause Friedreich's ataxia (FRDA) occurs in a genetic locus defined within the FXN gene, known as the FRDA instability region. RNA-guided nucleases (RGNs) can be used to excise the instability region in FRDA patient cells. This approach requires: 1) a guide RNA sequence and RGN that can be programmed to target alleles in the human genome; and 2) a delivery method for the RGN and guide sequence. In particular, many nucleases used for genome editing (e.g., the commonly used Cas9 nuclease (SpCas9) from Streptococcus pyogenes) are too large to be packaged into an adeno-associated virus (AAV) vector when the length of the SpCas9 gene and guide RNA is considered in addition to other genetic elements required for a functional expression cassette. This makes methods using SpCas9 more difficult.

[0335] Certain RNA-guided nucleases of the invention (e.g., LPG10134, LPG10145, LPG10136, and LPG10139) are well suited for packaging with guide RNA into AAV vectors. The invention encompasses strategies using the RGNs of the invention in which regions of genomic instability are removed. This strategy is applicable to other diseases and conditions with similar genetic bases, such as Huntington's disease. Similar strategies using the RGNs of the invention can also be applied to similar diseases and conditions of non-human animals of agronomic or economic importance, including dogs, cats, horses, pigs, cattle, sheep, chickens, donkeys, snakes, ferrets, and fish including salmon and shrimp.

[0336] C. Modification of causal mutations by targeted mutagenesis

[0337] The RGNs of the invention may also introduce disruptive mutations that may result in beneficial effects.Genetic defects in genes encoding heme, particularly the beta globin chain (HBB gene), may be responsible for a number of diseases known as hemopathies, including sickle cell anemia and thalassemia.

[0338] In adults, heme is a heterotetramer that includes two alpha-globulin chains and two beta-globulin chains and four heme groups. In adults, the α2β2 tetramer is called heme A (HbA) or adult heme. Normally, the α and β globulin chains are synthesized in a ratio of approximately 1:1, and this ratio appears to be critical for heme and red blood cell (RBC) stability. In the developing fetus, a different form of heme (fetal heme (HbF)) is produced that has a higher binding affinity for oxygen than heme A, allowing oxygen to be delivered to the baby's system via the mother's bloodstream. Fetal heme also contains two alpha-globulin chains, but instead of adult beta-globulin chains, it has two fetal gamma-globulin chains (i.e., fetal heme is α2γ2). The regulation of the switch from producing gamma-globulins to producing beta-globulins is quite complex and primarily involves a downregulation of gamma globulin transcription and a simultaneous upregulation of beta globulin transcription. At about 30 weeks of gestation, fetal gamma globulin synthesis begins to decline, while beta globulin production increases. By about 10 months of age, the hemoglobin of the newborn is almost entirely alpha 2 beta 2, although some HbF persists into adulthood (approximately 1-3% of total hemoglobin). In most patients with hemoglobinopathies, the genes encoding gamma globulin are still present, but expression is relatively low due to normal gene repression that occurs near delivery as described above.

[0339] Sickle cell disease is caused by the V6E mutation (GAG to GTG at the DNA level) in the beta globin gene (HBB), in which the heme produced is called "heme S" or "HbS". Under hypoxic conditions, HbS molecules aggregate and form fibrillar precipitates. These aggregates cause RBC abnormalities or "sickling", resulting in a loss of cell flexibility. Sickled RBCs are no longer able to squeeze into the microvascular bed and can cause vaso-occlusive crises in sickle cell patients. In addition, sickled RBCs are more fragile than normal RBCs and are prone to hemolysis, ultimately causing anemia in patients.

[0340] Treatment and management of sickle cell patients is a lifelong issue that involves antibiotic therapy, pain management, and fluids during acute episodes. One approach is the use of hydroxyurea, which works in part by increasing the production of gamma globulins. However, the long-term side effects of chronic hydroxyurea therapy remain unknown, and treatment can produce adverse side effects and may have varying effects between patients. Despite the improvements in the efficacy of sickle cell treatments, life expectancy for patients remains only in the mid to late 50s, and the morbidity associated with the disease has a profound impact on the patient's quality of life.

[0341] The thalassemias (alpha thalassemia and beta thalassemia) are also heme-related diseases and usually involve reduced expression of the globin chains. This can occur via mutations in the regulatory regions of the gene or from mutations in the globin coding sequences, resulting in reduced expression or reduced levels or functional globin. Treatment of thalassemia usually involves blood transfusions and iron chelation therapy. Bone marrow transplants can also be used to treat people with thalassemia major if a suitable donor can be found, but this approach can have significant risks.

[0342] One approach that has been proposed for treating sickle cell disease (SCD) and beta thalassemia is to increase the expression of gamma globulin so that HbF functionally replaces the abnormal adult heme. As described above, treatment of SCD patients with hydroxyurea is considered partially successful due to its effect in increasing gamma globulin 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 incorporated herein by reference). Increasing the expression of HbF involves identifying genes whose products play a role in the regulation of gamma globulin expression. One such gene is BCL11A. BCL11A encodes a zinc finger protein expressed in adult erythroid precursor cells, and downregulation of its expression results in increased expression of gamma globin (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., U.S. Patent Publication No. 2011 / 0182867, incorporated herein by reference), but this technology has several potential disadvantages, including that complete knockdown may not be achieved, delivery of such RNA may be problematic, and the RNA must be present continuously and multiple treatments may be required throughout life.

[0343] The RGNs of the invention (e.g., LPG10134, LPG10145, LPG10136, or LPG10139) can be used to target the BCL11A enhancer region to disrupt expression of BCL11A, thereby increasing gamma globin expression. This targeted disruption can be achieved by non-homologous end joining (NHEJ), whereby the RGNs of the invention target specific sequences within the BCL11A enhancer region, causing double-strand breaks, and the cell's mechanical repair of the breaks often introduces deleterious mutations simultaneously. Similar to that described for other disease targets, the RGNs of the invention have advantages over other known RGNs due to their relatively small size, enabling the packaging of the expression cassette for the RGN and its guide RNA in a single AAV vector for in vivo delivery. Similar strategies using the RGNs of the invention can also be applied to similar diseases and conditions in humans and non-human animals of agronomic or economic importance.

[0344] XI. Including Genetically Modified Cells

[0345] Provided herein are cells and organisms comprising a target sequence of interest that has been modified using a RGN, crRNA, and / or tracrRNA-mediated process as described herein. In some of these embodiments, the RGN comprises an amino acid sequence as described in any one of SEQ ID NOs: 1-12, or an active variant or fragment thereof. In various embodiments, the guide RNA comprises a CRISPR repeat sequence, and the CRISPR repeat sequence comprises a nucleotide sequence as described in any one of SEQ ID NOs: 13-24, or an active variant or fragment thereof. In a particular embodiment, the guide RNA comprises a tracrRNA, and the tracrRNA comprises a nucleotide sequence as described in any one of 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.

[0346] The modified cells can be eukaryotic (e.g., mammalian, plant, insect, avian cells) or prokaryotic. Also provided are organelles and embryos comprising at least one nucleotide sequence, which has been modified by methods utilizing RGN, crRNA and / or tracrRNA as described herein. Genetically modified cells, organisms, organelles and embryos can be heterozygous or homozygous for the modified nucleotide sequence.

[0347] The chromosomal modification of cells, organisms, organelles or embryos can result in expression of a protein product or an integrated sequence that changes (up or down), inactivates or changes. In those embodiments where the chromosomal modification results in gene inactivation or expression of a non-functional protein product, the genetically modified cells, organisms, organelles or embryos are referred to as "knockouts". The knockout expression type can be a deletion mutation (i.e., the deletion of at least one Nucleotide), an insertion mutation (i.e., the insertion of at least one Nucleotide) or a meaningless mutation (i.e., the replacement of at least one Nucleotide so that a stop codon is introduced).

[0348] Alternatively, chromosomal modification of cells, organisms, organelles or embryos can produce "knock-in", which is caused by the chromosomal integration of the nucleotide sequence encoding the protein. In some of these embodiments, the coding sequence is integrated into the chromosome, making the chromosomal sequence encoding the wild-type protein inactive, but expressing the exogenously introduced protein.

[0349] In some embodiments, the chromosomal modification results in the production of variant protein products. The expressed variant protein product may have at least one amino acid substitution and / or at least one amino acid addition or deletion. When compared to the wild-type protein, the variant protein product encoded by the altered chromosomal sequence may exhibit modified characteristics or activities, including but not limited to altered enzymatic activity or substrate specificity.

[0350] In yet other embodiments, chromosomal modifications can result in altered protein expression patterns. As non-limiting examples, chromosomal changes in regulatory regions that control protein product expression can result in overexpression or downregulation of protein products or altered tissue or temporal expression patterns.

[0351] The cells that have been modified can be grown into organisms, such as plants, according to conventional methods. For example, see McCormick et al. (1986) Plant Cell Reports 5: 81-84. Then, these plants can be grown and pollinated with the same modified strain or different strains, and the resulting hybrids have genetic modifications. The present invention provides genetically modified seeds. Progeny, variants and mutants of regenerated plants are also included within the scope of the present invention, provided that these parts include genetic modifications. Further provided are processed plant products or by-products that maintain genetic modifications, for example, including soybean meal.

[0352] The methods provided herein can be used to modify any plant species, including but not limited to monocots and dicots. Examples of plants of interest include but are not limited to corn (maize), sorghum, wheat, sunflower, tomato, crucifers, pepper, potato, cotton, rice, soybean, beet, sugarcane, tobacco, barley and rapeseed, Brassica napus, alfalfa, rye, millet, safflower, peanut, sweet potato, cassava, coffee, coconut, pineapple, citrus, cocoa, tea, banana, avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond, oat, vegetables, ornamental plants and conifers.

[0353] Vegetables include, but are not limited to, tomatoes, lettuces, mung beans, beans, peas, and members of the genus Cucumis, such as cucumbers, cantaloupes, and cantaloupes. Ornamental plants include, but are not limited to, azaleas, hydrangeas, hibiscus, roses, tulips, narcissus, petunias, carnations, Christmas reds, and chrysanthemums. Preferably, the plants of the present invention are crops (e.g., corn, sorghum, wheat, sunflowers, tomatoes, cruciferous plants, peppers, potatoes, cotton, rice, soybeans, beets, sugarcane, tobacco, barley, rapeseed, etc.).

[0354] 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, Klebsiella, Streptomyces, Rhizobium, Escherichia, Pseudomonas, Salmonella, Shigella, Vibrio, Yersinia, Mycoplasma, Agrobacterium, Lactobacillus).

[0355] 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, amoeba, algae and yeast. In some embodiments, cells modified by the methods disclosed herein include hematopoietic cells (e.g., cells of the immune system) including but not limited to B cells, T cells, natural killer (NK) cells, rich potential stem cells, induced rich potential stem cells, chimeric antigen receptor T (CAR-T) cells, monocytes, macrophages and dendritic cells.

[0356] Modified cells can be introduced into an organism. In the case of autologous cell transplantation, these cells may have been derived from the same organism (e.g., a human), wherein the cells are modified in an ex vivo method. Alternatively, in the case of allogeneic cell transplantation, the cells are derived from another organism (e.g., another human) in the same species.

[0357] XII. Kit

[0358] 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, e.g., for in vitro or in vivo DNA or RNA editing. In some embodiments, the kits include instructions for designing and using suitable gRNAs for targeted editing of nucleic acid sequences.

[0359] In some embodiments, the pharmaceutical composition can be provided as a pharmaceutical kit, which includes (a) a container containing a composition of the present disclosure in freeze-dried form and (b) a second container containing a pharmaceutically acceptable diluent (e.g., sterile water) for injection. The pharmaceutically acceptable diluent can be used to dissolve back or dilute the freeze-dried compound of the present disclosure. Optionally, associated with such a container can be a notice in a form prescribed by a government agency regulating the manufacture, use or sale of a drug or biological product, which reflects approval for human administration by the manufacturing, use or sales agency.

[0360] The articles "a" and "an" are used herein to refer to one or more than one (ie, at least one) of the grammatical object of the article. As an example, "polypeptide" means one or more polypeptides.

[0361] All publications and patent applications mentioned in the specification are indicative of the level of ordinary skill in the art to which the present disclosure pertains. All publications and patent applications are incorporated herein by reference as if each individual publication or patent application was specifically and individually indicated to be incorporated herein by reference.

[0362] 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 may be practiced within the scope of the appended embodiments.

[0363] Non-limiting examples include:

[0364] 1. A nucleic acid molecule comprising a polynucleotide encoding an RNA-guided nuclease (RGN) polypeptide, wherein the polynucleotide comprises a nucleotide sequence encoding the 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;

[0365] Among them, when combined with a guide RNA (gRNA) that can hybridize with a target DNA sequence, the RGN polypeptide can bind to the target DNA sequence in an RNA-guided sequence-specific manner.

[0366] 2. The nucleic acid molecule of embodiment 1, wherein the polynucleotide encoding the RGN polypeptide is operably linked to a promoter that is heterologous to the polynucleotide.

[0367] 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.

[0368] 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.

[0369] 5. The nucleic acid molecule of any one of embodiments 1 to 4, wherein the RGN polypeptide is capable of cleaving a target DNA sequence upon binding.

[0370] 6. The nucleic acid molecule of embodiment 5, wherein the RGN polypeptide is capable of producing double-strand breaks.

[0371] 7. The nucleic acid molecule of embodiment 5, wherein the RGN polypeptide is capable of producing single-strand breaks.

[0372] 8. The nucleic acid molecule of any one of embodiments 1 to 4, wherein the RGN polypeptide is nuclease-inactive or is a nickase.

[0373] 9. The nucleic acid molecule of any one of embodiments 1 to 5, 7 and 8, wherein the RGN polypeptide is operably linked to a lead editing polypeptide.

[0374] 10. The nucleic acid molecule of embodiment 9, wherein the lead editing polypeptide comprises a DNA polymerase.

[0375] 11. The nucleic acid molecule of embodiment 9, wherein the lead editing polypeptide comprises a reverse transcriptase.

[0376] 12. The nucleic acid molecule of any one of embodiments 1 to 8, wherein the RGN polypeptide is operably linked to a base editing polypeptide.

[0377] 13. The nucleic acid molecule of Example 12, wherein the base editing polypeptide is a deaminase.

[0378] 14. The nucleic acid molecule of embodiment 13, wherein the deaminase is cytosine deaminase or adenine deaminase.

[0379] 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.

[0380] 16. The nucleic acid molecule of embodiment 13, wherein the deaminase has at least 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 377-448.

[0381] 17. The nucleic acid molecule of any one of embodiments 1 to 16, wherein the RGN polypeptide comprises one or more nuclear localization signals.

[0382] 18. The nucleic acid molecule of any one of embodiments 1 to 17, wherein the RGN polypeptide is codon-optimized for expression in eukaryotic cells.

[0383] 19. The nucleic acid molecule of any one of embodiments 1 to 18, wherein the target DNA sequence is positioned adjacent to a pre-spacer adjacent motif (PAM).

[0384] 20. A vector comprising the nucleic acid molecule of any one of embodiments 1 to 19.

[0385] 21. The vector of Example 20, further comprising at least one nucleotide sequence encoding a gRNA capable of hybridizing with a target DNA sequence.

[0386] 22. The vector of embodiment 21, wherein the guide RNA is selected from the group consisting of:

[0387] a) a guide RNA comprising:

[0388] i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 13; and

[0389] ii) tracrRNA having at least 90% sequence identity to SEQ ID NO: 25;

[0390] wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1;

[0391] b) a guide RNA comprising:

[0392] i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 14; and

[0393] ii) tracrRNA having at least 90% sequence identity to SEQ ID NO: 26;

[0394] wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:2;

[0395] c) a guide RNA comprising:

[0396] i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 15; and

[0397] ii) tracrRNA having at least 90% sequence identity to SEQ ID NO: 27;

[0398] wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:3;

[0399] d) a guide RNA comprising:

[0400] i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 16; and

[0401] ii) tracrRNA having at least 90% sequence identity to SEQ ID NO: 28;

[0402] wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:4;

[0403] e) a guide RNA comprising:

[0404] i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 17; and

[0405] ii) tracrRNA having at least 90% sequence identity to SEQ ID NO: 29;

[0406] wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:5;

[0407] f) a guide RNA comprising:

[0408] i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 18; and

[0409] ii) tracrRNA having at least 90% sequence identity to SEQ ID NO: 30;

[0410] wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:6;

[0411] g) a guide RNA comprising:

[0412] i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 19; and

[0413] ii) tracrRNA having at least 90% sequence identity to SEQ ID NO: 31;

[0414] wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:7;

[0415] h) a guide RNA comprising:

[0416] i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 20; and

[0417] ii) tracrRNA having at least 90% sequence identity to SEQ ID NO: 32;

[0418] wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:8;

[0419] i) a guide RNA comprising:

[0420] i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 21; and

[0421] ii) tracrRNA having at least 90% sequence identity to SEQ ID NO: 33;

[0422] wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:9;

[0423] j) a guide RNA comprising:

[0424] i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 22; and

[0425] ii) tracrRNA having at least 90% sequence identity to SEQ ID NO: 34;

[0426] wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 10;

[0427] k) a guide RNA comprising:

[0428] i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 23; and

[0429] ii) tracrRNA having at least 90% sequence identity to SEQ ID NO: 35;

[0430] wherein the RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 11; and

[0431] 1) a guide RNA comprising:

[0432] i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 24; and

[0433] ii) tracrRNA having at least 90% sequence identity to SEQ ID NO: 36;

[0434] The RGN polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:12.

[0435] 23. The vector of embodiment 21, wherein the guide RNA is selected from the group consisting of:

[0436] a) a guide RNA comprising:

[0437] i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 13; and

[0438] ii) tracrRNA having at least 95% sequence identity to SEQ ID NO: 25;

[0439] wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1;

[0440] b) a guide RNA comprising:

[0441] i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 14; and

[0442] ii) tracrRNA having at least 95% sequence identity to SEQ ID NO: 26;

[0443] wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:2;

[0444] c) a guide RNA comprising:

[0445] i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 15; and

[0446] ii) tracrRNA having at least 95% sequence identity to SEQ ID NO: 27;

[0447] wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:3;

[0448] d) a guide RNA comprising:

[0449] i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 16; and

[0450] ii) tracrRNA having at least 95% sequence identity to SEQ ID NO: 28;

[0451] wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:4;

[0452] e) a guide RNA comprising:

[0453] i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 17; and

[0454] ii) tracrRNA having at least 95% sequence identity to SEQ ID NO: 29;

[0455] wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:5;

[0456] f) a guide RNA comprising:

[0457] i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 18; and

[0458] ii) tracrRNA having at least 95% sequence identity to SEQ ID NO: 30;

[0459] wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:6;

[0460] g) a guide RNA comprising:

[0461] i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 19; and

[0462] ii) tracrRNA having at least 95% sequence identity to SEQ ID NO: 31;

[0463] wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:7;

[0464] h) a guide RNA comprising:

[0465] i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 20; and

[0466] ii) tracrRNA having at least 95% sequence identity to SEQ ID NO: 32;

[0467] wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:8;

[0468] i) a guide RNA comprising:

[0469] i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 21; and

[0470] ii) tracrRNA having at least 95% sequence identity to SEQ ID NO: 33;

[0471] wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:9;

[0472] j) a guide RNA comprising:

[0473] i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 22; and

[0474] ii) tracrRNA having at least 95% sequence identity to SEQ ID NO: 34;

[0475] wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 10;

[0476] k) a guide RNA comprising:

[0477] i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 23; and

[0478] ii) tracrRNA having at least 95% sequence identity to SEQ ID NO: 35;

[0479] wherein the RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 11; and

[0480] 1) a guide RNA comprising:

[0481] i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 95% sequence identity to SEQ ID NO: 24; and

[0482] ii) tracrRNA having at least 95% sequence identity to SEQ ID NO: 36;

[0483] The RGN polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:12.

[0484] 24. The vector of embodiment 21, wherein the guide RNA is selected from the group consisting of:

[0485] a) a guide RNA comprising:

[0486] i) a CRISPR RNA comprising a CRISPR repeat sequence having 100% sequence identity to SEQ ID NO: 13; and

[0487] ...

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; in, When combined with a guide RNA (gRNA) that is capable of hybridizing to a target DNA sequence, the RGN polypeptide is capable of binding to the target DNA sequence in an RNA-guided, sequence-specific manner.

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 the RGN polypeptide is operably linked to a promoter heterologous to the polynucleotide.

4. The nucleic acid molecule of 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 producing a double-strand break.

6. The nucleic acid molecule of claim 4, wherein the RGN polypeptide is capable of producing single-strand breaks.

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 lead editing polypeptide.

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

10. The nucleic acid molecule of claim 8, wherein the lead 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 cytosine deaminase or adenine deaminase.

14. The nucleic acid molecule of claim 12, wherein the deaminase has at least 90% sequence identity to the amino acid sequence of any one 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 eukaryotic cells.

18. The nucleic acid molecule according to any one of claims 1 to 17, wherein the target DNA sequence is positioned 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. The vector according to claim 19, further comprising at least one nucleotide sequence encoding the gRNA capable of hybridizing with the target DNA sequence.

21. The vector of claim 20, wherein the guide RNA is selected from the group consisting of: a) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 13; as well as ii) 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 guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 14; and ii) 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 guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 15; and ii) 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 guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 16; and ii) 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 guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 17; and ii) 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 guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 18; and ii) 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 guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 19; and ii) 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: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 20; and ii) 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: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 21; and ii) 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 guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 22; and ii) 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 guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 23; and ii) 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 1) a guide RNA comprising: i) a CRISPR RNA comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 24; and ii) 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.

22. The vector of claim 20, wherein the guide RNA is selected from the group consisting of: a) a guide RNA comprising: 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; and ii) tracrRNA, having 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 guide RNA comprising: 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; and 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; 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 guide RNA comprising: 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; and 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; 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 guide RNA comprising: 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; and 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; 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 guide RNA comprising: 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; and 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; 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 guide RNA comprising: 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; and 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; 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 guide RNA comprising: 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; and 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; 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 guide RNA comprising: 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; and 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; 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 guide RNA comprising: 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; and 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; 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 guide RNA comprising: 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; and 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; 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 guide RNA comprising: 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; and 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; 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 1) a guide RNA comprising: 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; and 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; 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.

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

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

25. The vector according to 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. A method for producing an RGN polypeptide, comprising: The cell of claim 26 is cultured under conditions whereby the RGN polypeptide is expressed.

28. A method for producing a RGN polypeptide, comprising introducing into a cell a heterologous nucleic acid molecule comprising a nucleotide sequence encoding 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; in, When combined with a guide RNA (gRNA) that is capable of hybridizing to a target DNA sequence, the RGN polypeptide is capable of binding to the target DNA sequence in an RNA-guided, sequence-specific manner; and culturing the cell under conditions where the RGN polypeptide is expressed.

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. 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 a RGN ribonucleoprotein complex.

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

33. 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 in, When combined with a guide RNA (gRNA) that is capable of hybridizing to a target DNA sequence of a DNA molecule, the RGN polypeptide is capable of binding to the target DNA sequence in an RNA-guided, sequence-specific manner.

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 of the RGN polypeptide produces a double-stranded break.

38. The RGN polypeptide of claim 36, wherein single-strand breaks result from cleavage of the RGN polypeptide.

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 lead editing polypeptide.

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

42. The RGN polypeptide of claim 40, wherein the lead 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. The RGN polypeptide of claim 44, wherein the deaminase is a cytosine deaminase or an adenine deaminase.

46. ​​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 positioned 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 the RGN polypeptide according to any one of claims 33 to 49, and the guide RNA bound to the RGN polypeptide.

51. A system for binding 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 the 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, or a polynucleotide comprising a nucleotide sequence encoding said 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.

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

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. A system for binding 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 the 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.

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: 1-12.

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 that is 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 with each other in nature.

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

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

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

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

62. The system of any one of claims 51 to 61, wherein the gRNA is selected from the group consisting of: a) comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 13, and a gRNA comprising 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) comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 19, and a gRNA comprising 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 1) comprising a CRISPR repeat sequence having at least 90% sequence identity to SEQ ID NO: 24, and a gRNA comprising 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 system of any one of claims 51 to 61, wherein the gRNA is selected from the group consisting of: 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 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 1) 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 gRNA comprising 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. The system of any one of claims 51 to 63, wherein the target DNA sequence is positioned 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. 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. The system of claim 66, wherein the cleavage produces a double-strand break.

68. The system of claim 66, wherein the cleavage produces a single strand 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 lead editing polypeptide.

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

72. The system of claim 70, wherein the lead 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. The system of claim 74, wherein the deaminase is cytosine deaminase or adenine deaminase.

76. 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. 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 the amino acid sequence of any one of SEQ ID NOs: 377-448.

78. The system of 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, wherein the system further comprises 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 pharmaceutically acceptable carrier.

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

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

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

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

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

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

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. The method of claim 90 or 91, wherein the method is performed 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 of any one of claims 90 to 92, wherein the guide RNA or the RGN polypeptide further comprises an expression regulator, thereby allowing regulation of expression of the target DNA sequence.

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

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

97. The method of claim 95, wherein the lead 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 allowing modification of the target DNA sequence.

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

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

101. The method of 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. A method for cleaving and / or modifying a target DNA sequence of a DNA molecule, comprising contacting the DNA molecule with: 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) one or more guide RNAs capable of targeting the RGN of (a) to the target DNA sequence; The one or more guide RNAs hybridize to the target DNA sequence, thereby guiding the RGN polypeptide to bind to the target DNA sequence, and cutting and / or modification of the target DNA sequence occurs.

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 double-strand breaks are produced by cleavage of the RGN polypeptide.

105. The method of claim 102 or 103, wherein single-strand breaks are produced by cleavage of the RGN polypeptide.

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

107. The method of claim 106, wherein the lead editing polypeptide comprises a DNA polymerase.

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

109. The method of claim 102 or 103, wherein the RGN polypeptide is nuclease-inactive or is 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. The method of claim 110, wherein the deaminase is cytosine deaminase or 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. The method of claim 102 or 103, wherein the modified target DNA sequence comprises insertion of heterologous DNA into the target DNA sequence.

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

116. 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. 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. 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. The method of claim 119, wherein the sgRNA further comprises an extension comprising an editing template for lead editing.

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

122. The method of any one of claims 83 to 121, wherein: a) the RGN has at least 90% sequence identity with SEQ ID NO: 1, the guide RNA comprises a crRNA repeat sequence having at least 90% sequence identity with SEQ ID NO: 13, and a tracrRNA having at least 90% sequence identity with SEQ ID NO: 25; b) the RGN has at least 90% sequence identity with SEQ ID NO: 2, the guide RNA comprises a crRNA repeat sequence having at least 90% sequence identity with SEQ ID NO: 14, and a tracrRNA having at least 90% sequence identity with SEQ ID NO: 26; c) the RGN has at least 90% sequence identity with SEQ ID NO: 3, the guide RNA comprises 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) the RGN has at least 90% sequence identity with SEQ ID NO: 4, the guide RNA comprises 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) the RGN has at least 90% sequence identity with SEQ ID NO: 5, the guide RNA comprises a crRNA 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; f) the RGN has at least 90% sequence identity with SEQ ID NO: 6, the guide RNA comprises 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) the RGN has at least 90% sequence identity with SEQ ID NO: 7, the guide RNA comprises 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: 31; h) the RGN has at least 90% sequence identity with SEQ ID NO: 8, the guide RNA comprises 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) the RGN has at least 90% sequence identity with SEQ ID NO: 9, the guide RNA comprises a crRNA repeat sequence having at least 90% sequence identity with SEQ ID NO: 21, and a tracrRNA having at least 90% sequence identity with SEQ ID NO: 33; j) the RGN has at least 90% sequence identity with SEQ ID NO: 10, the guide RNA comprises a crRNA repeat sequence having at least 90% sequence identity with SEQ ID NO: 22, and a tracrRNA having at least 90% sequence identity with SEQ ID NO: 34; k) the RGN has at least 90% sequence identity to SEQ ID NO: 11, 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 1) the RGN has at least 90% sequence identity with SEQ ID NO: 12, the guide RNA comprises a crRNA repeat sequence having at least 90% sequence identity with SEQ ID NO: 24, and a tracrRNA having at least 90% sequence identity with SEQ ID NO:

36.

123. The method of any one of claims 83 to 121, wherein: 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, 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, 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, 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, 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, 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, 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, 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, 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, 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, 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, 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 1) 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, 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. The method of any one of claims 83 to 123, wherein the target DNA sequence is intracellular.

125. The method of claim 124, further comprising: The cells are cultured under conditions where 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 cells comprising the modified target DNA sequence are selected.

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

127. A pharmaceutical composition comprising the cell according to any one of claims 26, 81 and 126 and a pharmaceutically acceptable carrier.

128. A method of treating a disease, disorder or symptom, the method comprising administering to a subject in need of treatment 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 causal mutation and the effective amount of the pharmaceutical composition corrects the causal mutation.

130. The method of claim 128 or 129, wherein the individual is at risk for developing the disease, the disorder, or the symptoms.

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, wherein the CRISPR repeat sequence comprises a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 13-24; in, When the guide RNA is combined with an RNA-guided nuclease (RGN) polypeptide, the guide RNA can hybridize with a target DNA sequence via the spacer sequence of the crRNA in a sequence-specific manner, and the guide RNA comprises: a) the crRNA; and b) a transcriptionally activated CRISPR RNA (tracrRNA) that hybridizes to the CRISPR repeat sequence of the crRNA.

132. A nucleic acid molecule according to claim 131, wherein the polynucleotide encoding crRNA is operably linked to a promoter heterologous to the polynucleotide.

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 a nucleic acid molecule comprising the polynucleotide encoding the crRNA according to any one of claims 131 to 133.

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

136. The vector of claim 135, wherein 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: 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 1) 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.

137. The vector of claim 135, wherein the tracrRNA is selected from the group consisting of: 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 1) 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 of any one of claims 134 to 137, wherein the vector further comprises a polynucleotide encoding the RGN polypeptide.

139. The vector of claim 138, wherein 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: 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 1) 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.

140. The vector of claim 138, wherein the RGN polypeptide is selected from the group consisting of: 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 transcriptionally activated CRISPR RNA (tracrRNA) or a polynucleotide encoding a tracrRNA, wherein the tracrRNA comprises a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 25-36; in, When the guide RNA is combined with an RNA-guided nuclease (RGN) polypeptide, the guide RNA can hybridize with a target DNA sequence via the spacer sequence of the crRNA in a sequence-specific manner, and the guide RNA comprises: 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.

142. The nucleic acid molecule of claim 141, wherein the polynucleotide encoding 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. A vector comprising a nucleic acid molecule comprising the polynucleotide encoding the tracrRNA according to any one of claims 141 to 143.

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

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

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.

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