DNA modifying enzymes and their active fragments and variants, and methods of use

Abstract

Compositions and methods are provided that include a deaminase for targeted nucleic acid editing. The compositions include a deaminase polypeptide. Also provided are compositions that include a DNA-binding polypeptide and a fusion protein of the invention. The fusion protein includes an RNA-guided nuclease fused to a deaminase, optionally complexed with a guide RNA. The compositions also include a nucleic acid molecule that encodes the deaminase or the fusion protein. Vectors and host cells that include a nucleic acid molecule that encodes the deaminase or the fusion protein are also provided.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 164,273, filed March 22, 2021, which is incorporated by reference in its entirety.

[0002] Declaration of array list The Sequence Listing accompanying this application has been provided in ASCII format in lieu of a paper copy and is incorporated herein by reference. This ASCII copy, entitled L103438_1240WO_0134_1_SL.txt, is 1,310,113 bytes in size, was created on Mar. 22, 2022, and has been submitted electronically via EFS-Web.

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

[0004] Targeted genome editing or targeted genome modification is rapidly becoming an important tool for basic and applied research. Early methods involved engineering nucleases (such as meganucleases, zinc finger fusion proteins, or TALENs), which required the creation of chimeric nucleases with engineered programmable sequence-specific DNA binding domains specific for each particular target sequence. RNA-guided nucleases (RGNs) (such as the CRISPR-Cas proteins of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) bacterial system) allow the nuclease to target a particular sequence by complexing it with a guide RNA that specifically hybridizes to the particular target sequence. The creation of target-specific guide RNAs is less costly and more efficient than the creation of chimeric nucleases for each target sequence. Such RNA-guided nucleases can be used to edit genomes by introducing sequence-specific double-strand breaks that can be repaired through error-prone non-homologous end joining (NHEJ) to introduce mutations at specific genomic locations, or heterologous DNA can be introduced at those genomic sites through homology-directed repair.

[0005] In addition, RGNs are useful for targeted DNA editing approaches. Targeted nucleic acid sequence editing (e.g., targeted cleavage) can introduce specific modifications into genomic DNA, allowing a very precise approach to study gene function and gene expression. RGNs can also be used to create chimeric proteins that combine the RNA-guided activity of RGNs with DNA-modifying enzymes (e.g., deaminases) for targeted base editing. Targeted editing can be used to target genetic diseases in humans or introduce agriculturally beneficial mutations into crop genomes. The development of genome editing tools provides new approaches to mammalian therapeutics and agricultural biotechnology based on gene editing. Summary of the Invention

[0006] Compositions and methods for modifying a target DNA molecule are provided. The compositions are utilized to modify a target DNA molecule of interest. The compositions provided include a deaminase polypeptide. Also provided are fusion proteins comprising a nucleic acid molecule-binding polypeptide (e.g., a DNA-binding polypeptide) and a deaminase polypeptide, and ribonucleoprotein complexes comprising a fusion protein comprising an RNA-guided nuclease and a deaminase polypeptide and a ribonucleic acid. The compositions provided also include nucleic acid molecules encoding the deaminase polypeptide or fusion proteins, and vectors and host cells comprising the nucleic acid molecules. The methods disclosed herein relate to binding to a target sequence of interest in a target DNA molecule of interest and modifying the target DNA molecule of interest.

[0007] In a first aspect, the present disclosure provides a polypeptide having deaminase activity, comprising an amino acid sequence selected from the group consisting of: a) an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 2 and 7 to 12; and b) an amino acid sequence having at least 95% identity to SEQ ID NO: 4 or 6.

[0008] In some embodiments of the above aspects, the polypeptide comprises an amino acid sequence that has at least 95% sequence identity to any one of SEQ ID NOs: 2 and 7-12. In some embodiments, the polypeptide comprises an amino acid sequence that has 100% sequence identity to any one of SEQ ID NOs: 2, 4, and 6-12. In some embodiments, the polypeptide is isolated.

[0009] In another aspect, the disclosure provides a nucleic acid molecule comprising a polynucleotide encoding a deaminase polypeptide, wherein the deaminase is encoded by a nucleotide sequence selected from the group consisting of: a) a nucleotide sequence having at least 80% identity to any one of SEQ ID NOs: 114-119; b) a nucleotide sequence having at least 95% identity to any one of SEQ ID NOs: 109, 111, and 113; c) a nucleotide sequence encoding an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 2 and 7-12; and d) a nucleotide sequence encoding an amino acid sequence having at least 95% identity to SEQ ID NO: 4 or 6.

[0010] In some embodiments of the above aspects, the deaminase is encoded by a nucleotide sequence having at least 90% identity to any one of SEQ ID NOs: 114-119. In some embodiments, the deaminase is encoded by a nucleotide sequence having at least 95% identity to any one of SEQ ID NOs: 114-119. In some embodiments, the deaminase is encoded by a nucleotide sequence having 100% identity to any one of SEQ ID NOs: 109, 111, and 113-119. In some embodiments, the deaminase polypeptide has an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 2 and 7-12. In some embodiments, the deaminase polypeptide has an amino acid sequence having 100% identity to any one of SEQ ID NOs: 2, 4, and 6-12.

[0011] In some embodiments of the above aspects, the nucleic acid molecule further comprises a heterologous promoter operably linked to the polypeptide. In some embodiments, the nucleic acid molecule is isolated.

[0012] In another aspect, the disclosure provides a vector comprising the above-described nucleic acid molecule.

[0013] In another aspect, the disclosure provides a cell comprising the nucleic acid molecule or vector described above. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a human cell. In some embodiments, the human cell is an immune cell. In some embodiments, the immune cell is a stem cell. In some embodiments, the stem cell is an induced pluripotent stem cell. In some embodiments, the eukaryotic cell is an insect cell or an avian cell. In some embodiments, the eukaryotic cell is a fungal cell. In some embodiments, the eukaryotic cell is a plant cell.

[0014] In another aspect, the present disclosure provides a plant or seed comprising the plant cell described above.

[0015] In another aspect, the disclosure provides a pharmaceutical composition comprising a polypeptide, a nucleic acid molecule, a vector, or a cell of embodiment 11, comprising a pharma- ceutical acceptable carrier. In some embodiments, the pharma-ceutical acceptable carrier is heterologous to the polypeptide or the nucleic acid molecule. In some embodiments, the pharma-ceutical acceptable carrier is non-naturally occurring.

[0016] In another aspect, the present disclosure provides a method for producing a deaminase, comprising culturing the above-described cell under conditions in which the deaminase is expressed.

[0017] In another aspect, the disclosure provides a method of making a deaminase comprising introducing the nucleic acid molecule or vector described above into a cell and culturing the cell under conditions in which the deaminase is expressed.

[0018] In some embodiments of the above aspects, the method further includes purifying the deaminase.

[0019] In another aspect, the disclosure provides a fusion protein comprising a DNA binding polypeptide and a deaminase having an amino acid sequence selected from the group consisting of: a) an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 2 and 7-12; and b) an amino acid sequence having at least 95% identity to SEQ ID NO: 4 or 6. In some embodiments, the deaminase has at least 95% identity to any one of SEQ ID NOs: 2 and 7-12. In some embodiments, the deaminase has 100% identity to any one of SEQ ID NOs: 2, 4, and 6-12.

[0020] In some embodiments of the above aspects, the deaminase is a cytosine deaminase. In some embodiments, the DNA-binding polypeptide is a meganuclease, a zinc finger fusion protein, or a TALEN; or a variant of a meganuclease, a zinc finger fusion protein, or a TALEN, and the nuclease activity is reduced or inhibited.

[0021] In some embodiments of the above aspects, the DNA binding polypeptide is an RNA-guided DNA binding polypeptide. In some embodiments, the RNA-guided DNA binding polypeptide is an RNA-guided nuclease (RGN) polypeptide. In some embodiments, the RGN is a type II or type V CRISPR-Cas polypeptide. In some embodiments, the RGN is an RGN nickase. In some embodiments, the RGN nickase has an inactive RuvC domain. In some embodiments, the RGN is a nuclease-inactive RGN. In some embodiments, the RGN has an amino acid sequence that has at least 90% sequence identity with any one of the RGN sequences in Table 1. In some embodiments, the RGN has an amino acid sequence that has at least 95% sequence identity with any one of the RGN sequences in Table 1. In some embodiments, the RGN has an amino acid sequence of any one of the RGN sequences in Table 1. In some embodiments, the RGN has an amino acid sequence that has at least 90% sequence identity with any one of SEQ ID NOs: 74, 82, 87, 106, and 107. In some embodiments, the RGN has an amino acid sequence that has at least 95% sequence identity to any one of SEQ ID NOs: 74, 82, 87, 106, and 107. In some embodiments, the RGN has an amino acid sequence of any one of SEQ ID NOs: 74, 82, 87, 106, and 107.

[0022] In some embodiments of the above aspects, the RGN nickase has an amino acid sequence that has at least 90% sequence identity to any one of SEQ ID NOs: 75, 88-98. In some embodiments, the RGN nickase has an amino acid sequence that has at least 95% sequence identity to any one of SEQ ID NOs: 75, 88-98. In some embodiments, the RGN nickase has an amino acid sequence that has at least 95% sequence identity to any one of SEQ ID NOs: 75, 88-98.

[0023] In some embodiments of the above aspects, the fusion protein further comprises at least one nuclear localization signal (NLS). In some embodiments, the deaminase is fused to the amino terminus of the DNA binding polypeptide. In some embodiments, the deaminase is fused to the carboxyl terminus of the DNA binding polypeptide. In some embodiments, the fusion protein further comprises a linker sequence between the DNA binding polypeptide and the deaminase. In some embodiments, the linker sequence has the amino acid sequence set forth as SEQ ID NO: 78 or 79.

[0024] In some embodiments of the above aspects, the fusion protein further comprises a uracil stabilizing protein (USP). In some embodiments, the USP has the sequence set forth as SEQ ID NO:81. In some embodiments, the fusion protein further comprises a linker sequence between the USP and the deaminase or the DNA binding polypeptide. In some embodiments, the linker sequence between the USP and the deaminase or the DNA binding polypeptide has the amino acid sequence set forth as SEQ ID NO:120.

[0025] In some embodiments of the above aspects, the fusion protein has an amino acid sequence of any one of SEQ ID NOs: 67, 68, 146, and 147.

[0026] In another aspect, the disclosure provides a nucleic acid molecule comprising a polynucleotide encoding a fusion protein comprising a DNA-binding polypeptide and a deaminase, wherein the deaminase is encoded by a nucleotide sequence selected from the group consisting of: a) a nucleotide sequence having at least 80% identity to any one of SEQ ID NOs: 114-119; b) a nucleotide sequence having at least 95% identity to any one of SEQ ID NOs: 109, 111, and 113; c) a nucleotide sequence encoding an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 2 and 7-12; and d) a nucleotide sequence encoding an amino acid sequence having at least 95% identity to SEQ ID NO: 4 or 6.

[0027] In some embodiments of the above aspects, the deaminase is encoded by a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 114-119. In some embodiments, the deaminase is encoded by a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 114-119. In some embodiments, the nucleotide sequence of the deaminase has 100% sequence identity to any one of SEQ ID NOs: 109, 111, and 113-119. In some embodiments, the nucleotide sequence of the deaminase encodes an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 2 and 7-12. In some embodiments, the nucleotide sequence of the deaminase encodes an amino acid sequence having 100% sequence identity to any one of SEQ ID NOs: 2, 4, 6-12.

[0028] In some embodiments of the above aspects, the deaminase is a cytosine deaminase. In some embodiments, the DNA-binding polypeptide is a meganuclease, a zinc finger fusion protein, or a TALEN; or a variant of a meganuclease, a zinc finger fusion protein, or a TALEN, and the nuclease activity is reduced or inhibited.

[0029] In some embodiments of the above aspects, the DNA-binding polypeptide is an RNA-guided DNA-binding polypeptide. In some embodiments, the RNA-guided DNA-binding polypeptide is an RNA-guided nuclease (RGN) polypeptide. In some embodiments, the RGN is a type II or type V CRISPR-Cas polypeptide. In some embodiments, the RGN is an RGN nickase. In some embodiments, the RGN nickase has an inactive RuvC domain. In some embodiments, the RGN is a nuclease-inactive RGN.

[0030] In some embodiments of the above aspects, the RGN has an amino acid sequence that has at least 90% sequence identity with any one of the RGN sequences in Table 1. In some embodiments, the RGN has an amino acid sequence that has at least 95% sequence identity with any one of the RGN sequences in Table 1. In some embodiments, the RGN has the amino acid sequence of any one of the RGN sequences in Table 1. In some embodiments, the RGN has an amino acid sequence that has at least 90% sequence identity with any one of SEQ ID NOs: 74, 82, 87, 106, and 107. In some embodiments, the RGN has an amino acid sequence that has at least 95% sequence identity with any one of SEQ ID NOs: 74, 82, 87, 106, and 107. In some embodiments, the RGN has the amino acid sequence of any one of SEQ ID NOs: 74, 82, 87, 106, and 107.

[0031] In some embodiments of the above aspects, the RGN nickase has an amino acid sequence that has at least 90% sequence identity to any one of SEQ ID NOs: 75, 88-98. In some embodiments, the RGN nickase has an amino acid sequence that has at least 95% sequence identity to any one of SEQ ID NOs: 75, 88-98. In some embodiments, the RGN nickase has an amino acid sequence that has at least 95% sequence identity to any one of SEQ ID NOs: 75, 88-98.

[0032] In some embodiments of the above aspects, the polypeptide encoding the fusion protein is operably linked at its 5' end to a promoter. In some embodiments, the polypeptide encoding the fusion protein is operably linked at its 3' end to a terminator. In some embodiments, the fusion protein comprises one or more nuclear localization signals.

[0033] In some embodiments of the above aspects, the fusion protein is codon-optimized for expression in eukaryotic cells. In some embodiments, the fusion protein is codon-optimized for expression in prokaryotic cells. In some embodiments, the deaminase is fused to the amino terminus of the DNA binding polypeptide. In some embodiments, the deaminase is fused to the carboxyl terminus of the DNA binding polypeptide.

[0034] In some embodiments of the above aspects, the fusion protein further comprises a linker sequence between the DNA binding polypeptide and the deaminase. In some embodiments, the linker sequence has the amino acid sequence set forth as SEQ ID NO:78 or 79. In some embodiments, the fusion protein further comprises a uracil stabilizing protein (USP). In some embodiments, the USP has the sequence set forth as SEQ ID NO:81. In some embodiments, the fusion protein further comprises a linker sequence between the USP and the deaminase or the DNA binding polypeptide. In some embodiments, the linker sequence between the USP and the deaminase or the DNA binding polypeptide has the amino acid sequence set forth as SEQ ID NO:120. In some embodiments, the fusion protein has the amino acid sequence of any one of SEQ ID NOs:67, 68, 146, and 147.

[0035] In another aspect, the present disclosure provides a vector comprising the above-mentioned nucleic acid molecule. In some embodiments, the vector further comprises at least one nucleotide sequence encoding a guide RNA (gRNA) that can hybridize to a target sequence. In some embodiments, the gRNA is a single guide RNA. In some embodiments, the gRNA is a dual guide RNA.

[0036] In another aspect, the present disclosure provides a cell comprising the above fusion protein. In some embodiments, the cell further comprises a guide RNA (gRNA). In some embodiments, the gRNA is a single guide RNA. In some embodiments, the gRNA is a dual guide RNA.

[0037] In another aspect, the disclosure provides a cell comprising the above-described nucleic acid molecule or vector.

[0038] In some embodiments of the above aspects, the cell is a prokaryotic cell. In some embodiments of the above aspects, the cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a human cell. In some embodiments, the human cell is an immune cell. In some embodiments, the immune cell is a stem cell. In some embodiments, the stem cell is an induced pluripotent stem cell. In some embodiments, the eukaryotic cell is an insect cell or an avian cell. In some embodiments, the eukaryotic cell is a fungal cell. In some embodiments, the eukaryotic cell is a plant cell.

[0039] In another aspect, the disclosure provides a plant or seed comprising the above-described cell.

[0040] In another aspect, the disclosure provides a pharmaceutical composition comprising a fusion protein, a nucleic acid molecule, a vector, or a cell as described above, together with a pharma- ceutically acceptable carrier.

[0041] In another aspect, the disclosure provides a method of making a fusion protein comprising culturing the above-described cells under conditions in which the fusion protein is expressed.

[0042] In another aspect, the disclosure provides a method of making a fusion protein comprising introducing the nucleic acid molecule or vector described above into a cell and culturing the cell under conditions in which the fusion protein is expressed.

[0043] In some embodiments of the above aspects, the method further comprises purifying the fusion protein.

[0044] In another aspect, the present disclosure provides a method for producing an RGN fusion ribonucleoprotein complex, comprising introducing into a cell the above-mentioned nucleic acid molecule and a nucleic acid molecule comprising an expression cassette encoding a guide RNA (gRNA), or the above-mentioned vector, and culturing the cell under conditions in which the fusion protein and the gRNA are expressed to form an RGN fusion ribonucleoprotein complex. In some embodiments, the method further comprises purifying the RGN fusion ribonucleoprotein complex.

[0045] In another aspect, the present disclosure provides a system for modifying a target DNA molecule comprising a target DNA sequence, the system comprising: a) a fusion protein, or a nucleotide sequence encoding the fusion protein, wherein the fusion protein comprises an RNA-guided nuclease polypeptide (RGN) and a deaminase, the deaminase having an amino acid sequence selected from the group consisting of: i) an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 2 and 7-12; and ii) an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 4 or 6; and b) one or more guide RNAs capable of hybridizing to the target DNA sequence, or one or more nucleotide sequences encoding the one or more guide RNAs; the one or more guide RNAs form a complex with the fusion protein, allowing the fusion protein to bind to the target DNA sequence and modify the target DNA molecule.

[0046] In some embodiments of the above aspects, the deaminase has an amino acid sequence that has at least 95% sequence identity to any one of SEQ ID NOs: 2 and 7-12. In some embodiments, the deaminase has an amino acid sequence that has 100% sequence identity to any one of SEQ ID NOs: 2, 4, and 6-12. In some embodiments, at least one of the nucleotide sequence encoding the one or more guide RNAs and the nucleotide sequence encoding the fusion protein is operably linked to a promoter.

[0047] In some embodiments of the above aspects, the target DNA sequence is a eukaryotic target DNA sequence. In some embodiments, the target DNA sequence is located adjacent to a protospacer adjacent motif (PAM) recognized by the RGN.

[0048] In some embodiments of the above aspects, the target DNA molecule is in a cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is a plant cell. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a human cell. In some embodiments, the human cell is an immune cell. In some embodiments, the immune cell is a stem cell. In some embodiments, the stem cell is an induced pluripotent stem cell. In some embodiments, the eukaryotic cell is an insect cell. In some embodiments, the cell is a prokaryotic cell.

[0049] In some embodiments of the above aspects, the RGN of the fusion protein is a type II or type V CRISPR-Cas polypeptide. In some embodiments, the RGN of the fusion protein has an amino acid sequence that has at least 90% sequence identity to any one of the RGN sequences in Table 1. In some embodiments, the RGN of the fusion protein has an amino acid sequence that has at least 95% sequence identity to any one of the RGN sequences in Table 1. In some embodiments, the RGN of the fusion protein has an amino acid sequence of any one of the RGN sequences in Table 1. In some embodiments, the RGN of the fusion protein has an amino acid sequence that has at least 90% sequence identity to any one of SEQ ID NOs: 74, 82, 87, 106, and 107. In some embodiments, the RGN of the fusion protein has an amino acid sequence that has at least 95% sequence identity to any one of SEQ ID NOs: 74, 82, 87, 106, and 107. In some embodiments, the RGN of the fusion protein has the amino acid sequence of any one of SEQ ID NOs: 74, 82, 87, 106, and 107.

[0050] In some embodiments of the above aspects, the RGN of the fusion protein is an RGN nickase. In some embodiments, the RGN nickase has an inactive RuvC domain. In some embodiments, the RGN nickase has an amino acid sequence that has at least 90% sequence identity to any one of SEQ ID NOs: 75, 88-98. In some embodiments, the RGN nickase has an amino acid sequence that has at least 95% sequence identity to any one of SEQ ID NOs: 75, 88-98. In some embodiments, the RGN nickase is any one of SEQ ID NOs: 75, 88-98. In some embodiments, the RGN of the fusion protein is a nuclease inactive RGN.

[0051] In some embodiments of the above aspects, the fusion protein comprises one or more nuclear localization signals. In some embodiments, the deaminase is fused to the amino terminus of the DNA binding polypeptide. In some embodiments, the deaminase is fused to the carboxyl terminus of the DNA binding polypeptide. In some embodiments, the fusion protein further comprises a linker sequence between the DNA binding polypeptide and the deaminase. In some embodiments, the linker sequence has the amino acid sequence set forth as SEQ ID NO: 78 or 79.

[0052] In some embodiments of the above aspects, the fusion protein further comprises a uracil stabilizing protein (USP). In some embodiments, the USP has the sequence set forth as SEQ ID NO:81. In some embodiments, the fusion protein further comprises a linker sequence between the USP and the deaminase or the DNA binding polypeptide. In some embodiments, the linker sequence between the USP and the deaminase or the DNA binding polypeptide has the amino acid sequence set forth as SEQ ID NO:120.

[0053] In some embodiments of the above aspects, the fusion protein has an amino acid sequence set forth as any one of SEQ ID NOs: 67, 68, 146, and 147. In some embodiments, the fusion protein is codon optimized for expression in a eukaryotic cell. In some embodiments, the nucleotide sequence encoding the one or more guide RNAs and the nucleotide sequence encoding the fusion protein are located on a single vector.

[0054] In another aspect, the present disclosure provides a ribonucleoprotein complex comprising the at least one guide RNA and the fusion protein of the above system.

[0055] In another aspect, the disclosure provides a cell comprising the above system or ribonucleoprotein complex. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a human cell. In some embodiments, the human cell is an immune cell. In some embodiments, the immune cell is a stem cell. In some embodiments, the stem cell is an induced pluripotent stem cell. In some embodiments, the eukaryotic cell is an insect cell or an avian cell. In some embodiments, the eukaryotic cell is a fungal cell. In some embodiments, the eukaryotic cell is a plant cell.

[0056] In another aspect, the present disclosure provides a plant or seed comprising the plant cell described above.

[0057] In another aspect, the disclosure provides a pharmaceutical composition comprising the above-described system, ribonucleoprotein complex, or cell, together with a pharma- ceutically acceptable carrier.

[0058] In another aspect, the disclosure provides a method for modifying a target DNA molecule comprising a target DNA sequence, comprising delivering the above-described system or ribonucleoprotein complex to the target DNA molecule or to a cell comprising the target DNA molecule.

[0059] In some embodiments of the above aspects, the modified target DNA molecule comprises a C>N mutation of at least one nucleotide in the target DNA molecule, where N is A, G, or T. In some embodiments, the modified target DNA molecule comprises a C>T mutation of at least one nucleotide in the target DNA molecule. In some embodiments, the modified target DNA molecule comprises a C>G mutation of at least one nucleotide in the target DNA molecule.

[0060] In another aspect, the present disclosure provides a method for modifying a target DNA molecule comprising a target sequence, comprising: a) assembling an RGN-deaminase complex by combining in vitro, under conditions suitable for the formation of the RGN-deaminase complex, i) one or more guide RNAs capable of hybridizing to the target DNA molecule; ii) a fusion protein comprising an RNA-guided nuclease polypeptide (RGN) and at least one deaminase, wherein the deaminase has an amino acid sequence selected from the group consisting of I) an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 2 and 7 to 12; and II) an amino acid sequence having at least 95% identity to SEQ ID NO: 4 or 6; and b) contacting the target DNA molecule, or a cell comprising the target DNA molecule, with the in vitro assembled RGN-deaminase complex, whereby the one or more guide RNAs hybridize to the target DNA molecule, thereby binding the fusion protein to the target DNA molecule and causing modification of the target DNA molecule.

[0061] In some embodiments of the above aspects, the deaminase has an amino acid sequence that has at least 95% sequence identity to any one of SEQ ID NOs: 2 and 7-12. In some embodiments, the deaminase has an amino acid sequence that has 100% sequence identity to any one of SEQ ID NOs: 2, 4, and 6-12.

[0062] In some embodiments, the modified target DNA molecule comprises a C>N mutation of at least one nucleotide in the target DNA molecule, where N is A, G, or T. In some embodiments, the modified target DNA molecule comprises a C>T mutation of at least one nucleotide in the target DNA molecule. In some embodiments, the modified target DNA molecule comprises a C>G mutation of at least one nucleotide in the target DNA molecule.

[0063] In some embodiments of the above aspects, the RGN of the fusion protein is a type II or type V CRISPR-Cas polypeptide. In some embodiments, the RGN of the fusion protein has an amino acid sequence that has at least 90% sequence identity to any one of the RGN sequences in Table 1. In some embodiments, the RGN of the fusion protein has an amino acid sequence that has at least 95% sequence identity to any one of the RGN sequences in Table 1. In some embodiments, the RGN of the fusion protein has an amino acid sequence of any one of the RGN sequences in Table 1. In some embodiments, the RGN of the fusion protein has an amino acid sequence that has at least 90% sequence identity to any one of SEQ ID NOs: 74, 82, 87, 106, and 107. In some embodiments, the RGN of the fusion protein has an amino acid sequence that has at least 95% sequence identity to any one of SEQ ID NOs: 74, 82, 87, 106, and 107. In some embodiments, the RGN of the fusion protein has the amino acid sequence of any one of SEQ ID NOs: 74, 82, 87, 106, and 107.

[0064] In some embodiments of the above aspects, the RGN of the fusion protein is an RGN nickase. In some embodiments, the RGN nickase has an inactive RuvC domain. In some embodiments, the RGN nickase has an amino acid sequence that has at least 90% sequence identity to any one of SEQ ID NOs: 75, 88-98. In some embodiments, the RGN nickase has an amino acid sequence that has at least 95% sequence identity to any one of SEQ ID NOs: 75, 88-98. In some embodiments, the RGN nickase is any one of SEQ ID NOs: 75, 88-98. In some embodiments, the RGN of the fusion protein is a nuclease inactive RGN.

[0065] In some embodiments of the above aspects, the fusion protein comprises one or more nuclear localization signals. In some embodiments, the deaminase is fused to the amino terminus of the DNA binding polypeptide. In some embodiments, the deaminase is fused to the carboxyl terminus of the DNA binding polypeptide. In some embodiments, the fusion protein further comprises a linker sequence between the DNA binding polypeptide and the deaminase. In some embodiments, the linker sequence has the amino acid sequence set forth as SEQ ID NO: 78 or 79.

[0066] In some embodiments of the above aspects, the fusion protein further comprises a uracil stabilizing protein (USP). In some embodiments, the USP has the sequence set forth as SEQ ID NO:81. In some embodiments, the fusion protein further comprises a linker sequence between the USP and the deaminase or the DNA binding polypeptide. In some embodiments, the linker sequence between the USP and the deaminase or the DNA binding polypeptide has the amino acid sequence set forth as SEQ ID NO:120.

[0067] In some embodiments of the above aspects, the fusion protein has an amino acid sequence set forth as any one of SEQ ID NOs: 67, 68, 146, and 147. In some embodiments, the target DNA sequence is a eukaryotic target DNA sequence. In some embodiments, the target DNA sequence is located adjacent to a protospacer adjacent motif (PAM).

[0068] In some embodiments of the above aspects, the target DNA molecule is in a cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is a plant cell. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a human cell. In some embodiments, the human cell is an immune cell. In some embodiments, the immune cell is a stem cell. In some embodiments, the stem cell is an induced pluripotent stem cell. In some embodiments, the eukaryotic cell is an insect cell. In some embodiments, the cell is a prokaryotic cell.

[0069] In some embodiments of the above aspects, the method further comprises selecting a cell that contains the modified DNA molecule.

[0070] In another aspect, the disclosure provides a cell comprising a modified target DNA according to the above method. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a human cell. In some embodiments, the human cell is an immune cell. In some embodiments, the immune cell is a stem cell. In some embodiments, the stem cell is an induced pluripotent stem cell. In some embodiments, the eukaryotic cell is an insect cell. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the eukaryotic cell is a plant cell.

[0071] In another aspect, the disclosure provides a plant or seed comprising the above-described cell.

[0072] In another aspect, the disclosure provides a pharmaceutical composition comprising the cells described above and a pharma- ceutically acceptable carrier.

[0073] In another aspect, the present disclosure provides a method for producing a genetically modified cell in which a causative mutation associated with a genetic disease has been corrected, the method comprising: a) introducing into the cell a fusion protein, or a polynucleotide encoding the fusion protein, wherein the fusion protein comprises an RNA-guided nuclease (RGN) and a deaminase, the deaminase having an amino acid sequence selected from the group consisting of: i) an amino acid sequence having at least 90% identity with any one of SEQ ID NOs: 2 and 7 to 12; and ii) an amino acid sequence having at least 95% identity with SEQ ID NO: 4 or 6; and b) one or more guide RNAs (gRNAs) capable of hybridizing to a target DNA sequence, or a polynucleotide encoding the gRNA, whereby the fusion protein and gRNA are directed to the genomic locus of the causative mutation and the genomic sequence is modified to remove the causative mutation.

[0074] In some embodiments of the above aspects, the polynucleotide encoding the fusion protein is operably linked to a promoter active in the cell. In some embodiments, the polynucleotide encoding the gRNA is operably linked to a promoter active in the cell.

[0075] In some embodiments of the above aspects, the deaminase has an amino acid sequence that has at least 95% sequence identity to any one of SEQ ID NOs: 2 and 7-12. In some embodiments, the deaminase has an amino acid sequence that has 100% sequence identity to any one of SEQ ID NOs: 2, 4, and 6-12. In some embodiments, the RGN of the fusion protein is a type II or type V CRISPR-Cas polypeptide.

[0076] In some embodiments of the above aspects, the RGN of the fusion protein has an amino acid sequence that has at least 90% sequence identity to any one of the RGN sequences in Table 1. In some embodiments, the RGN of the fusion protein has an amino acid sequence that has at least 95% sequence identity to any one of the RGN sequences in Table 1. In some embodiments, the RGN of the fusion protein has the amino acid sequence of any one of the RGN sequences in Table 1.

[0077] In some embodiments of the above aspects, the RGN of the fusion protein is an RGN nickase. In some embodiments, the RGN nickase has an inactive RuvC domain. In some embodiments, the RGN nickase has an amino acid sequence that has at least 90% sequence identity to any one of SEQ ID NOs: 75, 88-98. In some embodiments, the RGN nickase has an amino acid sequence that has at least 95% sequence identity to any one of SEQ ID NOs: 75, 88-98. In some embodiments, the RGN nickase is any one of SEQ ID NOs: 75, 88-98. In some embodiments, the RGN of the fusion protein is a nuclease inactive RGN.

[0078] In some embodiments of the above aspects, the fusion protein comprises one or more nuclear localization signals. In some embodiments, the deaminase is fused to the amino terminus of the DNA binding polypeptide. In some embodiments, the deaminase is fused to the carboxyl terminus of the DNA binding polypeptide. In some embodiments, the fusion protein further comprises a linker sequence between the DNA binding polypeptide and the deaminase. In some embodiments, the linker sequence has the amino acid sequence set forth as SEQ ID NO: 78 or 79.

[0079] In some embodiments of the above aspects, the fusion protein further comprises a uracil stabilizing protein (USP). In some embodiments, the USP has the sequence set forth as SEQ ID NO:81. In some embodiments, the fusion protein further comprises a linker sequence between the USP and the deaminase or the DNA binding polypeptide. In some embodiments, the linker sequence between the USP and the deaminase or the DNA binding polypeptide has the amino acid sequence set forth as SEQ ID NO:120.

[0080] In some embodiments of the above aspects, the fusion protein has an amino acid sequence set forth as any one of SEQ ID NOs: 67, 68, 146, and 147. In some embodiments, the genomic modification comprises introducing a C>T mutation of at least one nucleotide in the target DNA sequence. In some embodiments, the genomic modification comprises introducing a C>G mutation of at least one nucleotide in the target DNA sequence.

[0081] In some embodiments of the above aspects, the cell is an animal cell. In some embodiments, the animal cell is a mammalian cell. In some embodiments, the cell is from a dog, cat, mouse, rat, rabbit, horse, sheep, goat, cow, pig, or human.

[0082] In some embodiments of the above aspects, correction of the causative mutation comprises correction of a nonsense mutation. In some embodiments, the genetic disease is a disease listed in Table 23. In some embodiments, the gRNA further comprises a spacer sequence that targets any one of SEQ ID NOs: 122-144, or a complement thereof.

[0083] In another aspect, the disclosure provides a composition comprising: a) a fusion protein comprising a DNA-binding polypeptide and a cytosine deaminase, or a nucleic acid molecule encoding the fusion protein; and b) a second cytosine deaminase, or a nucleic acid molecule encoding the second deaminase, wherein the second deaminase has an amino acid sequence selected from the group consisting of: i) an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 2 and 7-12; and ii) an amino acid sequence having at least 95% identity to SEQ ID NO: 4 or 6.

[0084] In some embodiments of the above aspects, the second cytosine deaminase has a sequence that is at least 95% identical to any one of SEQ ID NOs: 2 and 7-12. In some embodiments, the second cytosine deaminase has a sequence that is 100% identical to any one of SEQ ID NOs: 2, 4, and 6-12.

[0085] In some embodiments of the above aspects, the first cytosine deaminase has an amino acid sequence selected from the group consisting of: a) an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 2 and 7-12; and b) an amino acid sequence having at least 95% identity to SEQ ID NO: 4 or 6. In some embodiments, the first cytosine deaminase has at least 95% identity to any one of SEQ ID NOs: 2 and 7-12. In some embodiments, the first cytosine deaminase has 100% identity to any one of SEQ ID NOs: 2, 4, and 6-12.

[0086] In some embodiments of the above aspects, the DNA-binding polypeptide is a meganuclease, a zinc finger fusion protein, or a TALEN; or a variant of a meganuclease, a zinc finger fusion protein, or a TALEN, and the nuclease activity is reduced or inhibited.In some embodiments, the DNA-binding polypeptide is an RNA-guided DNA-binding polypeptide.In some embodiments, the RNA-guided DNA-binding polypeptide is an RNA-guided nuclease (RGN) polypeptide.

[0087] In some embodiments of the above aspects, the RGN is an RGN nickase. In some embodiments, the RGN is a nuclease-inactive RGN.

[0088] In another aspect, the disclosure provides a vector comprising a nucleic acid molecule encoding a fusion protein and a nucleic acid molecule encoding a second cytosine deaminase, wherein the fusion protein comprises a DNA-binding polypeptide and a first cytosine deaminase, and the second cytosine deaminase has an amino acid sequence selected from the group consisting of: a) an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 2 and 7-12; and b) an amino acid sequence having at least 95% identity to SEQ ID NO: 4 or 6.

[0089] In some embodiments of the above aspects, the second cytosine deaminase has a sequence that is at least 95% identical to any one of SEQ ID NOs: 2 and 7-12. In some embodiments, the second cytosine deaminase has a sequence that is 100% identical to any one of SEQ ID NOs: 2, 4, and 6-12.

[0090] In some embodiments of the above aspects, the first cytosine deaminase has an amino acid sequence selected from the group consisting of: a) an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 2 and 7-12; and b) an amino acid sequence having at least 95% identity to SEQ ID NO: 4 or 6.

[0091] In some embodiments of the above aspects, the first cytosine deaminase has at least 95% sequence identity to any one of SEQ ID NOs: 2 and 7-12. In some embodiments, the first cytosine deaminase has 100% sequence identity to any one of SEQ ID NOs: 2, 4, and 6-12. In some embodiments, the DNA binding polypeptide is a meganuclease, zinc finger fusion protein, or TALEN; or a variant of a meganuclease, zinc finger fusion protein, or TALEN, wherein the nuclease activity is reduced or inhibited. In some embodiments, the DNA binding polypeptide is an RNA-guided DNA binding polypeptide. In some embodiments, the RNA-guided DNA binding polypeptide is an RNA-guided nuclease (RGN) polypeptide.

[0092] In some embodiments of the above aspects, the RGN is an RGN nickase. In some embodiments, the RGN is a nuclease-inactive RGN.

[0093] In another aspect, the disclosure provides a cell comprising the above-described vector.

[0094] In another aspect, the disclosure provides a cell comprising: a) a fusion protein comprising a DNA-binding polypeptide and a first cytosine deaminase; or a nucleic acid molecule encoding the fusion protein; and b) a second cytosine deaminase, or a nucleic acid molecule encoding the second deaminase, wherein the second deaminase has an amino acid sequence selected from the group consisting of: i) an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 2 and 7-12; and ii) an amino acid sequence having at least 95% identity to SEQ ID NO: 4 or 6.

[0095] In some embodiments of the above aspects, the second cytosine deaminase has a sequence that is at least 95% identical to any one of SEQ ID NOs: 2 and 7-12. In some embodiments, the second cytosine deaminase has a sequence that is 100% identical to any one of SEQ ID NOs: 2, 4, and 6-12.

[0096] In some embodiments of the above aspects, the first cytosine deaminase has an amino acid sequence selected from the group consisting of: a) an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 2 and 7-12; and b) an amino acid sequence having at least 95% identity to SEQ ID NO: 4 or 6. In some embodiments, the first cytosine deaminase has at least 95% identity to any one of SEQ ID NOs: 2 and 7-12. In some embodiments, the first cytosine deaminase has 100% identity to any one of SEQ ID NOs: 2, 4, and 6-12. In some embodiments, the DNA binding polypeptide is a meganuclease, zinc finger fusion protein, or TALEN; or is a variant of a meganuclease, zinc finger fusion protein, or TALEN, wherein the nuclease activity is reduced or inhibited. In some embodiments, the DNA binding polypeptide is an RNA-guided DNA binding polypeptide.

[0097] In some embodiments of the above aspects, the RNA-guided DNA-binding polypeptide is an RNA-guided nuclease (RGN) polypeptide. In some embodiments, the RGN is an RGN nickase. In some embodiments, the RGN is a nuclease-inactive RGN.

[0098] In another aspect, the disclosure provides a pharmaceutical composition comprising a pharma- ceutically acceptable carrier and the composition, vector, or cell described above.

[0099] In another aspect, the disclosure provides a method of treating a disease comprising administering to a subject in need thereof the fusion protein, nucleic acid molecule, vector, cell, system, ribonucleoprotein complex, composition, or pharmaceutical composition described above.

[0100] In some embodiments of the above aspects, the disease is associated with a causative mutation and the pharmaceutical composition corrects the causative mutation. In some embodiments, the disease is a disease listed in Table 23.

[0101] In another aspect, the disclosure provides the use of the fusion protein, nucleic acid molecule, vector, cell, system, ribonucleoprotein complex, or composition described above for treating a disease in a subject.

[0102] In some embodiments of the above aspects, the disease is associated with a causative mutation and the treating comprises correcting the causative mutation. In some embodiments, the disease is a disease listed in Table 23.

[0103] In another aspect, the present disclosure provides the use of a fusion protein, a nucleic acid molecule, a vector, a cell, a system, a ribonucleoprotein complex, or a composition for the manufacture of a drug useful for treating a disease. In some embodiments, the disease is associated with a causative mutation, and an effective amount of the drug corrects the causative mutation. In some embodiments, the disease is a disease listed in Table 23. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0104] Modifications and other embodiments of the inventions described herein will come to mind to those skilled in the art to which this invention pertains having the benefit of the teachings presented in the foregoing description. It is to be understood, therefore, that these inventions are not limited to the specific embodiments disclosed, and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0105] I. Overview

[0106] The present disclosure provides a fusion protein comprising a cytosine deaminase, a nucleic acid molecule binding polypeptide (such as a DNA binding polypeptide) and a deaminase polypeptide. In an embodiment, the DNA binding polypeptide is a sequence-specific DNA binding polypeptide in the sense that it binds to a target sequence with a greater frequency than it binds to a randomized background sequence. In some embodiments, the DNA binding polypeptide is or is derived from a meganuclease, a zinc finger fusion protein, or a TALEN. In some embodiments, the fusion protein comprises an RNA-guided DNA binding polypeptide and a deaminase polypeptide. In some embodiments, the RNA-guided DNA binding polypeptide is an RNA-guided nuclease (such as a CRISPR-Cas (e.g., Cas9) polypeptide) that binds to a guide RNA (also called gRNA), which in turn binds to a target nucleic acid sequence through strand hybridization.

[0107] The deaminase polypeptides disclosed herein can deaminate nucleobases (e.g., cytosine, etc.). Deamination of nucleobases by deaminases can lead to point mutations at the respective residue positions, which is referred to herein as "nucleic acid editing" or "base editing". Thus, fusion proteins comprising RNA-guided nuclease (RGN) polypeptides and deaminases can be used to enable targeted editing of nucleic acid sequences.

[0108] Such fusion proteins are useful for targeted editing of DNA in vitro, for example to generate genetically modified cells. These genetically modified cells can be plant or animal cells. Such fusion proteins can also be useful for introducing targeted mutations to correct genetic defects in mammalian cells ex vivo (e.g., correcting a genetic defect in cells obtained from a subject and then reintroducing the cells into the same or another subject); and for introducing targeted mutations in disease-associated genes in mammalian subjects (e.g., correcting a genetic defect or introducing deactivating mutations). Such fusion proteins can also be useful for introducing targeted mutations in plant cells (e.g., introducing beneficial or agriculturally valuable traits or alleles).

[0109] The terms "protein", "peptide" and "polypeptide" are used interchangeably herein and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. These terms refer to proteins, peptides, or polypeptides of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids in length. A protein, peptide, or polypeptide can refer to an individual protein or a group of proteins. One or more of the amino acids in a protein, peptide, or polypeptide can be modified, for example, by the addition of chemicals (e.g., carbohydrate groups, hydroxyl groups, phosphate groups, farnesyl groups, isofarnesyl groups, fatty acid groups, linkers, etc., for conjugation, functionalization, or other modification). A protein, peptide, or polypeptide can be a single molecule or a multi-molecular complex. A protein, peptide, or polypeptide can be simply a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide can be naturally occurring, recombinant, or synthetic, or any combination thereof.

[0110] Any of the proteins provided herein can be produced by any method known in the art. For example, the proteins provided herein can be produced by recombinant protein expression and purification, and are particularly suitable for fusion proteins that contain peptide linkers. Methods for recombinant protein expression and purification are well known and include those described in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)), the entire contents of which are incorporated herein by reference.

[0111] II. Deaminases

[0112] The term "deaminase" refers to an enzyme that catalyzes a deamination reaction. The deaminase of the present invention is a nucleobase deaminase, and the terms "deaminase" and "nucleobase deaminase" are used interchangeably herein. The deaminase can be a naturally occurring deaminase enzyme, or an active fragment or variant thereof. The deaminase can be active on single-stranded nucleic acids (such as ssDNA or ssRNA) or double-stranded nucleic acids (such as dsDNA or dsRNA). In some embodiments, the deaminase can only deaminate ssDNA and does not act on dsDNA.

[0113] The disclosed methods and compositions include a cytosine deaminase that catalyzes the hydrolytic deamination of cytosine, cytidine, or deoxycytidine to uracil. The cytosine deaminase may act on DNA or RNA, and typically acts on single-stranded nucleic acid molecules. In further embodiments, the cytosine deaminase is an Apolipoprotein B mRNA editing complex (APOBEC) family deaminase. In some embodiments, the deaminase is an APOBECl family deaminase. In some embodiments, the cytosine deaminase is an activation-induced cytidine deaminase (AID). In some embodiments, the deaminase is an ACF1 / ASE deaminase. Additional suitable deaminase enzymes will be apparent to one of skill in the art based on the present disclosure. A fusion protein comprising a DNA-binding polypeptide and a cytosine deaminase is referred to herein as a "C base editor," "cytosine base editor," or "CBE." CBEs can convert cytosine to uracil, which can then be converted to thymidine through DNA replication or repair. In some embodiments, CBEs convert cytosine to guanine or cytosine to adenine. Without being bound by any theory or mechanism of action, it is believed that the conversion of cytosine to guanine or adenine by a cytosine base editor is achieved by the activity of uracil DNA glycosylase during base excision repair of the uracil residue after deamination of the cytosine to uracil.

[0114] In some embodiments, the cytosine deaminase of the present disclosure is used in combination with adenine deaminase.In some embodiments, the adenine deaminase is an ADAT family deaminase or its variant.Deamination of adenine, adenosine, or deoxyadenosine produces inosine, which is processed by polymerase as guanine.Currently, there is no known natural adenine deaminase that deaminates adenine in DNA. Several methods have been used to evolve adenine deaminase (ADAT) proteins acting on tRNA to be active on DNA molecules in mammalian cells (Gaudelli et al., 2017; Koblan, LW et al., 2018, Nat Biotechnol 36, 843-846; Richter, MF et al., 2020, Nat Biotechnol, doi:10.1038 / s41587-020-0562-8, each of which is incorporated by reference in its entirety). One such method utilizes a bacterial selection assay, in which only cells capable of activating antibiotic resistance through an A:T>G:C conversion survive. In some embodiments, the compositions and methods of the present disclosure comprising the cytosine deaminase of the present disclosure further comprise an adenine deaminase as set forth in U.S. Provisional Patent Application Nos. 63 / 077,089, filed September 11, 2020, and 63 / 146,840, filed February 8, 2021, and PCT International Application No. PCT / US2021 / 049853, filed September 10, 2021 (each of which is incorporated by reference in its entirety into this specification).

[0115] The present invention relates to cytosine deaminase polypeptides identified from bacteria and cytosine deaminases generated through truncation of bacterial deaminases. Cytosine deaminases are disclosed herein and set forth as SEQ ID NOs: 2, 4, and 6-12. Deaminases of the invention can be used to edit DNA or RNA molecules. In some embodiments, deaminases of the invention can be used to edit ssDNA or ssRNA molecules. The cytosine deaminases described herein are useful as the sole deaminases or as components in fusion proteins. Fusion proteins comprising a DNA targeting polypeptide and a cytosine deaminase polypeptide are referred to herein as "C-based editors," "cytosine base editors," or "CBEs," and can be used for targeted editing of nucleic acid sequences.

[0116] A "base editor" is a fusion protein that includes a DNA targeting polypeptide (such as RGN) and a deaminase. A cytosine base editor (CBE) includes a DNA targeting protein (such as RGN) and a cytosine deaminase. The CBE functions through deamination of cytosine on a DNA target molecule to uracil, which is then converted to thymine during DNA replication or repair. In some embodiments, a cytosine deaminase of the present disclosure, or an active variant or fragment thereof, introduces a C>N mutation into a DNA molecule, where N is A, G, or T. In some embodiments, a cytosine deaminase of the present disclosure, or a fusion protein comprising the same, introduces a C>T mutation into a DNA molecule. In some embodiments, a cytosine deaminase of the present disclosure, or a fusion protein comprising the same, introduces a C>G mutation into a DNA molecule.

[0117] In embodiments in which the deaminase is targeted to a specific region of a nucleic acid molecule via fusion to a DNA-binding polypeptide, the mutation rate of cytosines in or adjacent to the target sequence to which the DNA-binding polypeptide binds can be measured using any method known in the art, including polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), or DNA sequencing.

[0118] To increase the efficiency of introducing a desired C>N (where N is A, T, or G) mutation (such as a C>T or C>G mutation) into a target DNA molecule, a deaminase of the disclosure, or an active variant or fragment thereof that retains deaminase activity, can be introduced into a cell as part of a deaminase-DNA binding polypeptide fusion and / or co-expressed with a DNA binding polypeptide-deaminase fusion. Deaminases of the disclosure have the amino acid sequence of any of SEQ ID NOs: 2, 4, and 6-12, or a variant or fragment thereof that retains deaminase activity. In some embodiments, the deaminase has an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 2, 4, and 6-12. In particular embodiments, the deaminase comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 2 and 7-12. In certain embodiments, the deaminase comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 4 or 6.

[0119] III. Nucleic Acid Binding Polypeptides

[0120] Some aspects of the present disclosure provide fusion proteins comprising a nucleic acid molecule binding polypeptide and a deaminase polypeptide. Although binding to an RNA molecule and targeted editing of an RNA molecule are contemplated by the present invention, in some embodiments, the nucleic acid molecule binding polypeptide of the fusion protein is a DNA binding polypeptide. Such fusion proteins are useful for targeted editing of DNA in vitro, ex vivo, or in vivo. These fusion proteins are active in mammalian cells and are useful for targeted editing of DNA molecules.

[0121] The term "fusion protein" as used herein refers to a hybrid polypeptide that contains protein domains from at least two different proteins. A fusion protein can contain two or more different domains (e.g., a DNA binding domain and a deaminase). In some embodiments, a fusion protein is complexed with or associated with a nucleic acid (e.g., RNA).

[0122] In some embodiments, the fusion protein of the present disclosure comprises a DNA-binding polypeptide. As used herein, the term "DNA-binding polypeptide" refers to any polypeptide capable of binding to DNA. In certain embodiments, the DNA-binding polypeptide portion of the fusion protein of the present disclosure binds to double-stranded DNA. In particular embodiments, the DNA-binding polypeptide binds to DNA in a sequence-specific manner. As used herein, the term "sequence-specific" or "sequence-specifically" refers to selective interaction with a particular nucleotide sequence.

[0123] Two polynucleotide sequences are considered to be substantially complementary when the two sequences hybridize to each other under stringent conditions. Similarly, a DNA-binding polypeptide is considered to bind sequence-specifically to a particular target sequence if the DNA-binding polypeptide binds to the sequence under stringent conditions. "Stringent conditions" or "stringent hybridization conditions" refers to conditions under which the two polynucleotide sequences bind to each other (or the polypeptide binds to its particular target sequence) with a detectability greater than other sequences (e.g., at least twice background). Stringent conditions are sequence-dependent and will be different in different environments. Typically, stringent conditions are conditions in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion (or other 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., more than 50 nucleotides). Stringent conditions can also be achieved by the addition of a destabilizing agent (such as formamide). Representative low stringency conditions include hybridization at 37° C. in a buffer solution of 30-35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate), followed by washing in 1×-2×SSC (20×SSC=3.0 M NaCl / 0.3 M trisodium citrate) at 50-55° C. Representative medium stringency conditions include hybridization at 37° C. in 40-45% formamide, 1.0 M NaCl, 1% SDS, followed by washing in 0.5×-1×SSC at 55-60° C. Representative high stringency conditions include hybridization at 37° C. in 50% formamide, 1 M NaCl, 1% SDS, followed by washing in 0.1×SSC at 60-65° C. Optionally, the washing buffer can contain about 0.1% to about 1% SDS. Hybridization times are generally less than about 24 hours, and usually from about 4 to about 12 hours. Wash times will be at least long enough to reach equilibrium.

[0124] Tm is the temperature at which 50% of a complementary target sequence hybridizes (under a defined ionic strength and pH) to a perfectly matched sequence. For DNA-DNA hybrids, Tm can be roughly calculated from Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284, as Tm=81.5°C+16.6 (logM)+0.41(%GC)-0.61(%form)-500 / L, where M is the number of moles of monovalent cations, %GC is the percentage of guanosine and cytosine nucleotides in the DNA, %form is the percentage of formamide in the hybridization solution, and L is the hybrid length in base pairs. In general, stringent conditions are selected to be approximately 5°C lower than the melting point (Tm) of a specific sequence and its complementary sequence at a defined ionic strength and pH. However, highly stringent conditions can utilize hybridization and / or washing at 1, 2, 3, or 4° C. below the melting temperature (Tm); moderately stringent conditions can utilize hybridization and / or washing at 6, 7, 8, 9, or 10° C. below the melting temperature (Tm); and low stringency conditions can utilize hybridization and / or washing at 11, 12, 13, 14, 15, or 20° C. below the melting temperature (Tm). One of skill in the art will understand that variations in stringency of hybridization and / or washing solutions are essentially described using the above formula, hybridization and washing compositions, and desired Tm.Comprehensive guides to nucleic acid hybridization can be found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience, New York). See Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, NY).

[0125] In one embodiment, the sequence-specific DNA-binding polypeptide is an RNA-guided DNA-binding polypeptide (RGDBP). As used herein, the terms "RNA-guided DNA-binding polypeptide" and "RGDBP" refer to a polypeptide that can bind to DNA through hybridization of an associated RNA molecule with a target DNA sequence.

[0126] In some embodiments, the DNA-binding polypeptide of the fusion protein is a nuclease (such as a sequence-specific nuclease). As used herein, the term "nuclease" refers to an enzyme that catalyzes the cleavage of phosphodiester bonds between nucleotides in a nucleic acid molecule. In some embodiments, the DNA-binding polypeptide is an endonuclease that can cleave phosphodiester bonds between nucleotides in a nucleic acid molecule, while in certain embodiments, the DNA-binding polypeptide is an exonuclease that can cleave nucleotides at either end (5' or 3') of the nucleic acid molecule. In some embodiments, the sequence-specific nuclease is selected from the group consisting of meganucleases, zinc finger nucleases, TAL-effector DNA binding domain-nuclease fusion proteins (TALENs), and RNA-guided nucleases (RGNs), or variants thereof with reduced or inhibited nuclease activity.

[0127] As used herein, the term "meganuclease" or "homing endonuclease" refers to an endonuclease that binds to a recognition site in double-stranded DNA of 12-40 bp in length. Non-limiting examples of meganucleases are those that belong to the LAGLIDADG family, which contains the conserved amino acid motif LAGLIDADG (SEQ ID NO: 85). The term "meganuclease" can refer to dimeric or single-stranded meganucleases.

[0128] As used herein, the term "zinc finger nuclease" or "ZFN" refers to a chimeric protein containing a zinc finger DNA binding domain and a nuclease domain.

[0129] As used herein, the term "TAL-effector DNA binding domain-nuclease fusion protein" or "TALEN" refers to a chimeric protein comprising a TAL effector DNA binding domain and a nuclease domain.

[0130] In certain embodiments, the DNA-binding polypeptide is capable of generating a single-stranded region in a double-stranded DNA molecule. An example of a single-stranded region is a single-stranded loop contained within an R-loop, which is a triple-stranded nucleic acid structure that includes a region of single-stranded DNA formed in a double-stranded DNA molecule resulting from hybridization of a complementary strand to a single-stranded RNA or DNA molecule. Cytosines within or adjacent to the single-stranded region of an R-loop can be deaminated by a cytosine deaminase with activity against single-stranded nucleic acids (e.g., ssDNA). In some of these embodiments, the DNA-binding polypeptide capable of generating an R-loop in a double-stranded DNA molecule is an RNA-guided DNA-binding polypeptide or an RGN nuclease. As used herein, the term "RNA-guided nuclease" or "RGN" refers to an RNA-guided DNA-binding polypeptide with nuclease activity. RGN is considered to be "RNA-guided". This is because the guide RNA forms a complex with the RNA-guided nuclease to bind the RNA-guided nuclease to the target sequence and, in some embodiments, introduces a single- or double-stranded break in the target sequence.

[0131] In one embodiment, RGN is a nickase RGN or a nuclease-inactive RGN. The term "RGN polypeptide" encompasses RGN polypeptides that cleave only one strand of a target nucleotide sequence, and are referred to herein as nickases. Such RGNs have a single functional nuclease domain. RGN nickases can be natural nickases or can be RGN proteins that naturally cleave both strands of a mutated double-stranded nucleic acid molecule in one or more nuclease domains, thereby reducing or eliminating the nuclease activity of these mutated domains, making them nickases.

[0132] In some embodiments, the nickase RGN of the fusion protein contains a mutation (e.g., a D10A mutation (where the amino acid numbering is based on the S. pyogenes Cas9 sequence shown as SEQ ID NO: 99)) that allows RGN to cleave only the non-base-edited target strand of a nucleic acid duplex (the strand that contains the PAM and base-pairs with the gRNA). A nickase containing the D10A mutation or an equivalent mutation renders the RuvC nuclease domain inactive and cleaves the targeted strand. The D10 nickase is unable to cleave the non-target strand of DNA, i.e., the strand in which base editing is desired. In these embodiments, the RGN nicks the target strand while the complementary non-target strand is modified by a deaminase. The cellular DNA repair machinery can repair the nicked target strand using the modified non-target strand as a template, thereby introducing mutations into the DNA.

[0133] Thus, in some embodiments, the nickase comprises an inactive RuvC domain. The RuvC domain has an RNase H-fold structure (see, e.g., Nishimasu et al. (2014) Cell 156(5): 935-949, which is incorporated by reference in its entirety). The RuvC domain of RGN is often a split RuvC domain that includes two or more non-adjacent regions in the linear amino acid sequence. For example, the RuvC domain of Streptococcus pyogenes Cas9 includes amino acid residues 1-59, 718-769, and 909-1098 of SEQ ID NO: 99. A non-limiting example of a mutation in the RuvC domain that inactivates its nuclease activity is the D10A mutation, which mutates the first aspartic acid residue in the split RuvC nuclease domain. The present application discloses several D10 nickase variants or homologous nickase variants of RGN described in which the RuvC domain is inactivated. nAPG07433.1 and nAPG08290.1 ​​(represented as SEQ ID NOs: 75 and 88, respectively) are nickase variants of APG07433.1 and APG08290.1, represented as SEQ ID NOs: 44 and 87, respectively, and are described in WO2019 / 23566, which is incorporated by reference in its entirety. nAPG07433.1-del and nAPG08290.1-del (represented as SEQ ID NOs: 97 and 98, respectively) are deletion mutants of APG07433.1 and APG08290.1, respectively, and are described in U.S. Provisional Application Nos. 63 / 077,089, filed September 11, 2020, and 63 / 146,840, filed February 8, 2021, and PCT International Application No. PCT / US2021 / 049853, filed September 10, 2021. nAPG00969 (shown as SEQ ID NO:89) and nAPG09748 (shown as SEQ ID NO:90) are nickase variants of APG00969 and APG09748, respectively, and are described in WO2020 / 139783, which is incorporated by reference in its entirety.nAPG06646 (represented as SEQ ID NO: 91) and nAPG09882 (represented as SEQ ID NO: 92) are nickase variants of APG06646 and APG09882, respectively, and are described in PCT Publication No. WO2021 / 030344, which are incorporated herein by reference in their entireties. nAPG03850, nAPG07553, nAPG055886, and nAPG01604 are represented as SEQ ID NOs: 93-96, respectively, and are nickase variants of APG03850, APG07553, APG055886, and APG01604, which are described in pending U.S. Provisional Application Nos. 63 / 014,970 and 63 / 077,211, and PCT Publication No. WO2021 / 21702, each of which is incorporated herein by reference in its entirety.

[0134] In some embodiments, the nickase RGN of the fusion protein comprises a mutation (e.g., an H840A mutation (where the amino acid numbering is based on the S. pyogenes Cas9 sequence shown as SEQ ID NO: 99)) that enables the RGN to cleave only the base-edited non-target strand of the nucleic acid duplex (the strand that does not contain a PAM and does not base-pair with the gRNA). In some of these embodiments, the nickase comprises an inactive HNH nuclease domain. The HNH nuclease domain of RGN has a ββα-metallofold (see, e.g., Nishimasu et al. 2014). The HNH nuclease domain of S. pyogenes Cas9 comprises, e.g., amino acid residues 775-908 of SEQ ID NO: 99. One non-limiting example of a mutation in the HNH domain that inactivates nuclease activity is the H840A mutation, which mutates the first histidine of the HNH nuclease domain. A deaminase with an inactivated HNH domain acts on the non-target strand.

[0135] Methods for inactivating the RuvC and / or HNH domains of RGN are known in the art and generally involve mutating the first aspartic acid in the split RuvC domain and / or the first histidine in the HNH domain. Typically, the aspartic acid or histidine residue is mutated to alanine. Other amino acid residues in the RuvC domain that can be mutated to inactivate the nuclease activity of this domain include Glu762, His983, and Asp986 (typically to alanine) (where the amino acid numbering is based on the S. pyogenes Cas9 sequence shown as SEQ ID NO: 99). Other amino acid residues in the HNH domain that can be mutated include D839 and N863 (typically to alanine) (where the amino acid numbering is based on the S. pyogenes Cas9 sequence shown as SEQ ID NO: 99).

[0136] In some embodiments, the RGN of the fusion protein is nuclease-dead. Mutated nuclease-inactive or "dead" RGN protein can be referred to herein as an RNA-guided DNA-binding polypeptide, or nuclease-inactive RGN, or nuclease-dead RGN. Methods for generating nuclease-inactive RGN are known in the art and generally involve mutating only or all of the nuclease domains of RGN to render the nuclease domain inactive. In embodiments where RGN contains only a single nuclease domain (e.g., RuvC domain), the nuclease-inactive variant will have at least one mutation in the RuvC domain, resulting in the inactivation of the RuvC nuclease domain. In embodiments where RGN contains more than one nuclease domain (e.g., RuvC domain and HNH domain), at least one mutation in each of the RuvC domain and the HNH domain renders both nuclease domains inactive.

[0137] One suitable representative nuclease-inactive RGN is the D10A / H840A Cas9 mutant (see, e.g., Qi et al., Cell. 2013; 152(5): 1173-83, the entire contents of which are incorporated herein by reference). In addition, suitable nuclease-inactive variants of other known RNA-guided nucleases (RGNs) can be defined (e.g., RGN APG08290.1 ​​or RGN APG07433.1, as disclosed in U.S. Patent Application Publication No. 2019 / 0367949, the entire contents of which are incorporated herein by reference, or nuclease-inactive variants of dAPG09298, set forth as SEQ ID NO: 83).

[0138] Other additional suitable representative nuclease-inactive RGN variants, non-limiting examples include the mutant domains D10A / D839A / H840A and D10A / D839A / H840A / N863A (see, e.g., Mali et al., Nature Biotechnology. 2013; 31(9): 833-838, the entire contents of which are incorporated herein by reference).

[0139] Additional suitable RGN proteins that are mutated to become nickases or inactive nucleases will be apparent to one of skill in the art based on this disclosure and knowledge in the field (e.g., RGNs disclosed in PCT Publication No. WO2019 / 236566, which is incorporated by reference in its entirety) and are therefore within the scope of this disclosure.

[0140] In some embodiments, an RGN nickase that retains nickase activity comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of SEQ ID NOs: 75 and 88-98.

[0141] Any method known in the art for introducing mutations into an amino acid sequence, such as PCR-mediated mutagenesis or site-directed mutagenesis, can be used to generate a nickase or nuclease-dead RGN. See, e.g., U.S. Application Publication No. 2014 / 0068797 and U.S. Patent No. 9,790,490; each of which is incorporated by reference in its entirety.

[0142] RNA-guided nucleases (RGNs) allow targeted manipulation of single sites in the genome, making them useful in the context of gene targeting for therapeutic and research applications. RNA-guided nucleases have been used for genome manipulation by promoting non-homologous end joining or homologous recombination in a variety of organisms, including mammals. RGNs include CRISPR-Cas proteins, or active variants or fragments thereof, which are RNA-guided nucleases that are directed to target sequences by guide RNAs (gRNAs) as part of the clustered regularly interspaced short palindromic repeats (CRISPR) RNA-guided nuclease system.

[0143] Some aspects of the disclosure provide a fusion protein comprising an RNA-guided DNA-binding polypeptide and a deaminase polypeptide, particularly a cytosine deaminase polypeptide. In some embodiments, the RNA-guided DNA-binding polypeptide is an RNA-guided nuclease (RGN). In further embodiments, the RNA-guided nuclease is a native CRISPR-Cas protein, or an active variant or fragment thereof. CRISPR-Cas systems are classified into class 1 or 2 systems. Class 2 systems contain a single effector nuclease and include types II, V, and VI. Class 1 and 2 systems are classified into multiple types (type I, type II, type III, type IV, type V, type VI), with some types further classified into subtypes (e.g., type II-A, type II-B, type II-C, type VV-B).

[0144] In an embodiment, the RGN is a naturally occurring type II CRISPR-Cas protein, or an active variant or fragment thereof. As used herein, the term "type II CRISPR-Cas protein," "type II CRISPR-Cas effector protein," or "type II RNA-guided nuclease" refers to an RGN that requires a trans-activating RNA (tracrRNA) and contains two nuclease domains (i.e., RuvC and HNH), each of which is responsible for cleaving a single strand of a double-stranded DNA molecule. In some embodiments, the present invention provides a fusion protein comprising a deaminase of the present disclosure fused to a Cas9 protein, such as Streptococcus pyogenes Cas9 (SpCas9) or SpCas9 nickase (the sequences of which are set forth as SEQ ID NOs: 99 and 100, respectively, and described in U.S. Patent Nos. 10,000,772 and 8,697,359, each of which is incorporated herein by reference in its entirety). In some embodiments, the invention provides a fusion protein comprising a deaminase of the disclosure fused to Streptococcus thermophilus Cas9 (StCas9) or StCas9 nickase (the sequences of which are set forth as SEQ ID NOs: 101 and 102, respectively, and are disclosed in U.S. Patent No. 10,113,167, which is incorporated by reference in its entirety). In some embodiments, the invention provides a fusion protein comprising a deaminase of the disclosure fused to Streptococcus aureus Cas9 (SaCas9) or SaCas9 nickase (the sequences of which are set forth as SEQ ID NOs: 103 and 104, respectively, and are disclosed in U.S. Patent No. 9,752,132, which is incorporated by reference in its entirety).

[0145] In some embodiments, the CRISPR-Cas protein is a naturally occurring type V CRISPR-Cas protein, or an active variant or fragment thereof. As used herein, the term "V-type CRISPR-Cas protein," "V-type CRISPR-Cas effector protein," or "V-type RNA-guided nuclease" refers to an RGN that cleaves dsDNA and contains a single RuvC nuclease domain or one split RuvC nuclease domain and lacks an HNH domain (Zetsche et al. 2015, Cell doi:10.1016 / j.cell.2015.09.038; Shmakov et al. 2017, Nat Rev Microbiol doi:10.1038 / nrmicro.2016.184; Yan et al. 2018, Science doi:10.1126 / science.aav7271; Harrington et al. 2018, Science doi:10.1126 / science.aav4294). In some embodiments, the fusion protein of the present disclosure comprises Cas12 (e.g., Cas12a). Note that Cas12a, also known as Cpf1, does not require tracrRNA, whereas other type V CRISPR-Cas proteins (such as Cas12b) do require tracrRNA. Most type V effectors can also target ssDNA (single-stranded DNA), often without the need for PAM (Zetsche et al., 2015; Yan et al., 2018; Harrington et al., 2018). The terms "V-type CRISPR-Cas protein" and "V-type RGN" encompass unique RGNs that contain a split RuvC nuclease domain (such as those disclosed in U.S. Provisional Application Nos. 62 / 955,014, filed December 30, 2019, and 63 / 058,169, filed July 29, 2020, and PCT International Application No. PCT / US2020 / 067138, filed December 28, 2020, the contents of each of which are incorporated by reference in their entirety herein).In some embodiments, the present invention provides a fusion protein comprising a deaminase of the present disclosure fused to either Francisella novicida Cas12a (FnCas12a), the sequence of which is set forth as SEQ ID NO:105 and which is disclosed in U.S. Patent No. 9,790,490, which is incorporated by reference in its entirety, or a nuclease-inactivated mutant of FnCas12a disclosed in U.S. Patent No. 9,790,490.

[0146] In some embodiments, the CRISPR-Cas protein is a naturally occurring type VI CRISPR-Cas protein, or an active variant or fragment thereof. As used herein, the term "type VI CRISPR-Cas protein," "type VI CRISPR-Cas effector protein," or "type VI RGN" refers to a CRISPR-Cas effector protein that does not require tracrRNA and contains two HEPN domains that cleave RNA. In some embodiments, the present invention provides a fusion protein that includes a deaminase of the present disclosure fused to Cas13.

[0147] In particular embodiments, the fusion proteins of the present disclosure comprise an RGN, or a nickase, or a nuclease-dead variant thereof, listed in Table 1. Guide RNA sequences (crRNA repeats and tracrRNA sequences) that can be used with each RGN in Table 1 are provided, as well as consensus PAM sequences. In an embodiment, the fusion protein comprises an active variant (capable of RNA-guided binding to a nucleic acid molecule) of an RGN listed in Table 1 that has between 80% and 99% or more (including, but not limited to, about 80% or more, 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) identity to any one of the amino acid sequences listed in Table 1. In particular embodiments, the fusion protein comprises an RGN having a sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the RGN amino acid sequence disclosed in Table 1. In other embodiments, the fusion protein comprises a fragment of an RGN listed in Table 1 (which differs by as few as 1-15 amino acid residues, e.g., as few as 1-10 (such as 6-10), as few as 5, as few as 4, as few as 3, as few as 2, or as few as 1 amino acid residue). In particular embodiments, the RGN comprises an N- or C-terminal truncation, which comprises at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or more amino acid deletions from the N- or C-terminus of the polypeptide. In some embodiments, the RGN comprises an internal deletion, which comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, or more amino acid deletions.

[0148] [Table 1-1] [Table 1-2]

[0149] 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 allow the associated RNA-guided nuclease to bind to the target nucleotide sequence in a sequence-specific manner. For CRISPR-Cas RGNs, each guide RNA is one or more RNA molecules (typically one or two) that can bind to the RGN and allow the guide RGN to bind to a specific target nucleotide sequence, and can also cleave the target nucleotide sequence if the RGN has nickase or nuclease activity. Guide RNAs include CRISPR RNAs (crRNAs) and, in some embodiments, trans-activating CRISPR RNAs (tracrRNAs). In some embodiments, a portion of the guide RNA comprises DNA nucleotides. In some embodiments, the guide RNA comprises an artificial non-natural nucleotide analogue or one or more nucleotides are chemically modified.

[0150] CRISPR RNA comprises a spacer sequence and a CRISPR repeat sequence. A "spacer sequence" is a nucleotide sequence that directly hybridizes to a target nucleotide sequence of interest. The spacer sequence is engineered to be fully or partially complementary to the target nucleotide sequence of interest. In various embodiments, the spacer sequence can comprise about 8 to about 30 or more nucleotides. For example, the spacer sequence can be about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30 or more nucleotides in length. In some embodiments, the spacer sequence is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleotides in length. In some embodiments, the spacer sequence is about 10 to about 26 nucleotides in length, or about 12 to about 30 nucleotides in length. In particular embodiments, the spacer sequence is about 30 nucleotides in length. In some embodiments, the spacer sequence is 30 nucleotides in length. In some embodiments, the degree of complementarity between a spacer sequence and its corresponding target sequence is between 50% and 99% or greater (including, but not limited to, about 50% or greater, about 60%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater) when optimally aligned using a suitable alignment algorithm. In particular embodiments, the degree of complementarity between a 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 greater when optimally aligned using a suitable alignment algorithm.In particular embodiments, the spacer sequence is free of secondary structure, which can be predicted using any suitable polynucleotide folding algorithm known in the art, non-limiting examples of which include mFold (see, e.g., Zuker and Stiegler (1981) Nucleic Acids Res. 9:133-148) and RNAfold (see, e.g., Gruber et al. (2008) Cell 106(1):23-24).

[0151] CRISPR RNA repeats contain nucleotide sequences that form a structure by themselves or in cooperation with hybridized tracrRNA, which is recognized by RGN molecules. In various embodiments, CRISPR RNA repeats contain about 8 to about 30 or more nucleotides. In particular embodiments, CRISPR RNA repeats contain 8 to 30 or more nucleotides. For example, CRISPR repeats can be about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30 or more nucleotides in length. In particular embodiments, 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 length. In some embodiments, the degree of complementarity between the CRISPR repeat sequence and its corresponding tracrRNA sequence is between 50% and 99% or greater (including but not limited to about 50% or greater, about 60%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater) when optimally aligned using a suitable alignment algorithm. In particular embodiments, the degree of complementarity between the 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 greater when optimally aligned using a suitable alignment algorithm.

[0152] In some embodiments, the guide RNA further comprises a tracrRNA molecule. The transactivating CRISPR RNA or tracrRNA molecule comprises a nucleotide sequence that includes a region of sufficient complementarity to hybridize to the CRISPR repeat sequence of the crRNA, referred to herein as the anti-repeat region. In some embodiments, the tracrRNA molecule further comprises a region with a secondary structure (e.g., a stem-loop) or forms a secondary structure when hybridized to its corresponding crRNA. In particular embodiments, the region of the tracrRNA that is fully or partially complementary to the CRISPR repeat structure is located at the 5' end of the molecule, and the 3' end of the tracrRNA includes a secondary structure. This region of secondary structure generally includes several hairpin structures, including a nexus hairpin found adjacent to the anti-repeat region. There is often a terminal hairpin at the 3' end of the tracrRNA, which can vary in structure and number, but often includes a GC-rich, Rho-independent transcription terminator hairpin followed by a string of U's at the 3' end. See, e.g., Briner et al. (2014) Molecular Cell 56:333-339, Briner and Barrangou (2016) Cold Spring Harb Protoc; doi: 10.1101 / pdb.top090902, and U.S. Application Publication No. 2017 / 0275648, each of which is incorporated by reference in its entirety.

[0153] In various embodiments, the anti-repeat sequence of the tracrRNA that is fully or partially complementary to the CRISPR repeat sequence comprises about 6 to about 30 or more nucleotides. For example, the region that base pairs between the tracrRNA anti-repeat sequence and the CRISPR repeat sequence can be about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30 or more nucleotides in length. In a particular embodiment, the region that base pairs between the tracrRNA anti-repeat sequence and the CRISPR repeat sequence is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides in length. In a particular embodiment, the anti-repeat sequence of the tracrRNA that is fully or partially complementary to the CRISPR repeat sequence is about 10 nucleotides in length. In a particular embodiment, the anti-repeat sequence of the tracrRNA that is fully or partially complementary to the CRISPR repeat sequence is 10 nucleotides in length. In some embodiments, the degree of complementarity between the CRISPR repeat sequence and its corresponding racrRNA anti-repeat sequence is between 50% and 99% or greater (including, but not limited to, about 50% or more, about 60%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater) when optimally aligned using a suitable alignment algorithm. In particular embodiments, the degree of complementarity between the CRISPR repeat sequence and its corresponding tracrRNA anti-repeat sequence is 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater when optimally aligned using a suitable alignment algorithm.

[0154] In various embodiments, the entire tracrRNA comprises from about 60 nucleotides to more than about 210 nucleotides. In particular embodiments, the entire tracrRNA comprises from about 60 nucleotides to more than 210 nucleotides. For example, 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 length. In particular embodiments, the tracrRNA is 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 150, 160, 170, 180, 190, 200, 210, or more nucleotides in length. In particular embodiments, the tracrRNA is about 100 to about 200 nucleotides in length, including about 95, about 96, about 97, about 98, about 99, about 100, about 105, about 106, about 107, about 108, about 109, and about 100 nucleotides in length. In particular embodiments, the tracrRNA is 100-110 nucleotides in length, including 95, 96, 97, 98, 99, 100, 105, 106, 107, 108, 109, and 110 nucleotides in length.

[0155] The guide RNA forms a complex with an RNA-guided DNA-binding polypeptide or an RNA-guided nuclease, binding the RNA-guided nuclease to the target sequence. When the guide RNA forms a complex with an RGN, the bound RGN introduces a single-stranded or double-stranded break at the location of the target sequence. After the target sequence is cleaved, the break can be repaired, so that the DNA sequence of the target sequence is modified during the repair process. Provided herein is a method of modifying a target sequence in the DNA of a host cell using a mutant variant of an RNA-guided nuclease (either a nuclease-inactive or a nickase) linked to a deaminase. A mutant variant of an RNA-guided nuclease that has inactivated or significantly reduced nuclease activity can be referred to as an RNA-guided DNA-binding polypeptide, since the polypeptide can bind to the target sequence but does not necessarily cleave the target sequence. An RNA-guided nuclease that can cleave only a single strand of a double-stranded nucleic acid molecule is referred to herein as a nickase.

[0156] The target nucleotide sequence bound by the RNA-guided DNA-binding polypeptide hybridizes to the guide RNA associated with RGDBP, and if RGDBP has nuclease activity (including nickase activity) (i.e., is an RGN), it can then cleave the target sequence.

[0157] The guide RNA can be a single guide RNA or a dual guide RNA system. A single guide RNA comprises a crRNA and optionally a tracrRNA on a single molecule of RNA, whereas a dual guide RNA system comprises a crRNA and a tracrRNA present on two separate RNA molecules and hybridized to each other through at least a portion of the CRISPR repeat sequence of the crRNA and at least a portion of the tracrRNA (which can be fully or partially complementary to the CRISPR repeat sequence of the crRNA). In some embodiments where the guide RNA is a single guide RNA, the crRNA and optionally the tracrRNA are separated by a linker nucleotide sequence.

[0158] In general, the linker nucleotide sequence is a sequence that does not contain complementary bases to avoid formation of secondary structures among or involving the nucleotides of the linker nucleotide sequence. In some embodiments, the linker nucleotide sequence between the crRNA and the tracrRNA is at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or more nucleotides in length. In particular embodiments, the linker nucleotide sequence between the crRNA and the tracrRNA is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more nucleotides in length. In particular embodiments, the linker nucleotide sequence of the single guide RNA is at least 4 nucleotides in length. In particular embodiments, the linker nucleotide sequence of the single guide RNA is 4 nucleotides in length.

[0159] In some embodiments, the guide RNA can be introduced into a target cell, organelle, or embryo as an RNA molecule. The guide RNA can be in vitro transcribed or chemically synthesized. In some embodiments, a nucleotide sequence encoding the guide RNA is introduced into a cell, organelle, or embryo. In some embodiments, the nucleotide sequence encoding the guide RNA is operably linked to a promoter (e.g., an RNA polymerase III promoter). The promoter can be a native promoter or a promoter heterologous to the nucleotide sequence encoding the guide RNA. In some embodiments, the promoter is selected from any one of the promoters disclosed in U.S. Provisional Application No. 63 / 209,660, filed June 11, 2021, which is incorporated by reference in its entirety, including SEQ ID NOs: 348-357, or an active variant or fragment thereof, including a promoter having a sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to any one of SEQ ID NOs: 348-357.

[0160] In various embodiments, the guide RNA is linked to an RNA-guided nuclease polypeptide and can be introduced into a target cell, organelle, or embryo as a ribonucleoprotein complex, as described herein.

[0161] The guide RNA directs the associated RNA-guided nuclease to a particular target nucleotide sequence of interest through hybridization of the guide RNA to the target nucleotide sequence. The target nucleotide sequence can include DNA, RNA, or a combination of both, and can be single-stranded or double-stranded. The target nucleotide sequence can be genomic DNA (i.e., chromosomal DNA), plasmid DNA, or an RNA molecule (e.g., messenger RNA, ribosomal RNA, transfer RNA, microRNA, small interfering RNA). The target nucleotide sequence can be bound to (and cleaved by) an RNA-guided nuclease in vitro or in a cell (and in some embodiments, cleaved by an RNA-guided DNA-binding polypeptide). The chromosomal sequence targeted by RGDBP can be a nuclear, plastid, or mitochondrial chromosomal sequence. In some embodiments, the target nucleotide sequence is unique in the target genome.

[0162] In some embodiments, the target nucleotide sequence is adjacent to a protospacer adjacent motif (PAM). The PAM is generally located within about 1 to about 10 nucleotides of the target nucleotide sequence, including about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 nucleotides from the target nucleotide sequence. In particular embodiments, the PAM is located within 1 to 10 nucleotides of the target nucleotide sequence, including about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 nucleotides from the target nucleotide sequence. Unless otherwise specified, the PAM is generally adjacent to the target nucleotide sequence at its 5' or 3' end. In some embodiments, the PAM is 3' to the target sequence. Generally, the PAM is a consensus sequence of about 2 to 6 nucleotides, but in particular embodiments, the PAM is 1, 2, 3, 4, 5, 6, 7, 8, 9, or more nucleotides in length.

[0163] PAM restricts which sequences a given RGDBP or RGN can target because the PAM of the RGDBP or RGN must be proximal to the target nucleotide sequence. When the RGN recognizes its corresponding PAM sequence, it can cleave the target nucleotide sequence at a specific cleavage site. As used herein, a cleavage site is comprised of two specific nucleotides in the target nucleotide sequence between which the nucleotide sequence is cleaved by the RGN. The cleavage site can include the first and second, second and third, third and fourth, fourth and fifth, fifth and sixth, seventh and eighth, or eighth and ninth nucleotides in the 5' or 3' direction from the PAM. Since the RGN can cleave the target nucleotide sequence to a sticky end, in some embodiments, the cleavage site is defined based on the distance of those two nucleotides from the PAM on the plus (+) strand of the polynucleotide and the distance of those two nucleotides from the PAM on the minus (-) strand of the polynucleotide.

[0164] RGDBP and RGN can be used to deliver fused polypeptide, polynucleotide, or small molecule payloads to specific genomic locations.

[0165] In embodiments where the DNA-binding polypeptide comprises a meganuclease, the target sequence can comprise a pair of inverted 9 base pair "half-sites" separated by 4 base pairs. In the case of a single-chain meganuclease, the N-terminal domain of the protein contacts the first half-site and the C-terminal domain of the protein contacts the second half-site. Cleavage by the meganuclease results in a 4 base pair 3' overhang. In embodiments where the DNA-binding polypeptide comprises a compact TALEN, the recognition sequence comprises a first CNNNGN sequence recognized by the I-TevI ​​domain, followed by a non-specific spacer 4-16 base pairs in length, followed by a second sequence 16-22 bp in length (this sequence typically has a 5' T base) recognized by the TAL-effector domain. In embodiments in which the DNA-binding polypeptide comprises a zinc finger, the DNA-binding domain typically recognizes an 18-bp recognition sequence that contains a pair of 9 base pair "half-sites" separated by 2-10 base pairs, and upon cleavage by the nuclease generates either a blunt end or a 5' overhang of variable length (often 4 base pairs).

[0166] IV. Fusion Proteins

[0167] In some embodiments, a DNA-binding polypeptide (e.g., nuclease-inactive RGN or nickase RGN) is operably linked to a deaminase of the present invention. In some embodiments, a DNA-binding polypeptide (e.g., nuclease-inactive RGN or nickase RGN) fused to a deaminase of the present invention can be targeted to a specific location (in some embodiments, a specific genomic locus) of a nucleic acid molecule (i.e., a target nucleic acid molecule) to alter the expression of a desired sequence. In some embodiments, binding of the fusion protein to the target sequence results in deamination of a nucleobase, resulting in the conversion of one nucleobase to another nucleobase. In some embodiments, binding of the fusion protein to the target sequence results in deamination of a nucleobase adjacent to the target sequence. The nucleobases adjacent to the target sequence that are deaminated and mutated using the disclosed compositions and methods can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 base pairs from the 5' or 3' end of the target sequence (to which the gRNA is bound) in the target nucleic acid molecule. Some aspects of the disclosure provide fusion proteins that include (i) a DNA-binding polypeptide (e.g., a nuclease-inactive RGN polypeptide or a nickase RGN polypeptide); (ii) a deaminase polypeptide; and, optionally, (iii) a second deaminase. The second deaminase can be the same as the first deaminase or a different deaminase. In some embodiments, both the first and second deaminases are cytosine deaminases of the invention.

[0168] The present disclosure provides fusion proteins of various configurations. In some embodiments, the deaminase polypeptide is fused to the N-terminus of the DNA-binding polypeptide (e.g., the RGN polypeptide). In some embodiments, the deaminase polypeptide is fused to the C-terminus of the DNA-binding polypeptide (e.g., the RGN polypeptide).

[0169] In some embodiments, the deaminase and the DNA-binding polypeptide (e.g., an RNA-guided DNA-binding polypeptide) are fused to each other via a peptide linker. The linker between the deaminase and the DNA-binding polypeptide (e.g., an RNA-guided DNA-binding polypeptide) can determine the editing window of the fusion protein, thereby increasing the deaminase specificity and reducing off-target mutations. To achieve optimal length and rigidity for deaminase activity for a particular application, the (GGGGS) n and (G) n From a highly flexible linker of the form (EAAAK) n and (XP) nLinkers of various lengths and flexibility can be used, ranging from a more rigid linker in the form of a nuclease. The term "linker" as used herein refers to a chemical group or molecule that connects two molecules or moieties (e.g., the binding domain and cleavage domain of a nuclease). In some embodiments, the linker joins an RNA-guided nuclease and a deaminase. In some embodiments, the linker joins a dead or inactive RGN and a deaminase. In further embodiments, the linker joins two deaminases. In some embodiments, the linker joins an RNA-guided nuclease and a USP. In some embodiments, the linker joins a deaminase and a USP. In an embodiment, the linker joins an RNA-guided nuclease deaminase fusion with a USP. Typically, the linker is located between or adjacent to two groups, molecules, or other moieties and is connected to each by a covalent bond, thereby connecting the two. In some embodiments, the linker is an amino acid or multiple amino acids (e.g., a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 3-100 amino acids in length, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated. In some embodiments, shorter linkers are preferred to reduce the overall size or length of the fusion protein or its coding sequence.

[0170] In some embodiments, the linker is (GGGGS) n , (G) n (EAAAK) n , or (XP) nor any combination thereof, where n is independently an integer between 1 and 30. In some embodiments, n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, and any combination thereof when more than one linker or more than one linker motif is present. Additional suitable linker motifs and linker configurations will be apparent to those of skill in the art. In some embodiments, suitable linker motifs and configurations include those described in Chen et al., 2013 (Adv Drug Deliv Rev. 65(10):1357-69, the entire contents of which are incorporated herein by reference). Additional suitable linker sequences will be apparent to those of skill in the art. In some embodiments, the linker sequence comprises the amino acid sequence shown as SEQ ID NO:78 or 79.

[0171] In some cases, cellular uracil DNA glycosylase (UDG) can recognize U:G heteroduplex DNA resulting from deamination of cytosine and catalyze the removal of uracil from DNA, leaving an abasic site, thereby initiating base-excision repair with the primary and common outcome being the reversion of the U:G pair to a C:G pair, although C>G or C>A mutations, as well as indel (insertion or deletion) formation, are known to occur. To prevent or reduce base excision repair by uracil DNA glycosylase to revert uracil generated by the cytosine base editor back to cytosine, in some embodiments the cytosine base editor fusion protein further comprises a uracil stabilizing polypeptide (USP) (such as a uracil DNA glycosylase inhibitor (UGI) or USP2).

[0172] As used herein, the terms "uracil stabilizing protein," "uracil stabilizing polypeptide," and "USP" refer to a polypeptide having uracil stabilizing activity. As used herein, the term "uracil stabilizing activity" refers to the ability of a molecule (e.g., a polypeptide) to increase the rate of mutation of at least one cytidine, deoxycytidine, or cytosine in a nucleic acid molecule to thymidine, deoxythymidine, or thymine by cytosine deaminase compared to the rate of mutation by cytosine deaminase in the absence of the molecule (e.g., a uracil stabilizing polypeptide). Without being bound by theory or mechanism of action, it is believed that USPs can function by maintaining the presence of uracil in single-stranded DNA generated through deamination of the bases cytidine, deoxycytidine, or cytosine for a sufficient period of time to allow replication to occur and introduce the desired C>T mutation. Uracil stabilizing activity may occur through inhibition of uracil DNA glycosylase, base excision repair pathways, or mismatch repair mechanisms.

[0173] Non-limiting examples of USPs that can be fused to a fusion protein comprising a cytosine deaminase of the present disclosure and a DNA-binding polypeptide of the present disclosure include uracil DNA glycosylase inhibitor (UGI), one example of which is set forth as SEQ ID NO: 86, and any one of the USPs disclosed in PCT Publication No. WO2021 / 217002, which is incorporated by reference herein in its entirety, including USP2, which is set forth as SEQ ID NO: 81.

[0174] In some embodiments, the deaminase-DBD fusion protein comprises a USP that is a wild-type USP, or an active fragment or variant thereof. For example, in some embodiments, the deaminase-DBD fusion protein comprises a USP comprising SEQ ID NO: 81 or 86, or an active fragment or variant thereof. In some embodiments, the USP fragment comprises an amino acid sequence that comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid sequence set forth as SEQ ID NO: 81 or 86. In some embodiments, the deaminase-DBD fusion protein comprises a USP that has a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to the USP set forth as SEQ ID NO: 81 or 86.

[0175] Additional suitable USP sequences will be known to those of skill in the art and include, for example, those published in Wang et al., 1989. J. Biol. Chem. 264: 1163-1171; Lundquist et al., 1997. J. Biol. Chem. 272: 21408-21419; Ravishankar et al., 1998. Nucleic Acids Res. 26:4880-4887; and Putnam et al., 1999. J. Mol. Biol. 287:331-346(1999), the entire contents of each of which are incorporated herein by reference.

[0176] In some embodiments, a linker joins the deaminase-DBD fusion protein to the USP. In some embodiments, a linker joins the deaminase to the USP. In some embodiments, the linker joining the USP to the deaminase or deaminase-DBD fusion protein is an amino acid or multiple amino acids (e.g., a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 3-100 amino acids in length, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated. In some embodiments, shorter linkers are preferred to reduce the overall size or length of the fusion protein or its coding sequence. In a particular embodiment, the linker joining the USP to the deaminase or deaminase-DBD fusion protein comprises the sequence shown as SEQ ID NO:120.

[0177] In some embodiments, the general architecture of the representative fusion proteins provided herein can have the following structures: [NH2]-[deaminase]-[DBP]-[COOH]; [NH2]-[DBP]-[deaminase]-[COOH]; [NH2]-[DBP]-[deaminase]-[deaminase]-[COOH]; [NH2]-[deaminase]-[DBP]-[deaminase]-[COOH]; [NH2]-[deaminase]-[DBP]-[deaminase]-[COOH]; [NH2]-[deaminase]-[DBP]-[deaminase]-[COOH]; [NH2]-[deaminase]-[DBP]-[USP]-[COOH]; [N [H2]-[DBP]-[deaminase]-[USP]-[COOH]; [NH2]-[USP]-[deaminase]-[DBP]-[COOH]; [NH2]-[USP]-[DBP]-[deaminase]-[COOH]; [NH2]-[deaminase]-[USP]-[DBP]-[COOH]; or [NH2]-[DBP]-[USP]-[deaminase]-[COOH], where DBP is a DNA binding polypeptide, USP is a uracil stabilizing polypeptide, NH2 is the N-terminus of the fusion protein, and COOH is the C-terminus of the fusion protein. In some embodiments, the fusion protein comprises three or more deaminase polypeptides.

[0178] In certain embodiments, the general architecture of the representative fusion proteins provided herein can have the following structures: [NH2]-[deaminase]-[RGN]-[COOH]; [NH2]-[RGN]-[deaminase]-[COOH]; [NH2]-[RGN]-[deaminase]-[deaminase]-[COOH]; [NH2]-[deaminase]-[RGN]-[deaminase]-[COOH]; or [NH2]-[deaminase]-[deaminase]-[RGN]-[COOH]; [NH2]-[deaminase]-[RGN]-[USP]-[COOH]; [NH 2]-[RGN]-[deaminase]-[USP]-[COOH];[NH2]-[USP]-[deaminase]-[RGN]-[COOH];[NH2]-[USP]-[RGN]-[deaminase]-[COOH];[NH2]-[deaminase]-[USP]-[RGN]-[COOH];or [NH2]-[RGN]-[USP]-[deaminase]-[COOH], where RGN is an RNA-guided nuclease, USP is a uracil-stabilizing polypeptide, NH2 is the N-terminus of the fusion protein, and COOH is the C-terminus of the fusion protein. In some embodiments, the fusion protein comprises three or more deaminase polypeptides.

[0179] In some embodiments, the fusion protein has the structure: [NH2]-[deaminase]-[nuclease inactive RGN]-[COOH];[NH2]-[deaminase]-[deaminase]-[nuclease inactive RGN]-[COOH];[NH2]-[nuclease inactive RGN]-[deaminase]-[COOH];[NH2]-[deaminase]-[nuclease inactive RGN]-[deaminase]-[COOH];[NH2]-[nuclease inactive RGN]-[deaminase]-[COOH];[NH2]-[nuclease inactive RGN]-[deaminase]-[deaminase]-[COOH];[NH2]-[deaminase [NH2]-[nuclease inactive RGN]-[USP]-[COOH];[NH2]-[nuclease inactive RGN]-[deaminase]-[USP]-[COOH];[NH2]-[USP]-[deaminase]-[nuclease inactive RGN]-[COOH];[NH2]-[USP]-[nuclease inactive RGN]-[deaminase]-[COOH];[NH2]-[USP]-[nuclease inactive RGN]-[COOH];[NH2]-[deaminase]-[USP]-[nuclease inactive RGN]-[COOH];or [NH2]-[nuclease inactive RGN]-[USP]-[deaminase]-[COOH]. It should be understood that "nuclease inactive RGN" refers to any RGN, including any CRISPR-Cas protein that has been mutated to be nuclease inactive. In some embodiments, the fusion protein comprises three or more deaminase polypeptides.

[0180] In some embodiments, the fusion protein has the structure: [NH2]-[deaminase]-[RGN nickase]-[COOH]; [NH2]-[deaminase]-[deaminase]-[RGN nickase]-[COOH]; [NH2]-[RGN nickase]-[deaminase]-[COOH]; [NH2]-[deaminase]-[RGN nickase]-[deaminase]-[COOH]; or [NH2]-[RGN nickase]-[deaminase]-[deaminase]-[COOH]; [NH2]-[deaminase]-[RGN nickase]-[deaminase]-[COOH]; [NH2]-[RGN nickase]-[USP]-[deaminase]-[USP]-[COOH]; [NH2]-[USP]-[deaminase]-[RGN nickase]-[COOH]; [NH2]-[USP]-[deaminase]-[RGN nickase]-[COOH]; [NH2]-[USP]-[RGN nickase]-[deaminase]-[COOH]; [NH2]-[USP]-[RGN nickase]-[deaminase]-[COOH]; [NH2]-[deaminase]-[USP]-[RGN nickase]-[COOH]; or [NH2]-[RGN nickase]-[USP]-[deaminase]-[COOH]. It should be understood that "RGN nickase" refers to any RGN (including any CRISPR-Cas protein) that has been mutated to be active as a nickase.

[0181] In some embodiments, the "-" used in the above general architecture indicates the presence of an optional linker sequence. In some embodiments, the fusion proteins provided herein do not include a linker sequence. In some embodiments, at least one of the optional linker sequences is present.

[0182] Other representative features that may be present are localization sequences (such as nuclear localization sequences, cytoplasmic localization sequences, transport sequences (such as nuclear transport sequences), or other localization sequences), as well as sequence tags useful for solubilization, purification, or detection of the fusion protein. Provided herein, non-limiting examples include suitable localization signal sequences and protein tag sequences, such as biotin carboxylase carrier protein (BCCP) tags, myc tags, calmodulin tags, FLAG tags (e.g., 3XFLAG tags), hemagglutinin (HA) tags, polyhistidine tags (also called histidine tags or His tags), maltose binding protein (MBP) tags, nus tags, glutathione-S-transferase (GST) tags, green fluorescent protein (GFP) tags, thioredoxin tags, S tags, Softags (e.g., Softag 1, Softag 3), streptags, biotin ligase tags, FlAsH® tags, V5 tags, and SBP tags. Additional suitable sequences will be apparent to those of skill in the art.

[0183] In an embodiment, the fusion protein of the present disclosure comprises at least one cell entry domain that facilitates the uptake of the fusion protein into cells. Cell entry domains are known in the art and generally comprise a stretch of positively charged amino acid residues (i.e., polycationic cell entry domains), a stretch of alternating polar and non-polar amino acid residues (i.e., amphipathic cell entry domains), or a stretch of hydrophobic amino acid residues (i.e., hydrophobic cell entry domains) (see, for example, Milletti F. (2012) Drug Discov Today 17:850-860). A non-limiting example of a cell entry domain is the transactivating transcription activator (TAT) from human immunodeficiency virus 1.

[0184] In some embodiments, the deaminase or fusion protein provided herein further comprises a nuclear localization sequence (NLS). The nuclear localization signal, plastid localization signal, mitochondrial localization signal, dual targeting localization signal, and / or cell entry domain can be located at the amino-terminus (N-terminus), carboxyl-terminus (C-terminus), or internal position of the fusion protein.

[0185] In some embodiments, the NLS is fused to the N-terminus of the fusion protein or deaminase. In some embodiments, the NLS is fused to the C-terminus of the fusion protein or deaminase. In some embodiments, the NLS is fused to the N-terminus of the deaminase of the fusion protein. In some embodiments, the NLS is fused to the C-terminus of the deaminase of the fusion protein. In some embodiments, the NLS is fused to the N-terminus of the DNA binding polypeptide (e.g., the RGN polypeptide) of the fusion protein. In some embodiments, the NLS is fused to the C-terminus of the DNA binding polypeptide (e.g., the RGN polypeptide) of the fusion protein. In some embodiments, the NLS is fused to the N-terminus of the deaminase polypeptide of the fusion protein. In some embodiments, the NLS is fused to the C-terminus of the deaminase polypeptide of the fusion protein. In some embodiments, the NLS is fused to the fusion protein via one or more linkers (non-limiting examples include SEQ ID NO: 148). In some embodiments, the NLS is fused to the fusion protein without a linker. In some embodiments, the NLS comprises the amino acid sequence of any one of the NLS sequences presented or referenced herein. In some embodiments, the NLS comprises the amino acid sequence set forth in SEQ ID NO: 76 or SEQ ID NO: 80. In some embodiments, the fusion protein or deaminase comprises SEQ ID NO: 76 at its N-terminus and SEQ ID NO: 80 at its C-terminus.

[0186] In some embodiments, the fusion proteins provided herein comprise the full-length sequence of a deaminase (e.g., any one of SEQ ID NOs: 2, 4, and 6-12). However, in some embodiments, the fusion proteins provided herein do not comprise the full-length sequence of a deaminase, but only a fragment thereof. For example, in some embodiments, the fusion proteins provided herein further comprise a DNA-binding polypeptide (e.g., an RNA-guided DNA-binding) domain and a deaminase domain.

[0187] In some embodiments, a fusion protein of the invention comprises a DNA binding polypeptide (e.g., RGN) and a deaminase, where the deaminase has an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any of SEQ ID NOs: 2, 4, and 6-12. Examples of such fusion proteins are described in the Examples section herein.

[0188] In some embodiments, the fusion protein comprises one deaminase polypeptide. In some embodiments, the fusion protein comprises at least two deaminase polypeptides operably linked directly or via a peptide linker. In some embodiments, the fusion protein comprises one deaminase polypeptide, and a second deaminase polypeptide is expressed simultaneously with the fusion protein.

[0189] Also provided herein is a ribonucleoprotein complex comprising a fusion protein comprising a deaminase, RGDBP, and a guide RNA (collectively also referred to as gRNA) as a single guide or dual guide RNA.

[0190] V. Nucleotides encoding deaminases, fusion proteins, and / or gRNAs

[0191] The present disclosure provides polynucleotides encoding the deaminase polypeptides of the present disclosure (SEQ ID NOs: 109, 111, and 113-119). The present disclosure further provides polynucleotides encoding fusion proteins comprising a deaminase and a DNA-binding polypeptide (e.g., a meganuclease, a zinc finger fusion protein, or a TALEN). The present disclosure further provides polynucleotides encoding fusion proteins comprising a deaminase and an RNA-guided DNA-binding polypeptide. Such RNA-guided DNA-binding polypeptides can be RGN or RGN variants. Protein variants can be nuclease-inactive or nickases. RGN can be CRISPR-Cas proteins, or active variants or fragments thereof. SEQ ID NOs: 74 and 75 are non-limiting examples of RGN and nickase RGN variants, respectively. Examples of CRISPR-Cas nucleases are well known in the art, and similar corresponding mutations can create mutant variants that are also nickases or nuclease inactive.

[0192] One embodiment of the present invention provides a polynucleotide encoding a fusion protein comprising RGDBP and a deaminase as described herein (SEQ ID NOs: 2, 4, and 6-12, or variants thereof). In some embodiments, a second polynucleotide encodes a guide RNA that is required by RGDBP to target a nucleotide sequence of interest. In some embodiments, the guide RNA and the fusion protein are encoded by the same polynucleotide.

[0193] Use of the term "polynucleotide" is not intended to limit the disclosure to polynucleotides that contain DNA, although such DNA polynucleotides are contemplated. One of skill in the art will recognize that polynucleotides can include ribonucleotides (RNA) (e.g., mRNA) and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogs. Polynucleotides disclosed herein include sequences in all forms, non-limiting examples of which include single-stranded forms, double-stranded forms, stem and loop structures, circular forms (including, for example, circular RNA), and the like.

[0194] One embodiment of the present invention is a nucleic acid molecule comprising a sequence at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any of SEQ ID NOs: 109, 111, and 113-119, wherein the nucleic acid molecule encodes a deaminase having cytosine deaminase activity. The nucleic acid molecule may further comprise a heterologous promoter or terminator. The nucleic acid molecule may encode a fusion protein, wherein the encoded deaminase is operably linked to a DNA binding polypeptide, optionally to a second deaminase, and optionally to a USP. In some embodiments, the nucleic acid molecule encodes a fusion protein, wherein the encoded deaminase is operably linked to an RGN, optionally to a second deaminase, and optionally to a USP.

[0195] In some embodiments, nucleic acid molecules comprising polynucleotides encoding the deaminases of the invention are codon-optimized for expression in an organism of interest. A "codon-optimized" coding sequence is a polynucleotide that encodes a sequence with a codon usage designed to mimic the preferred codon usage or transcription conditions of a particular host cell. One or more codons are changed at the nucleic acid level, but the translated amino acid sequence is unchanged, resulting in increased expression in that particular host cell or organism. The entire nucleic acid molecule can be codon-optimized or in part. Codon tables and other references providing preferred information for a wide range of organisms are available in the art (see, e.g., Campbell and Gowri (1990) Plant Physiol. 92:1-11 for plant-preferred codon usage). Methods are available in the art for synthesizing plant-preferred genes. See, e.g., U.S. Patent Nos. 5,380,831 and 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference.

[0196] In some embodiments, a polynucleotide encoding a deaminase, fusion protein, and / or gRNA described herein is provided in an expression cassette and expressed in vitro or in a cell, organelle, embryo, or organism of interest. The cassette can include 5' and 3' regulatory sequences operably linked to the polynucleotide encoding the deaminase, and / or to the fusion protein comprising the deaminase, the RNA-guided DNA-binding polypeptide, and optionally a second deaminase, and / or to the gRNA provided herein that allows expression of the polynucleotide. The cassette can additionally include at least one additional gene or genetic element for co-transformation into the organism. When additional genes or elements are included, these elements are operably linked. The term "operably linked" is intended to mean a functional linkage between two or more elements. For example, an operable linkage between a promoter and a coding region of interest (e.g., a region encoding a deaminase, an RNA-guided DNA-binding polypeptide, and / or a gRNA) is an operable linkage that allows expression of the coding region of interest. Operably linked elements may be contiguous or discontinuous. When operably linked is used to mean the joining of two protein coding regions, it is assumed that the coding regions are in the same reading frame. In some embodiments, additional genes or elements are provided on multiple expression cassettes. For example, a nucleotide sequence encoding a deaminase of the present disclosure can be present on one expression cassette, either alone or as a component of a fusion protein, while a nucleotide sequence encoding a gRNA can be present on another expression cassette. Another example can have a nucleotide sequence encoding only a deaminase of the present disclosure on a first expression cassette, a second expression cassette encoding a fusion protein containing the deaminase, and a nucleotide sequence encoding a gRNA on a third expression cassette.The expression cassette is provided with multiple restriction and / or recombination sites for inserting a polynucleotide under the transcriptional control of the regulatory region. An expression cassette containing a selectable marker gene can also be present.

[0197] The expression cassette comprises, in the 5'→3' direction of transcription, a transcriptional (and in some embodiments translational) initiation region (i.e., promoter) functional in the organism of interest, a polynucleotide encoding the deaminase of interest, and a transcriptional (and in some embodiments translational) termination region (i.e., termination region). The promoter of the present invention can direct or drive the expression of a coding sequence in a host cell. These regulatory regions (e.g., promoter, transcriptional regulatory region, and transcriptional termination region) can be endogenous or heterologous to the host cell or to each other. As used herein, "heterologous" with respect to a sequence is a sequence that is derived from a foreign species or, if derived from the same species, is substantially modified by human intervention from its natural form in the composition and / or genomic locus. As used herein, a chimeric gene comprises, as a coding sequence, a coding sequence operably linked to a transcriptional initiation region heterologous to the coding sequence.

[0198] Convenient termination regions (eg, the octopine synthase termination region and the nopaline synthase termination region) are available from the Ti plasmid of A. tumefaciens. Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144;Proudfoot (1991) Cell 64:671-674;Sanfacon et al. (1991) Genes Dev. 5:141-149;Mogen et al. (1990) Plant Cell 2:1261-1272;Munroe et al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic Acids Res. 15:9627-9639.

[0199] Non-limiting examples of additional regulatory signals include transcription initiation sites, operators, activators, enhancers, other regulatory elements, ribosome binding sites, start codons, termination signals, etc. See, e.g., U.S. Patent Nos. 5,039,523 and 4,853,331; EPO0480762A2; Sambrook et al. (1992) Molecular Cloning: A Laboratory Manual, ed. Maniatis et al. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) (hereafter referred to as "Sambrook 11"); Davis et al., eds. (1980) Advanced Bacterial Genetics (Cold Spring Harbor Laboratory Press), Cold Spring Harbor, NY, and references cited therein.

[0200] In preparing an expression cassette, various DNA fragments are manipulated to provide DNA sequences in the proper orientation and, if necessary, in the proper reading frame. To this end, joining of DNA fragments with adapters or linkers or other manipulations to provide convenient restriction sites, remove excess DNA, remove restriction sites, etc. To this end, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions (e.g., transitions and transversions) can be included.

[0201] Numerous promoters can be used to practice the present invention. The promoter can be selected based on the desired outcome. The nucleic acid can be combined with a constitutive promoter, an inducible promoter, a developmental stage specific promoter, a cell type specific promoter, a tissue selective promoter, a tissue specific promoter, or other promoter and expressed in the organism of interest. See, e.g., WO 99 / 43838 and the promoters described in U.S. Patent 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.

[0202] For expression in plants, constitutive promoters include the CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell 2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); and MAS (Velten et al. (1984) EMBO J. 3:2723-2730).

[0203] Examples of inducible promoters are the Adh1 promoter, which is inducible by hypoxic or cold stress, the Hsp70 promoter, which is inducible by heat stress, and the PPDK and peptcarboxylase promoters, which are inducible by light. Chemically inducible promoters are also useful, such as the safener-induced In2-2 promoter (U.S. Patent No. 5,364,780), the auxin-induced, tapetum-specific but also active in callus Axig1 promoter (PCT US01 / 22169), steroid-responsive promoters (e.g., the estrogen-induced ERE promoter and glucocorticoid-inducible promoters in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 and McNellis et al. (1998) Plant J. 14(2):247-257), and tetracycline-inducible and tetracycline-repressible promoters (e.g., Gatz et al. (1991) Mol. Gen. Genet. 227:229-237 and U.S. Patent Nos. 5,814,618 and 5,789,156, which are incorporated herein by reference.

[0204] In some embodiments, tissue-specific or tissue-selective promoters can be used to express the expression construct in a particular tissue. In some embodiments, the tissue-specific or tissue-selective promoter is active in plant tissue. Examples of promoters under developmental control in plants include promoters that selectively initiate transcription in a given tissue (e.g., leaves, roots, fruits, seeds, or flowers). A "tissue-specific" promoter is a promoter that initiates transcription only in a given tissue. Tissue-specific expression is distinct from constitutive expression of a gene and is the result of several levels of gene regulatory interactions. Therefore, promoters from homologous or closely related plant species can be selectively used to achieve efficient and reliable expression of transgenes in a particular tissue. In some embodiments, the expression includes a tissue-selective promoter. A "tissue-selective" promoter is a promoter that selectively initiates transcription, but not necessarily exclusively in a given tissue or only in a given tissue.

[0205] In some embodiments, the nucleic acid molecule encoding the deaminase described herein comprises a cell type specific promoter. A "cell type specific" promoter is a promoter that drives expression primarily in some types of cells in one or more organs. Some examples of plant cells in which a cell type specific promoter that functions in a plant can be primarily active include, for example, BELT cells, roots, vascular cells in leaves, stem cells, and stem cells. The nucleic acid molecule can also comprise a cell type selective promoter. A "cell type selective" promoter is a promoter that drives expression primarily in some types of cells, usually in one or more organs, but not necessarily entirely in or only in a given tissue. Some examples of plant cells in which a cell type selective promoter that functions in a plant can be selectively active include, for example, BELT cells, roots, vascular cells in leaves, stem cells, and stem cells.

[0206] In some embodiments, the nucleic acid sequence encoding the deaminase, fusion protein, and / or gRNA is operably linked to a promoter sequence recognized by a phage RNA polymerase, for example for in vitro mRNA synthesis. In such embodiments, the in vitro transcribed RNA can be purified and used in the methods described herein. For example, the promoter sequence can be a T7 promoter sequence, a T3 promoter sequence, or an SP6 promoter sequence, or variations of the T7 promoter sequence, the T3 promoter sequence, or the SP6 promoter sequence. In such embodiments, the expressed protein and / or RNA can be purified and used in the methods of genome modification described herein.

[0207] In certain embodiments, the polynucleotide encoding the deaminase, fusion protein, and / or gRNA is 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 some embodiments, the sequence encoding the deaminase or fusion protein is linked to a sequence encoding at least one nuclear localization signal, at least one cell entry domain, and / or at least one signal peptide capable of translocating the protein to a specific intracellular location, as described elsewhere herein.

[0208] In some embodiments, the polynucleotides encoding the deaminase, fusion protein, and / or gRNA are present in one vector or multiple vectors. "Vector" refers to a polynucleotide composition for transferring, delivering, or introducing a nucleic acid into a host cell. Suitable vectors include plasmid vectors, phagemids, cosmids, artificial / mini-chromosomes, transposons, and viral vectors (e.g., lentiviral vectors, adeno-associated viral vectors, baculoviral vectors). In some embodiments, the vectors contain additional expression control sequences (e.g., enhancer sequences, Kozak sequences, polyadenylation sequences, transcription termination sequences), selectable marker sequences (e.g., antibiotic resistance genes), origins of replication, and the like. Additional information can be found in "Current Protocols in Molecular Biology" Ausubel et al., John Wiley & Sons, New York, 2003, or "Molecular Cloning: A Laboratory Manual" Sambrook & Russell, Cold Spring Harbor Press, Cold Spring Harbor, NY, 3rd edition, 2001.

[0209] In some embodiments, the vector contains a selection marker gene for selecting transformed cells. The selection marker gene is used to select transformed cells or tissues. Marker genes include genes that code for antibiotic resistance, such as genes that code for neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes that confer resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinone, and 2,4-dichlorophenoxyacetate (2,4-D).

[0210] In some embodiments, the expression cassette or vector comprising a sequence encoding a fusion protein comprising an RNA-guided DNA-binding polypeptide (such as RGN) further comprises a sequence encoding a gRNA. In some embodiments, the sequence encoding the gRNA is operably linked to at least one transcription control sequence for expressing the gRNA in an organism or host cell of interest. For example, the polynucleotide encoding the gRNA can be operably linked to a promoter sequence recognized by RNA polymerase III (Pol III). Non-limiting examples of suitable Pol III promoters include mammalian U6, U3, H1, and 7SL RNA promoters, and rice U6 and U3 promoters.

[0211] As shown, an expression construct comprising a nucleotide sequence encoding a deaminase, a fusion protein, and / or a gRNA can be used to transform an organism of interest. The method of transformation involves introducing a nucleotide construct into the organism of interest. By "introducing" it is meant that the nucleotide construct is introduced into a host cell, allowing the construct to access the interior of the host cell. The method of the present invention does not require a particular method of introducing a nucleotide construct into a host organism, only that the nucleotide construct is able to access the interior of at least one cell of the host organism. In some embodiments, an mRNA encoding a deaminase or a fusion protein is introduced into the host cell. In some embodiments, the fusion protein comprises RGDBP, an mRNA encoding the fusion protein is introduced into a cell and a gRNA is introduced into the cell. The host cell can be a eukaryotic or prokaryotic cell. In particular embodiments, the eukaryotic cell is a plant cell, a mammalian cell, or an insect cell. Methods for introducing nucleotide constructs into plants and other host cells are known in the art, non-limiting examples of which include stable transformation methods, transient transformation methods, and virus-mediated methods.

[0212] These methods result in transformed organisms, such as plants, including whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, propagules, and embryos and progeny thereof. Plant cells can be differentiated or undifferentiated (e.g., callus, suspension culture cells, protoplasts, leaf cells, root cells, phloem cells, pollen).

[0213] "Transgenic organism" or "transformed organism" or "stably transformed" organism or cell or tissue refers to an organism into which a polynucleotide encoding the deaminase of the present invention has been incorporated or integrated. It is known that other exogenous or endogenous nucleic acid sequences or DNA fragments can also be incorporated into a host cell. Agrobacterium-mediated transformation and biolistic-mediated transformation remain the two main approaches used to transform plant cells. However, host cell transformation can be achieved by infection, transfection, microinjection, electroporation, microprojection, biolistic or particle bombardment, electroporation, silica / carbon fiber, ultrasound-mediated, PEG-mediated, calcium phosphate co-precipitation, polycation DMSO technique, DEAE-dextran procedure, and virus-mediated, liposome-mediated, etc. Virus-mediated introduction of polynucleotides encoding deaminases, fusion proteins, and / or gRNAs includes retrovirus-, lentivirus-, adenovirus-, and adeno-associated virus-mediated introduction and expression, as well as the use of caulimoviruses (e.g., cauliflower mosaic virus), geminiviruses (e.g., bean yellow mosaic virus, or corn streak virus), and RNA plant viruses (e.g., tobacco mosaic virus).

[0214] Transformation protocols, as well as protocols for introducing polypeptide or polynucleotide sequences into plants, may vary depending on the type of host cell targeted for transformation (e.g., monocotyledonous or dicotyledonous cells). Transformation methods are known in the art, and include those described in U.S. Patent Nos. 8,575,425; 7,692,068; 8,802,934; and 7,541,517, each of which is incorporated herein by reference. Rakoczy-Trojanowska, M. (2002) Cell Mol Biol Lett. 7:849-858;Jones et al. (2005) Plant Methods 1:5;Rivera et al. (2012) Physics of Life Reviews 9:308-345;Bartlett et al. (2008) Plant Methods 4:1-12;Bates, GW (1999) Methods in Molecular Biology 111:359-366;Binns and Thomashow (1988) Annual Reviews in Microbiology 42:575-606;Christou, P. (1992) The Plant Journal 2:275-281;Christou, P. (1995) Euphytica 85:13-27;Tzfira et al. (2004) TRENDS in Genetics 20:375-383; Yao et al. (2006) Journal of Experimental Botany 57:3737-3746; Zupan and Zambryski (1995) Plant Physiology 107: 1041-1047; Jones et al. (2005) Plant Methods 1:5;

[0215] Transformation can result in stable or transient integration of a nucleic acid into a cell. "Stable transformation" means that a nucleotide construct introduced into a host cell can be integrated into the genome of the host cell and inherited by its progeny. "Transient transformation" means that a polynucleotide is introduced into a host cell but is not integrated into the genome of the host cell.

[0216] Methods for transforming chloroplasts are known in the art. See, for example, 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. This method relies on particle gun delivery of DNA containing a selectable marker, which is then targeted to the plastid genome through homologous recombination. In addition, plastid transformation can be achieved by cross-activating plastid-borne silent transgenes through tissue-selective expression of a nuclear-encoded plastid-derived RNA polymerase. Such a system is reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91:7301-7305.

[0217] The transformed cells can be conventionally grown in transgenic organisms (such as plants). See, e.g., McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants are then grown and pollinated with the same transformed strain or different strains, and the resulting hybrids are identified that have the deaminase or fusion protein polynucleotide. After growth for two or more generations to confirm stable maintenance and inheritance of the deaminase or fusion protein polynucleotide, seeds can be harvested and the presence of the deaminase or fusion protein polynucleotide confirmed. In this manner, the present invention provides transformed seeds (also referred to as "transgenic seeds") in which the nucleotide construct of the present invention (e.g., the expression cassette of the present invention) is stably integrated into the genome.

[0218] In some embodiments, the transformed cells are introduced into an organism. These cells may be derived from that organism, in which case the cells are transformed by an ex vivo approach.

[0219] The sequences provided herein can be used to transform any plant species, non-limiting examples of which include monocotyledonous and dicotyledonous plants. Non-limiting examples of plants of interest include corn (maize), sorghum, wheat, sunflower, tomato, cruciferous plants, pepper, potato, cotton, rice, soybean, sugar beet, sugarcane, tobacco, barley, and rapeseed, Brassica species, alfalfa, rye, millet, safflower, peanut, sweet potato, cassava, coffee, coconut, pineapple, citrus, cocoa, tea, banana, avocado, fig, guava, mango, olive, papaya, cashew nut, macadamia nut, almond, oat, vegetables, ornamental plants, and conifers.

[0220] Non-limiting examples of vegetables include tomatoes, lettuce, green peas, lima beans, peas, and members of the genus Cucumis (such as cucumbers, cantaloupes, and muskmelons). Non-limiting examples of ornamental plants include azaleas, hydrangeas, hibiscus, roses, tulips, daffodils, petunias, carnations, poinsettias, and chrysanthemums. The plants of the present invention are preferably crop plants (e.g., corn, sorghum, wheat, sunflowers, tomatoes, crucifers, peppers, potatoes, cotton, rice, soybeans, sugar beets, sugarcane, tobacco, barley, rapeseed, etc.).

[0221] As used herein, the term plant includes plant cells, plant protoplasts, plant cell tissue cultures capable of regenerating plants, plant callus, populations of plants, and plant cells that are intact within a plant or are parts of a plant (such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, kernels, ears, cobs, husks, stems, roots, root caps, anthers, etc.). Grain refers to mature seeds produced by agricultural growers for purposes other than multiplication or reproduction. Progeny, variants, and mutants of regenerated plants are also within the scope of the invention, provided that these parts contain the introduced polynucleotide. Additionally, processed plant products or by-products (e.g., soybean meal) carrying the sequences disclosed herein are also provided.

[0222] In some embodiments, the polynucleotides encoding the deaminase, fusion protein, and / or gRNA are used to transform any eukaryotic species. Non-limiting examples of eukaryotic species include animals (e.g., mammals, insects, fish, birds, and reptiles), fungi, amoebae, algae, and yeast. In some embodiments, the polynucleotides encoding the deaminase, fusion protein, and / or gRNA are used to transform any prokaryotic species. Non-limiting examples of prokaryotic species include archaea and bacteria (e.g., Bacillus species, Klebsiella species, Streptomyces species, Rhizobium species, Escherichia species, Pseudomonas species, Salmonella species, Shigella species, Vibrio species, Yersinia species, Mycoplasma species, Agrobacterium species, and Lactobacillus species).

[0223] In some embodiments, conventional viral and non-viral based gene transfer methods are used to introduce nucleic acids into mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding the deaminase or fusion protein of the invention, and optionally gRNA, to cells in culture or in a host organism. Non-viral vector delivery systems include DNA plasmids, RNA (e.g., transcripts of vectors described herein), naked nucleic acid, and nucleic acid complexed with a delivery vehicle (e.g., liposomes). Viral vector delivery systems include DNA and RNA viruses, which have episomal or integrated genomes after delivery to cells. Non-limiting examples include vectors using caulimoviruses (e.g., cauliflower mosaic virus), geminiviruses (e.g., pea yellow mosaic virus, or corn streak virus), and RNA plant viruses (e.g., tobacco mosaic virus). For reviews of gene therapy procedures, see Anderson, Science 256: 808- 813 (1992);Nabel & Feigner, TIBTECH 11:211-217 (1993);Mitani & Caskey, TIBTECH 11:162-166 (1993);Dillon, TIBTECH 11:167-175 (1993);Miller, Nature 357:455-460 (1992);Van Brunt, Biotechnology 6(10): 1149-1154 (1988);Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995);Kremer & Perricaudet, British Medical Bulletin 51(1):31-44 (1995); Current Topics in Microbiology and Immunology, Doerfler and Bohm (eds) (1995); and Yu et al., Gene Therapy 1:13-26 (1994).

[0224] Non-viral methods of delivery of nucleic acids include lipofection, Agrobacterium-mediated transformation, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid complexes, naked DNA, artificial virions, and drug-enhanced uptake of DNA. Lipofection has been described, for example, in U.S. Patent Nos. 5,049,386; 4,946,787; and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91 / 17424; WO 91 / 16024. Delivery can be to cells (e.g., in vitro or ex vivo administration) or to target tissues (e.g., in vivo administration). The preparation of lipid:nucleic acid complexes, including the preparation of targeted liposomes (such as immunolipid complexes), is well known to those of 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 Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820). (1992); see U.S. Patent Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787.

[0225] The use of RNA or DNA virus-based systems for nucleic acid delivery takes advantage of the highly evolved methods for directing viruses to specific cells in the body and transporting the viral payload to the nucleus. Viral vectors can be administered directly to patients (in vivo). Alternatively, viral vectors can be used to treat cells in vitro and these modified cells can be optionally administered to patients (ex vivo). Traditional virus-based systems could include retroviral, lentiviral, adenoviral, adeno-associated and herpes simplex virus vectors for gene delivery. Integration into the host genome is possible using retroviral, lentiviral and adeno-associated virus gene delivery methods, often resulting in long-term expression of the inserted transgene. In addition, high gene delivery efficiency has been observed in many different cell types and target tissues.

[0226] The tropism of retroviruses can be altered by incorporating foreign envelope proteins and expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that can transduce or infect non-dividing cells and typically produce high viral titers. The choice of retroviral gene delivery system may therefore depend on the target tissue. Retroviral vectors consist of cis-acting long terminal repeats and have the capacity to package foreign sequences up to 6-10 kb. A minimal cis-acting LTR is sufficient for vector replication and packaging, and this vector can be used to integrate therapeutic genes into target cells and permanently express the transgene. Widely used retroviral vectors include vectors based on murine leukemia virus (MuLV), gibbon leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol. 66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992); Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol. 63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991); PCT / US94 / 05700).

[0227] For applications in which transient expression is preferred, an adenovirus-based system can be used. Adenovirus-based vectors are capable of very high gene transfer efficiency in many cell types and do not require cell division. High titers and high expression levels have been obtained using such vectors. The vectors can be produced in large quantities in a relatively simple system. Adeno-associated virus ("AAV") vectors can also be used to introduce target nucleic acids into cells, for example, for in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Patent No. 4,797,368; WO 93 / 24641; Katin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994)). The construction of recombinant AAV vectors has been described in numerous 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 & Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., J. Virol. 63:03822-3828 (1989). Packaging cells are typically used to form viral particles capable of infecting host cells. Such cells include 293 cells (which package adenovirus) and ψJ2 or PA317 cells (which package retrovirus).

[0228] Viral vectors used in gene therapy are usually produced by generating cell lines that package nucleic acid vectors into viral particles. The vectors typically contain minimal viral sequences necessary for packaging and subsequent integration into the host, with other viral sequences being replaced by an expression cassette for the polynucleotide to be expressed. Missing viral functions are typically supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only have the ITR sequences from the AAV genome necessary for packaging and integration into the host genome. The viral DNA is packaged into a cell line that contains a helper plasmid that encodes the other AAV genes (i.e., rep and cap) but lacks the ITR sequences.

[0229] The cell line can also be infected with adenovirus as a helper. The helper virus promotes the replication of the AAV vector and the expression of AAV genes from the helper plasmid. The helper plasmid is not packaged in large amounts because it lacks ITR sequences. Contamination with adenovirus can be reduced, for example, by heat treatment, to which adenovirus is more sensitive than AAV. Additional methods of delivering nucleic acids to cells are known to those of skill in the art. See, for example, U.S. Patent Application Publication No. 2003 / 0087817, incorporated herein by reference.

[0230] Ideally, the coding sequence of the RGN-deaminase fusion protein of the present invention and the corresponding guide RNA targeting the fusion protein can all be packaged into a single AAV vector. The generally accepted size limit for AAV vectors is 4.7 kb, but larger sizes can be considered at the cost of reduced packing efficiency. To ensure that expression cassettes for both the fusion protein and its corresponding guide RNA can fit into the AAV vector, new active deletion variants of RGN (such as those shown as SEQ ID NOs: 97, 98, 106, and 107) or active deletion variants of deaminase (such as those described herein as SEQ ID NOs: 2, 4, and 6) can be used. In addition to shortening the amino acid sequence and thus the coding sequence of RGN and / or the deaminase of the fusion protein, the peptide linker connecting RGN and deaminase can also be shortened. The USP (if present) and the linker connecting the USP and the RGN-deaminase fusion protein can be shortened. Finally, genetic elements (such as promoters, enhancers, and / or terminators) can also be engineered through deletion analysis to determine the minimal size each requires to function. The present invention also teaches methods of using said fusion proteins for targeted base editing in vitro via AAV vector delivery.

[0231] In some embodiments, host cells are transiently or non-transiently transfected with one or more vectors described herein. In some embodiments, cells are transfected as they naturally occur in a subject. In some embodiments, the transfected cells are taken from a subject.

[0232] In some embodiments, the transfected cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is an animal cell (e.g., mammalian, insect, fish, bird, and reptile). In some embodiments, the transfected cell is a human cell. In some embodiments, the transfected cell is a cell of hematopoietic origin, such as an immune cell (i.e., a cell of the innate or adaptive immune system), including, but not limited to, B cells, T cells, natural killer (NK) cells, pluripotent stem cells, induced pluripotent stem cells, chimeric antigen receptor T (CAR-T) cells, monocytes, macrophages, and dendritic cells.

[0233] In some embodiments, the cells are derived from cells (such as a cell line) taken from a subject. In some embodiments, the cells or cell lines are prokaryotic cells. In some embodiments, the cells or cell lines are eukaryotic cells. In further embodiments, the cells or cell lines are derived from insect, avian, plant, or fungal species. In some embodiments, the cells or cell lines can be mammalian (e.g., human, monkey, mouse, cow, pig, goat, hamster, rat, cat, or dog, etc.). A variety of cell lines for tissue culture are known in the art. Non-limiting examples of cell lines include: C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLaS3, Huhl, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panel, PC-3, TFl, CTLL-2, CIR, Rat6, CVI, RPTE, AlO, T24, 182 , A375, ARH-77, Calul, SW480, SW620, SKOV3, SK-UT, CaCo2, P388Dl, SEM-K2, WEHI-231, HB56, T IB55, 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 epithelium, BALB / 3T3 mouse embryonic fibroblasts, 3T3 Swiss, 3T3-Ll, 132-d5 human fetal fibroblasts; 10.1 mouse fibroblasts, 293-T, 3T3, 721, 9L, A2780, A2780ADR, A2 780cis, A172, A20, A253, A431, A-549, ALC, B16, B35, BCP-I cells, BEAS-2B, bEnd.3, BHK-21, BR 293, BxPC3, C3H-10Tl / 2, C6 / 36, Cal-27, CHO, CHO-7, CHO-IR, CHO-Kl, CHO-K2, CHO-T, CHO Dhfr- / -, COR-L23, COR-L23 / CPR, COR-L235010, CORL23 / R23, COS-7, COV-434, CML Tl, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3, EMT6 / AR1, EMT6 / AR10.0, FM3, H1299, H69, HB54, HB55, HCA 2, HEK-293, HeLa, Hepalclc7, HL-60, HMEC, HT-29, lurkat, lY cells, K562 cells, Ku812, KCL22, KGl, KYOl, LNCap, Ma-Mel 1-48, MC-38, MCF-7, MCF-l0A, MDA-MB-231, MDA-MB-468, MDA-MB-435, MDCKII, MDCKII, MOR / 0.2R, MONO-MAC 6, MTD-lA, MyEnd, NCI-H69 / CPR, NCI-H69 / LX10, NCI-H69 / LX20, NCI-H69 / LX4, NIH-3T3, NALM-1, NW-145, OPCN / OPCT cell line, Peer, PNT-lA / PNT 2, RenCa, RIN-5F, RMA / RMAS, Saos-2 adipose, 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 utilized from a variety of sources known to those of skill in the art (see, for example, American Type Culture Collection (ATCC), Manassas, Va.).

[0234] In some embodiments, cells transfected with one or more vectors described herein are used to establish new cell lines that contain one or more vector-derived sequences. In some embodiments, cells transiently transfected with the fusion proteins and optionally gRNAs described herein, or ribonucleoprotein complexes of the present invention, and modified through the activity of the fusion proteins or ribonucleoprotein complexes, are used to establish new cell lines that contain modifications but completely lack 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 an insect. In further embodiments, the insect is a pest (such as a mosquito or a tick). In some embodiments, the insect is a plant pest (such as a corn rootworm or an armyworm). In some embodiments, the transgenic animal is an avian (such as a chicken, turkey, goose, or duck). In some embodiments, the transgenic animal is a mammal (such as a human, mouse, rat, hamster, monkey, ape, rabbit, pig, cow, horse, goat, sheep, cat, or dog).

[0236] VI. Polypeptide and Polynucleotide Variants and Fragments

[0237] The present disclosure provides cytosine deaminases active against DNA molecules having the amino acid sequences shown as SEQ ID NOs: 2, 4, and 6-12, active variants or fragments thereof, and polynucleotides encoding same.

[0238] The activity of the variants or fragments may be altered compared to the polynucleotide or polypeptide of interest, but the variants and fragments should retain the function of the polynucleotide or polypeptide of interest, e.g., the variants or fragments may have enhanced activity, decreased activity, a different spectrum of activity, or some other altered activity compared to the polynucleotide or polypeptide of interest.

[0239] Fragments and variants of the deaminases of the invention that have cytosine deaminase activity will retain said activity when they are part of a fusion protein further comprising a DNA binding polypeptide or a fragment thereof.

[0240] The term "fragment" refers to a portion of a polynucleotide or polypeptide sequence of the invention. A "fragment" or "biologically active portion" includes a polynucleotide that comprises a sufficient number of contiguous nucleotides to retain biological activity (i.e., deaminase activity on nucleic acids). A "fragment" or "biologically active portion" includes a polypeptide that comprises a sufficient number of contiguous amino acid residues to retain biological activity. Fragments of deaminases disclosed herein include those that are shorter than the full-length sequence due to the use of alternative downstream start sites. In some embodiments, the biologically active portion of a deaminase is a polypeptide that comprises, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, or more contiguous amino acid residues of any of SEQ ID NOs: 2, 4, and 6-12, or a variant thereof. Such biologically active portions can be prepared by recombinant techniques and assessed for activity.

[0241] In general, "variant" refers to a substantially similar sequence. In the context of polynucleotides, variants include deletions and / or additions of one or more nucleotides at one or more internal sites within the naturally occurring polynucleotide, and / or substitutions of one or more nucleotides at one or more sites within the naturally occurring polynucleotide. As used herein, a "natural" or "wild-type" polynucleotide or polypeptide includes a naturally occurring nucleotide sequence or amino acid sequence, respectively. In the context of polynucleotides, conservative variants include sequences that, due to the degeneracy of the genetic code, encode the naturally occurring amino acid sequence of the gene of interest. Such naturally occurring allelic variants can be identified using well-known molecular biology techniques, such as polymerase chain reaction (PCR) and hybridization techniques, as outlined below. Variant polynucleotides also include polynucleotides of synthetic origin (e.g., generated by site-directed mutagenesis) that still encode the polypeptide or polynucleotide of interest. Generally, a variant of a particular polynucleotide disclosed herein will have a sequence that is at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to that particular polynucleotide when examined using sequence alignment programs and parameters described elsewhere herein.

[0242] Variants of a particular polynucleotide (i.e., a reference polynucleotide) disclosed herein can also be assessed by comparing the percentage of sequence identity between the polypeptides encoded by the variant polynucleotide and the polypeptides encoded by the reference polynucleotide. The percentage of sequence identity between any two polypeptides can be calculated using sequence alignment programs and parameters described elsewhere herein. When any given pair of polynucleotides disclosed herein is assessed by comparing the percentage of sequence identity shared by the two polypeptides encoded by the polynucleotides, the percentage of sequence identity between the two encoded polypeptides is at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity.

[0243] In particular embodiments, the polynucleotides of the present disclosure encode a cytosine deaminase that comprises an amino acid sequence that is at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to the amino acid sequence of any of SEQ ID NOs:2, 4, and 6-12.

[0244] Biologically active variants of cytosine deaminase of the invention may differ by as few as 1-15 amino acid residues, as few as 1-10, for example as few as 6-10, as few as 5, as few as 4, as few as 3, as few as 2, or as few as 1 amino acid residue. In particular embodiments, these polypeptides include N- or C-terminal truncations, which may include at least the deletion of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or more amino acids from the N- or C-terminus of the polypeptide. In some embodiments, the polypeptides contain internal deletions, which can include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, or more amino acid deletions.

[0245] In some embodiments, the biologically active polypeptide variant of SEQ ID NO:2 does not include amino acid residues 1-12 or 195-230 of SEQ ID NO:1. In certain embodiments, the biologically active variant of SEQ ID NO:4 does not include amino acid residues 1-12 or 198-201 of SEQ ID NO:3. In particular embodiments, the biologically active polypeptide variant of SEQ ID NO:6 does not include amino acid residues 1-15 of SEQ ID NO:5. In certain embodiments, the deaminase has an amino acid sequence at least 90% identical to SEQ ID NO:2 and does not include amino acid residues 1-12 or 195-230 of SEQ ID NO:1. In some embodiments, the deaminase has an amino acid sequence at least 95% identical to SEQ ID NO:4 and does not include amino acid residues 1-12 or 198-201 of SEQ ID NO:3. In some embodiments, the deaminase has an amino acid sequence at least 95% identical to SEQ ID NO:6 and does not include amino acid residues 1-15 of SEQ ID NO:5.

[0246] It is recognized that the deaminases presented herein can be modified to generate variant proteins and polynucleotides. Human-designed changes can be introduced by applying site-directed mutagenesis techniques. In some embodiments, naturally occurring, but as yet unknown or as yet unidentified, polynucleotides and / or polypeptides related in structure and / or function to the sequences disclosed herein can also be identified that are within the scope of the present invention. Conservative amino acid substitutions that do not alter the function of a polypeptide, such as a cytosine deaminase, can be made in non-conserved regions. In some embodiments, modifications are made that improve the cytosine deaminase activity of the deaminase.

[0247] Variant polynucleotides and variant proteins also encompass sequences and proteins resulting from mutagenesis and recombination procedures, such as DNA shuffling. Such procedures can be used to engineer one or more of the different deaminases disclosed herein (e.g., 2, 4, and 6-12) to generate new cytosine deaminases with desired properties. In this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides that contain sequence regions with substantially identical sequences and thus are capable of homologous recombination in vitro or in vivo. For example, using this approach, sequence motifs encoding domains of interest can be shuffled between the deaminase sequences presented herein and other deaminase genes subsequently identified to produce sequences with improved properties of interest (e.g., K in the case of the enzyme). mBy using such methods, new genes encoding proteins (with increased activity) can be obtained. Strategies for such DNA shuffling are known in the art. See, e.g., Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S. Patent Nos. 5,605,793 and 5,837,458. A "shuffled" nucleic acid is a nucleic acid generated by a shuffling procedure (e.g., any of the shuffling procedures set forth herein). A shuffled nucleic acid is generated by recombining (physically or virtually), e.g., artificially, and optionally recursively, two or more nucleic acids (or character strings). Generally, the shuffling process employs one or more screening steps to identify nucleic acids of interest. This screening step can be performed before or after any recombination step. In some (but not all) shuffling embodiments, it is desirable to perform multiple rounds of recombination before selection to increase the diversity of the pool to be screened. The entire recombination and selection process is repeated, optionally recursively. Depending on the context, shuffling can refer to the entire recombination and selection process, or just the recombination portion of the entire process.

[0248] As used herein, "sequence identity" or "match" in the context of two polynucleotide or polypeptide sequences refers to residues that are the same in the two sequences when aligned for maximum correspondence over a specified comparison window. When percentage sequence identity is used in the context of proteins, it is recognized that residue positions that are not the same often differ in conservative amino acid substitutions, where an amino acid residue is replaced with another amino acid residue of similar chemical properties (e.g., charge or hydrophobicity), and thus the functional properties of the molecule are not altered. When sequences differ with respect to conservative substitutions, the percentage of sequence identity can be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ in such conservative substitutions are said to have "sequence similarity" or "similarity". Means for making this adjustment are well known to those of skill in the art. Typically, this involves scoring conservative substitutions as partial mismatches rather than complete mismatches, thereby increasing the percentage of sequence identity. Thus, for example, if matching amino acids are given a score of 1 and non-conserved substitutions are given a score of 0, then conserved substitutions are given a score between 0 and 1. Conserved substitution scores are calculated, for example, as implemented in the program PC / GENE (Intelligenetics, Mountain View, CA).

[0249] As used herein, "percentage of sequence identity" refers to a value obtained by comparing two sequences that are optimally aligned over a comparison window. In this case, the portion of the polynucleotide sequence that is within the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. This percentage is calculated by obtaining the number of matched positions by determining the number of positions where the nucleic acid base or amino acid residue is the same in both sequences, dividing this number of matched positions by the total number of positions within the comparison window, and multiplying by 100 to obtain the percentage of sequence identity.

[0250] Unless otherwise specified, the sequence identity / similarity values ​​presented herein refer to values ​​obtained using GAP Version 10 (with the following parameters: for nucleotide sequences, % identity and % similarity obtained using a GAP Weight of 50, a Length Weight of 3, and the nwsgapdna.cmp scoring matrix; for amino acid sequences, % identity and % similarity obtained using a GAP Weight of 8, a Length Weight of 2, and the BLOSUM62 scoring matrix), or any equivalent program. By "equivalent program" is meant any sequence comparison program that produces alignments with the same nucleotide or amino acid residue matches and the same percentage of sequence identity for any two sequences in question when compared to corresponding alignments produced by GAP Version 10.

[0251] Two sequences are "optimally aligned" when the two sequences are aligned using a predefined amino acid substitution matrix (e.g., BLOSUM62), gap existence penalty, and gap extension penalty to determine a similarity score, and the highest possible score is reached for the pair of sequences. Amino acid substitution matrices and their use to quantify the similarity between two sequences are well known in the art and are described, for example, in Dayhoff et al. (1978) "Models of Evolutionary Change in Proteins" in "Atlas of Protein Sequence and Structure," Vol. 5, Suppl. 3 (ed. MO Dayhoff), pp. 345-352, and Henikoff et al. (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919. The BLOSUM62 matrix is ​​often used as the default scoring substitution matrix in sequence alignment protocols. A gap existence penalty is imposed for introducing a single amino acid gap into one of the aligned sequences, and a gap extension penalty is imposed for introducing an additional empty amino acid position into each existing gap. The alignment is defined by the amino acid positions in each sequence where the alignment begins and ends, and optionally one or more gaps are inserted in one or both sequences to obtain the highest possible score. Optimal alignment and scoring can be achieved manually, but the process can be facilitated by computer-implemented alignment algorithms such as Gapped BLAST 2.0, described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402, and publicly available at the National Center for Biotechnology Information Website (www.ncbi.nlm.nih.gov).Optimal alignments, including multiple alignments, can be prepared, for example, using PSI-BLAST (described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402 and available at www.ncbi.nlm.nih.gov).

[0252] For an amino acid sequence that is optimally aligned with a reference sequence, an amino acid residue "corresponds" to the position in the reference sequence that it pairs with in the alignment. The "position" is represented by the number of each amino acid that is sequentially identified based on its position relative to the N-terminus in the reference sequence. In general, the number of an amino acid residue in a test sequence, when simply counted from the N-terminus, is not necessarily the same as the number of the corresponding position in the reference sequence, since there are deletions, insertions, truncations, fusions, etc. that must be considered when determining optimal alignment. For example, if there is a deletion in the aligned test sequence, there is no amino acid that corresponds to the position of the deletion site in the reference sequence. If there is an insertion in the aligned test sequence, the insertion does not correspond to any amino acid position in the reference sequence. If there is a truncation or fusion, there may be a series of amino acids in the reference sequence or aligned sequence that do not correspond to any amino acid in the corresponding sequence.

[0253] VII. Antibodies

[0254] Also encompassed are antibodies to the deaminases, fusion proteins, or ribonucleoproteins comprising the deaminases of the invention (including those having an amino acid sequence as set forth in any one of SEQ ID NOS: 2, 4, and 6-12), or active variants or fragments thereof. Methods for producing antibodies are well known in the art (see, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; U.S. Patent No. 4,196,265). These antibodies can be used in kits for detecting and isolating the deaminases, or fusion proteins, or ribonucleoproteins comprising the deaminases described herein. Thus, the disclosure provides kits that include antibodies that specifically bind to the polypeptides or ribonucleoproteins described herein (including, e.g., polypeptides comprising a sequence at least 85% identical to any one of SEQ ID NOS: 2, 4, and 6-12).

[0255] VIII. SYSTEMS AND RIBONUCLEOPROTEIN COMPLEXES FOR BINDING AND / OR MODIFYING TARGET SEQUENCES OF INTEREST AND METHODS FOR MAKING SAME

[0256] The present disclosure provides a system for targeting a nucleic acid sequence and modifying the target nucleic acid sequence. In some embodiments, the RNA-guided DNA-binding polypeptide (such as RGN) and gRNA are important for directing the ribonucleoprotein complex to the nucleic acid sequence of interest; the deaminase polypeptide fused to RGDBP is important for modifying the targeted nucleic acid sequence from C to N. In some embodiments, the deaminase converts C to T. In some embodiments, the deaminase converts C to G. After the guide RNA hybridizes to the target sequence of interest, it also forms a complex with the RNA-guided DNA-binding polypeptide, thereby binding the RNA-guided DNA-binding polypeptide to the target sequence. The RNA-guided DNA-binding polypeptide is part of a fusion protein that also includes a deaminase as described herein. In some embodiments, the RNA-guided DNA-binding polypeptide is an RGN (such as Cas9). Other examples of RNA-guided DNA-binding polypeptides include RGNs (such as those described in International Patent Application Publication Nos. WO2019 / 236566 and WO2020 / 139783, each of which is incorporated by reference in its entirety). In some embodiments, the RNA-guided DNA-binding polypeptide is a type II CRISPR-Cas polypeptide, or an active variant or fragment thereof. In some embodiments, the RNA-guided DNA-binding polypeptide is a type V CRISPR-Cas polypeptide, or an active variant or fragment thereof. In some embodiments, the RNA-guided DNA-binding polypeptide is a type VI CRISPR-Cas polypeptide. In some embodiments, the DNA-binding polypeptide of the fusion protein does not require an RNA guide (such as a zinc finger nuclease, TALEN, or meganuclease polypeptide). In some embodiments, the nuclease activity of the DNA-binding polypeptide is partially or completely inactivated.In further embodiments, the RNA-guided DNA-binding polypeptide comprises the amino acid sequence of RGN (such as APG07433.1 (SEQ ID NO: 74)), or an active variant or fragment thereof (such as nickase nAPG07433.1 (SEQ ID NO: 75) or other nickase RGN variants (SEQ ID NOs: 75 and 88-98)).

[0257] In some embodiments, the system for binding and modifying a target sequence of interest presented herein is a ribonucleoprotein complex that is at least one molecule of RNA bound to at least one protein. The ribonucleoprotein complex presented herein comprises at least one guide RNA as an RNA component and a fusion protein comprising a deaminase of the present invention and an RNA-guided DNA-binding polypeptide as a protein component. In some embodiments, the ribonucleoprotein complex is purified from cells or organisms cultured under conditions that allow expression of the fusion protein and the guide RNA after transformation with a polynucleotide encoding the fusion protein and the guide RNA.

[0258] Methods for producing a deaminase, fusion protein, or fusion protein ribonucleoprotein complex are provided. Such methods include culturing cells comprising a nucleotide sequence encoding a deaminase, fusion protein, and, in some embodiments, a nucleotide sequence encoding a guide RNA, under conditions in which the deaminase or fusion protein (and, in some embodiments, the guide RNA) is expressed. The deaminase, fusion protein, or fusion ribonucleoprotein can then be purified from a lysate of the cultured cells.

[0259] Methods for purifying the deaminase, fusion protein, or fusion ribonucleoprotein complex from a lysate of a biological sample are known in the art (e.g., size-exclusion and / or affinity chromatography, 2D-PAGE, HPLC, reverse phase chromatography, immunoprecipitation). In particular methods, the deaminase or fusion protein is recombinantly produced and contains a purification tag to aid in its purification. Non-limiting examples of purification tags include glutathione-S-transferase (GST), chitin-binding protein (CBP), maltose-binding protein, thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tags, myc, AcV5, AU1, AU5, E, ECS, E2, FLAG, HA, nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV-G, 6xHis, biotin carboxyl carrier protein (BCCP), and calmodulin. Generally, the tagged deaminase, fusion protein, or fusion ribonucleoprotein complex is purified using immunoprecipitation or other similar methods known in the art.

[0260] An "isolated" or "purified" polypeptide, or a biologically active portion thereof, is substantially or essentially free from components that normally accompany or interact with the polypeptide found in its natural environment. Thus, an isolated or purified polypeptide is substantially free of other cellular material or culture medium when produced by recombinant techniques, and is substantially free of chemical precursors or other compounds when chemically synthesized. Proteins that are substantially free of cellular material include protein preparations that contain less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating proteins. When the protein of the invention, or a biologically active portion thereof, is produced recombinantly, optimally, the culture medium contains less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or chemicals without the protein of interest.

[0261] A particular method presented herein for binding to and / or cleaving a target sequence of interest involves the use of an in vitro assembled ribonucleoprotein complex. The in vitro assembly of the ribonucleoprotein complex can be performed using any method known in the art, in which an RGDBP polypeptide, or a fusion protein comprising the same, is contacted with a guide RNA under conditions that allow the RGDBP polypeptide, or a fusion protein comprising the same, to bind to the guide RNA. As used herein, "contact," "contacting," or "contacted" refers to bringing together components of a desired reaction and placing them under conditions suitable for carrying out the desired reaction. The RGDBP polypeptide, or a fusion protein comprising the same, can be purified from a biological sample, cell lysate, or medium, transcribed in vitro, or chemically synthesized. The guide RNA can be produced through in vitro transcription or chemical synthesis and purified from a biological sample, cell lysate, or medium. The RGDBP polypeptide, or a fusion protein containing it, can be contacted with a guide RNA in a solution (e.g., a buffered saline solution) to assemble a ribonucleoprotein complex in vitro.

[0262] IX. Methods of Modifying Target Sequences

[0263] The present disclosure provides a method for modifying a target nucleic acid molecule of interest (e.g., a target DNA molecule). The method comprises delivering a fusion protein comprising a DNA-binding polypeptide and at least one deaminase of the present invention, or a polynucleotide encoding the same, to the target sequence, or a cell, organelle, or embryo comprising the target sequence. In some embodiments, the method comprises delivering a system comprising at least one guide RNA or a polynucleotide encoding the same, and at least one fusion protein comprising at least one deaminase of the present invention and an RNA-guided DNA-binding polypeptide, or a polynucleotide encoding the same, to the target sequence, or a cell, organelle, or embryo comprising the target sequence. In some embodiments, the fusion protein comprises any one of the amino acid sequences of SEQ ID NOs: 2, 4, and 6-12, or an active variant or fragment thereof.

[0264] In some embodiments, the method includes contacting a DNA molecule with (a) a fusion protein comprising a deaminase and an RNA-guided DNA-binding polypeptide (such as a nuclease-inactive RGN or a nickase RGN); and (b) a gRNA that directs the fusion protein of (a) to a target nucleotide sequence of the DNA molecule; the DNA molecule is then contacted with the fusion protein and the gRNA in an effective amount and under conditions suitable for deamination of the nucleobase. In some embodiments, the target DNA molecule comprises a sequence associated with a disease or disorder, and the deamination of the nucleobase results in a sequence not associated with the disease or disorder. In some embodiments, the disease or disorder affects an animal. In further embodiments, the disease or disorder affects a mammal, such as a human, cow, horse, dog, cat, goat, sheep, pig, monkey, rat, mouse, or hamster. In some embodiments, the target DNA sequence is within an allele of a crop plant, and the speciality of this allele for the trait of interest results in a plant with less agricultural value. Deamination of nucleobases results in alleles that improve the trait, increasing the agricultural value of the plant.

[0265] In embodiments where the method includes delivering a polynucleotide encoding a guide RNA and / or a fusion protein, the cell or embryo can then be cultured under conditions where the guide RNA and / or fusion protein is expressed. In various embodiments, the method includes contacting the target sequence with a ribonucleoprotein complex comprising a gRNA and a fusion protein (comprising a deaminase and an RNA-guided DNA-binding polypeptide of the invention). In some embodiments, the method includes introducing a ribonucleoprotein complex of the invention into a cell, organelle, or embryo that contains the target sequence. The ribonucleoprotein complex of the invention can be purified from a biological sample, recombinantly produced and then purified, or assembled in vitro as described herein. In embodiments where the ribonucleoprotein complex that is contacted with the target sequence or cell organelle or embryo is assembled in vitro, the method can further include assembling the complex in vitro prior to contacting with the target sequence, cell, organelle, or embryo.

[0266] The purified or in vitro assembled ribonucleoprotein complexes of the invention can be introduced into cells, organelles, or embryos using any method known in the art, non-limiting examples of which include electroporation. In some embodiments, a fusion protein comprising a deaminase and an RNA-guided DNA-binding polypeptide of the invention and a polynucleotide encoding or comprising a guide RNA is introduced into a cell, organelle, or embryo using any method known in the art, such as electroporation.

[0267] When the guide RNA is delivered to or contacts a target sequence or a cell, organelle, or embryo containing the target sequence, the fusion protein binds to the target sequence in a sequence-specific manner. The target sequence can then be modified by the deaminase of the fusion protein. In some embodiments, the fusion protein binds to the target sequence and modifies the nucleotides adjacent to the target sequence. The nucleic acid bases adjacent to the target sequence that are modified by the deaminase can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 base pairs from the 5' or 3' end of the target sequence. The fusion protein comprising the deaminase of the present invention and an RNA-guided DNA-binding polypeptide can introduce a C>N mutation at a specific position in the targeted DNA molecule. In some embodiments, the fusion protein introduces a C>T mutation at a specific position in the targeted DNA molecule. In certain embodiments, the fusion protein introduces a C>G mutation at a specific position in the targeted DNA molecule.

[0268] Methods for measuring binding of a fusion protein to a target sequence are known in the art and include chromatin immunoprecipitation assays, gel mobility shift assays, DNA pull-down assays, reporter assays, and microplate capture and detection assays. Similarly, methods for measuring cleavage or modification of a target sequence are known in the art and include in vitro or in vivo cleavage assays, in which cleavage is confirmed using PCR, sequencing, or gel electrophoresis, with or without an appropriate label (e.g., radioisotope, fluorescent substance) attached to the target sequence to facilitate detection of degradation products. In some embodiments, a nick-triggered exponential amplification reaction (NTEXPAR) assay is used (see, e.g., 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).

[0269] In some embodiments, the method comprises the use of an RNA-binding DNA-guided polypeptide as part of a fusion protein complexed with two or more guide RNAs. The two or more guide RNAs can target different regions of a single gene or target multiple genes. This multiple targeting allows the deaminase of the fusion protein to modify nucleic acid, thereby introducing multiple mutations into the target nucleic acid molecule of interest (e.g., genome).

[0270] In embodiments where the method includes the use of an RNA-guided nuclease (RGN), such as a nickase RGN, i.e. capable of cleaving only a single strand of a double-stranded polynucleotide (e.g., nAPG07433.1 (SEQ ID NO: 75 or SEQ ID NO: 88-98)), the method can include introducing two different RGNs or RGN variants that target the same or overlapping target sequence 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 along with a second RGN nickase that cleaves only the minus (-) strand of a double-stranded polynucleotide. In some embodiments, two different fusion proteins are provided, each of which then contains a different RGN with a different PAM recognition sequence, allowing a greater diversity of nucleotide sequences to be targeted for mutation.

[0271] Those skilled in the art will understand that any of the methods disclosed herein can be used to target a single target sequence or multiple target sequences. Thus, the method includes the use of a fusion protein that includes a single RNA-guided DNA-binding polypeptide in combination with multiple different guide RNAs that can target multiple different sequences in a single gene and / or multiple genes. The deaminase of the fusion protein will then introduce mutations into each of the targeted sequences. Also encompassed herein are methods in which multiple different guide RNAs are introduced in combination with multiple different RNA-guided DNA-binding polypeptides. Such RNA-guided DNA-binding polypeptides can be multiple RGNs or RGN variants. These guide RNAs and guide RNA / fusion protein systems can target multiple different sequences in a single gene and / or multiple genes.

[0272] In some embodiments, a fusion protein comprising an RNA-guided DNA-binding polypeptide and a deaminase polypeptide of the invention can be used to generate mutations in a targeted gene or a targeted region in a gene of interest. In some embodiments, the fusion proteins of the invention can be used for saturation mutagenesis of a target gene or a region of a target gene of interest, followed by high-throughput forward genetic screening to identify novel mutations and / or phenotypes. In some embodiments, the fusion proteins described herein can be used to generate mutations in targeted genomic locations that may or may not contain coding DNA sequences. Libraries of cell lines generated by the targeted mutagenesis described above can also be useful for studying gene function or gene expression.

[0273] X. Target Polynucleotide

[0274] In one aspect, the present invention provides a method for modifying a target polynucleotide in a eukaryotic cell in vivo, ex vivo, or in vitro. In some embodiments, the method includes sampling a cell or a population of cells from a human or non-human animal or plant (including microalgae) and modifying the cell or cells. Cultivation can be performed ex vivo at any stage. The cell or cells can even be reintroduced into the non-human animal or plant (including microalgae).

[0275] Plant breeders exploit natural variability to combine highly useful genes in search of desirable qualities, such as yield, quality, uniformity, hardiness, and resistance to pathogens. These desirable qualities include growth, photoperiod preference, temperature conditions, date of onset of flowering or reproductive development, fatty acid content, insect resistance, disease resistance, nematode resistance, fungal resistance, herbicide resistance, tolerance to various environmental factors, such as drought, heat, humidity, cold, wind, and adverse soil conditions, including high salinity. Sources of these useful genes include native or exotic species, heirloom species, wild plant relatives, and induced mutations, such as treating plant material with mutagenizing agents. The present invention provides plant breeders with a new tool for inducing mutations. Thus, the skilled artisan can analyze genomes to search for useful genes and use the present invention to induce increases in useful genes in varieties with desired characteristics or traits more precisely than previous mutagenizing agents, thus accelerating and improving plant breeding programs.

[0276] The target polynucleotide for the deaminase or fusion protein of the invention can be any polynucleotide, endogenous or exogenous to the eukaryotic cell. For example, the target polynucleotide can be a polynucleotide present in the nucleus of the eukaryotic cell. In some embodiments, the target polynucleotide is a sequence that encodes a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or junk DNA). In some embodiments, the target sequence for the fusion protein of the invention is associated with a PAM (protospacer adjacent motif); i.e., a short sequence recognized by an RNA-guided DNA-binding polypeptide. While the exact sequence and length required for the PAM will vary depending on the RNA-guided DNA-binding polypeptide used, the PAM is typically a 2-5 base pair sequence adjacent to the protospacer (i.e., the target sequence).

[0277] The target polynucleotides of the fusion proteins of the present invention may include a number of disease-associated genes and polynucleotides, as well as genes and polynucleotides involved in signal transduction biochemical pathways. Examples of target polynucleotides include sequences (e.g., genes or polynucleotides involved in signal transduction biochemical pathways) that are involved in signal transduction biochemical pathways. Examples of target polynucleotides include disease-associated genes or polynucleotides. A "disease-associated" gene or polynucleotide refers to any gene or polynucleotide that gives rise to an abnormal level or abnormal form of transcription or translation product in cells derived from disease-affected tissues when compared to non-disease control tissues or cells. It may be a gene that becomes expressed at an abnormally high level or at an abnormally low level, where the altered expression correlates with the development and / or progression of the disease. A disease-associated gene also refers to a gene that has a mutation or genetic variation that is directly responsible for the origin of the disease (e.g., a causative mutation) or that is in linkage equilibrium with a gene that is responsible for the origin of the disease. The transcription or translation product may be known or unknown, and may be at normal or abnormal levels.

[0278] Non-limiting examples of disease-associated genes that can be targeted using the methods and compositions of the present disclosure are provided in Table 23. In some embodiments, the disease-associated genes targeted are those disclosed in Table 23 that have a T>C mutation or a G>C mutation. Additional examples of disease-associated genes and polynucleotides are available on the World Wide Web from the McKusick-Nathans Institute of Medical Genetics, Johns Hopkins University (Baltimore, MD) and the National Center for Biotechnology Information, National Library of Medicine (Bethesda, MD).

[0279] In some embodiments, the method comprises contacting a DNA molecule comprising a target DNA sequence with a DNA-binding polypeptide-deaminase fusion protein of the invention, where the DNA molecule is contacted with the fusion protein in an effective amount and under conditions suitable for deamination of the nucleobase. In some embodiments, the method comprises contacting a DNA molecule comprising a target DNA sequence with (a) an RGN-deaminase fusion protein of the invention; and (b) a gRNA that directs the fusion protein of (a) to a target nucleotide sequence of the DNA molecule; where the DNA molecule is contacted with the fusion protein and the gRNA in an effective amount and under conditions suitable for deamination of the nucleobase. In some embodiments, the target DNA sequence comprises a sequence associated with a disease or disorder, and the 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 in an allele of a crop, where a particular allele of a trait of interest results in a plant with less agricultural value. The deamination of the nucleobase results in an allele that improves the trait, increasing the agricultural value of the plant.

[0280] In some embodiments, the target DNA sequence contains a disease or disorder-associated point mutation, T>C or G>C, and deamination of the mutant C base results in a sequence that is not associated with the disease or disorder, in some embodiments, deamination corrects the point mutation in the disease or disorder-associated sequence.

[0281] In some embodiments, the disease or disorder associated sequence encodes a protein, and deamination introduces a stop codon into the disease or disorder associated sequence, resulting in truncation of the encoded protein. In some embodiments, the contacting is performed in vivo in a subject likely to have the disease or disorder, a subject with the disease or disorder, or a subject diagnosed with the disease or disorder. In some embodiments, the disease or disorder 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 disease.

[0282] XI. Pharmaceutical Compositions and Methods of Treatment

[0283] Provided herein is a method for treating disease in a subject in need of treatment.The method comprises administering to the subject in need thereof an effective amount of the fusion protein of the present disclosure, or the polynucleotide encoding it, the gRNA of the present disclosure, or the polynucleotide encoding it, the fusion protein system of the present disclosure, or the cell modified by any one of these compositions, or the cell comprising any one of these compositions.

[0284] In some embodiments, the treatment involves in vivo gene editing by administering to a subject in need of treatment a fusion protein, gRNA, or fusion protein system of the present disclosure, or a polynucleotide encoding the same. In some embodiments, the treatment involves ex vivo gene editing, where cells are genetically modified ex vivo with a fusion protein, gRNA, or fusion protein system of the present disclosure, or a polynucleotide encoding the same, and then the modified cells are administered to the subject. In some embodiments, the genetically modified cells are derived from a subject, and then the modified cells are administered to the subject. The transplanted cells are referred to herein as autologous. In some embodiments, the genetically modified cells are derived from a different subject (i.e., a donor) of the same species as the subject to which the modified cells are administered (i.e., the recipient), and the transplanted cells are referred to herein as allogeneic. In some examples described herein, the cells can be expanded in culture and then administered to a subject in need thereof.

[0285] In some embodiments, the disease treated with the compositions of the present disclosure is a disease that can be treated with immunotherapy (such as chimeric antigen receptor (CAR) T cells). A non-limiting example of such a disease includes cancer.

[0286] In some embodiments, the deamination of the target nucleobase results in the correction of a genetic abnormality or a point mutation that leads to loss of function of a gene product. In some embodiments, the genetic abnormality is related to a disease or disorder, such as a lysosomal disease or a metabolic disease, such as type I diabetes. Thus, in some embodiments, the disease treated with the composition of the present disclosure is related to a sequence that is mutated to treat the disease or disorder or to alleviate symptoms associated with the disease or disorder (i.e., a sequence that causes the disease or disorder or causes symptoms associated with the disease or disorder).

[0287] In some embodiments, the disease treated with the composition of the present disclosure is associated with a causative mutation. As used herein, a "causative mutation" refers to a specific nucleotide, nucleotides, or nucleotide sequence in a genome that contributes to the severity or presence of a disease or disorder in a subject. Correction of the causative mutation leads to the amelioration of at least one symptom resulting from the disease or disorder. In some embodiments, correction of the causative mutation leads to the amelioration of at least one symptom resulting from the disease or disorder. In some embodiments, the causative mutation is adjacent to a PAM site that is recognized by an RGDBP (e.g., RGN) fused to a deaminase disclosed herein. The causative mutation can be corrected using a fusion polypeptide comprising an RGDBP (e.g., RGN) and a deaminase of the present disclosure. Non-limiting examples of diseases associated with causative mutations include cystic fibrosis, Niemann-Pick disease, diseases caused by splice site disruption, and diseases listed in Table 23. Additional non-limiting examples of genes and mutations associated with disease are available on the World Wide Web from the McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, MD) and the National Center for Biotechnology Information, National Library of Medicine (Bethesda, MD).

[0288] In some embodiments, the methods provided herein are utilized to introduce deactivating point mutations into genes or alleles that encode gene products associated with a disease or disorder. For example, in some embodiments, methods are provided herein that use fusion proteins to introduce deactivating point mutations into cancer genes (e.g., in the treatment of proliferative diseases). The deactivating mutations, in some embodiments, can result in a premature stop codon in the coding sequence, resulting in the expression of a truncated gene product (e.g., a truncated protein that lacks the function of the full-length protein). In some embodiments, the goal of the methods provided herein is to restore the function of a dysfunctional gene by genome editing. The fusion proteins provided herein can be validated as in vitro gene editing-based human therapeutics, for example, by correcting disease-associated mutations in human cell cultures. One skilled in the art will recognize that the fusion proteins provided herein (e.g., fusion proteins comprising an RNA-guided DNA-binding polypeptide and a deaminase polypeptide) can be used to correct any single T>C or G>C point mutation. Deamination of the C to T or G mutation leads to correction of the mutation.

[0289] As used herein, "treatment" or "treat" or "alleviate" or "ameliorate" are used interchangeably. These terms refer to an approach to obtain beneficial or desired results, non-limiting examples of which include therapeutic benefit and / or preventive benefit. Therapeutic benefit refers to any therapeutically significant improvement of one or more diseases, conditions, or symptoms being treated, or any therapeutically significant effect on one or more diseases, conditions, or symptoms being treated. To obtain preventive benefit, the composition can be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more physiological symptoms of a disease, even if the disease, condition, or symptom has not yet manifested.

[0290] The term "effective amount" or "therapeutically effective amount" refers to an amount of an agent sufficient to obtain a beneficial or desired result. A therapeutically effective amount can vary depending on one or more of the subject and the disease state being treated, the subject's weight and age, the severity of the disease state, the mode of administration, and the like, but can be readily determined by one of ordinary skill in the art. The specific dosage can vary depending on one or more of the specific agent selected, the dosing regimen followed, whether it is administered in combination with other compounds, the timing of administration, and the delivery system by which the agent is carried.

[0291] The term "administration" refers to placing an active ingredient in a subject by some method or route that results in at least partial localization of the introduced active ingredient at a desired site (such as a site of injury or repair) to produce a desired effect. In embodiments in which cells are administered, the cells can be administered by any suitable route to deliver the cells to a desired location in a subject, such that at least a portion of the transplanted cells, or components of the cells, remain viable at that location. The survival period of the cells after administration to a subject can be as short as a few hours (e.g., 24 hours), to as long as a few days, to as long as several years, or even the lifetime of the patient, i.e., long-term engraftment. 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 (such as an intraperitoneal or intravenous route).

[0292] In some embodiments, the administration comprises administration by viral delivery. In some embodiments, the administration comprises administration by electroporation. In some embodiments, the administration comprises administration by nanoparticle delivery. In some embodiments, the administration comprises administration by liposomal delivery. Any effective route of administration can be used to administer the pharmaceutical compositions described herein in effective amounts. In some embodiments, the administration comprises administration by a method selected from the group consisting of intravenous administration, subcutaneous administration, intramuscular administration, oral administration, rectal administration, administration by aerosol, parenteral administration, ocular administration, pulmonary administration, transdermal administration, vaginal administration, aural administration, nasal administration, and topical administration, or any combination thereof. In some embodiments, administration by injection or infusion is utilized to deliver the cells.

[0293] As used herein, the term "subject" refers to any individual for whom diagnosis, treatment, or therapy is desired. In some embodiments, the subject is an animal. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0294] The effectiveness of treatment can be determined by a skilled clinician. However, a treatment is considered to be "effective treatment" if any one or all of the signs or symptoms of a disease or disorder are altered in a beneficial manner (e.g., at least a 10% reduction) or other clinically accepted symptoms or markers of the disease are improved or enhanced. Efficacy can also be measured by the absence of deterioration (e.g., the progression of the disease is stopped or at least delayed) as assessed by the need for hospitalization or medical intervention. Methods for measuring these indicators are known to those skilled in the art. Treatment includes (1) inhibition of the disease (e.g., stopping or slowing the progression of symptoms); or (2) palliating the disease (e.g., inducing regression of symptoms); and (3) preventing or reducing the likelihood of the symptoms progressing.

[0295] Pharmaceutical compositions are provided that comprise an RGN polypeptide of the present disclosure, or a polynucleotide encoding same, a gRNA of the present disclosure, or a polynucleotide encoding same, a deaminase of the present disclosure, or a polynucleotide encoding same, a fusion protein of the present disclosure, a system of the present disclosure (such as a system comprising a fusion protein), or a cell comprising any of said RGN polypeptide, or a polynucleotide encoding RGN, said gRNA, or a polynucleotide encoding gRNA, a polynucleotide encoding the fusion protein, or any of said systems, and a pharma- ceutical composition comprising a pharma-ceutical acceptable carrier.

[0296] As used herein, a "pharmaceutical acceptable carrier" refers to a material that does not significantly irritate an organism and does not impair the activity and properties of the active ingredient (e.g., the deaminase or fusion protein, or the nucleic acid molecule encoding it). The carrier must be of sufficiently high purity and sufficiently low toxicity to be suitable for administration to the subject being treated. The carrier can be inert or have pharmaceutical benefits. In some embodiments, the pharmaceutical 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 pharmaceutical composition comprises a pharmaceutical acceptable non-natural carrier. In some embodiments, the pharmaceutical acceptable carrier and the active ingredient are heterologous, since they are not found together in nature.

[0297] The pharmaceutical compositions used in the disclosed methods can be formulated with suitable carriers, excipients, and other agents to provide suitable transport, delivery, tolerance, etc. Many suitable formulations are known to those skilled in the art. See, for example, Remington, The Science and Practice of Pharmacy (21st ed. 2005). Non-limiting examples include sterile diluents (such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents); antimicrobial agents (such as benzyl alcohol or methylparabens); antioxidants (such as ascorbic acid or sodium bisulfite); chelating agents (such as ethylenediaminetetraacetic acid); buffers (such as acetates, citrates, or phosphates), and agents to adjust isotonicity (such as sodium chloride or dextrose). A specific carrier for intravenous administration is saline or phosphate buffered saline (PBS). Pharmaceutical compositions for oral or parenteral use can be prepared in unit dose dosage forms suitable for one dose of active ingredient. Such dosage forms in unit dose form include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. These compositions may also contain adjuvants, including preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, etc. It may also be desirable to include isotonic agents, for example, sugars, sodium chloride, etc. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0298] In some embodiments of administering cells modified with the disclosed RGN, gRNA, deaminase, fusion protein, system (including systems that include fusion proteins), or polynucleotides encoding same to a subject, the cells are administered as a suspension containing a medicament acceptable base. Those skilled in the art will recognize that the medicament acceptable base used in the cell composition will not contain buffers, compounds, cryopreservatives, preservatives, or other agents in amounts that substantially interfere with the survival of the cells delivered to the subject. The formulation containing the cells may, for example, contain an osmotic buffer that allows the integrity of the cell membrane to be maintained, and may optionally contain nutrients to maintain cell survival or enhance cell retention after administration. Such formulations and suspensions are known to those skilled in the art and / or can be adapted for use with the cells described herein using routine experimentation.

[0299] The cell composition may also be emulsified or provided as a liposomal 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 pharma- ceutically acceptable and compatible with the active ingredients, in amounts suitable for use in the therapeutic methods described herein.

[0300] Additional agents included in the cell compositions can include pharma- ceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the polypeptide) formed with inorganic acids (e.g., hydrochloric acid or phosphoric acid) or organic acids (e.g., acetic acid, tartaric acid, mandelic acid, etc.). Salts formed with the free carboxyl groups can also be derived from inorganic bases (e.g., sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide) and organic bases (e.g., isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, etc.).

[0301] Modifying causative mutations using base editing

[0302] One example of a genetic disease that may be correctable using the RGN-deaminase fusion protein-based approach of the present invention is Niemann-Pick disease type C. Niemann-Pick disease (NPC) is an autosomal recessive lysosomal storage disease caused by mutations in the NPC1 or NPC2 genes (the sequence of the NPC1 gene is shown as SEQ ID NO: 121), which results in abnormal accumulation of cholesterol and glycosphingolipids (GSLs). Patients with NPC typically present between the ages of 4 and 7. Major symptoms include liver and lung disease, hypotonia, dysphagia, psychomotor retardation, cerebellar ataxia, progressive cognitive impairment, dementia, and other neurological dysfunction. One common variant associated with early-onset neurological disease onset is NM_000271.5(NPC1):c.3182T>C (p.Ile1061Thr) in exon 21, which can be corrected using cytosine base editing. The present invention also discloses potential target sequences for directing the fusion proteins of the present invention to various disease-causing mutations, including the NM_000271.5 (NPC1):c.3182T>C (p.I1061T) mutation in exon 21 known to cause Niemann-Pick disease type C.

[0303] XII. Cells containing polynucleotide genetic modifications

[0304] Provided herein are cells and / or organisms comprising a target nucleic acid molecule of interest modified using methods mediated by a fusion protein, optionally with a gRNA, as described herein. In some embodiments, the fusion protein comprises a deaminase polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 2, 4, and 6-12, or an active variant or fragment thereof. In some embodiments, the fusion protein comprises a cytosine deaminase comprising an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 2, 4, and 6-12. In some embodiments, the fusion protein comprises a deaminase and a DNA-binding polypeptide (e.g., an RNA-guided DNA-binding polypeptide). In further embodiments, the fusion protein comprises a deaminase and an RGN or a variant thereof, such as APG07433.1 (SEQ ID NO: 74) or its nickase variant nAPG07433.1 (SEQ ID NO: 75). In some embodiments, the fusion protein comprises a deaminase and a Cas9 or a variant thereof, such as dCas9 or nickase Cas9. In some embodiments, the fusion protein comprises a nuclease inactive variant or nickase variant of a Type II CRISPR-Cas polypeptide. In some embodiments, the fusion protein comprises a nuclease inactive variant or nickase variant of a Type V CRISPR-Cas polypeptide. In some embodiments, the fusion protein comprises a nuclease inactive variant or nickase variant of a Type VI CRISPR-Cas polypeptide.

[0305] The modified cells can be eukaryotic (e.g., mammalian, plant, insect, avian) or prokaryotic. Organelles and embryos containing at least one nucleotide sequence modified by the methods using the fusion proteins described herein are also provided. The genetically modified cells, organisms, organelles, and embryos can be heterozygous or homozygous for the modified nucleotide sequence. The mutation introduced by the deaminase of the fusion protein can result in altered expression (up- or down-regulation), inactivation, or expression of the protein product or the incorporated sequence. If the mutation results in gene inactivation or expression of a non-functional protein product, the genetically modified cell, organism, organelle, or embryo is called a "knockout." The knockout phenotype can be the result of a deletion mutation (i.e., deletion of at least one nucleotide), an insertion mutation (i.e., insertion of at least one nucleotide), or a nonsense mutation (i.e., replacement of at least one nucleotide to introduce a stop codon).

[0306] In some embodiments, a variant protein product is produced as a result of the mutation introduced by the deaminase of the fusion protein. The expressed variant protein product may have at least one amino acid substitution and / or at least one amino acid addition or deletion. The variant protein product may exhibit altered characteristics or activities when compared to the wild-type protein, including, non-limiting examples, altered enzymatic activity or substrate specificity.

[0307] In some embodiments, the expression pattern of the protein is altered as a result of the mutation introduced by the deaminase of the fusion protein. As a non-limiting example, a mutation in a regulatory region that controls the expression of a protein product may result in the protein product being overexpressed or downregulated, or may result in a change in the tissue or temporal expression pattern.

[0308] The transformed cells can be conventionally grown into transgenic organisms (such as plants). See, e.g., McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants can then be grown and pollinated with the same transformed line or with a different line to produce hybrids carrying the genetic modification. Genetically modified seeds are provided by the invention. Progeny, variants, and mutants of the regenerated plants are also within the scope of the invention, provided that these portions contain the genetic modification. Also provided are processed plant products or by-products, including soybean meal, that carry the sequences disclosed herein.

[0309] The methods presented herein can be used to modify any plant species, including non-limiting examples of monocotyledonous and dicotyledonous plants.Non-limiting examples of plants of interest include corn (maize), sorghum, wheat, sunflower, tomato, cruciferous plants, pepper, potato, cotton, rice, soybean, sugar beet, sugarcane, tobacco, barley, and rapeseed, Brassica species, alfalfa, rye, millet, safflower, peanut, sweet potato, cassava, coffee, coconut, pineapple, citrus, cocoa, tea, banana, avocado, fig, guava, mango, olive, papaya, cashew nut, macadamia nut, almond, oat, vegetables, ornamental plants, and conifers.

[0310] Non-limiting examples of vegetables include tomatoes, lettuce, green peas, lima beans, peas, and members of the genus Cucumis (such as cucumbers, cantaloupes, and muskmelons). Non-limiting examples of ornamental plants include azaleas, hydrangeas, hibiscus, roses, tulips, daffodils, petunias, carnations, poinsettias, and chrysanthemums. The plants of the present invention are preferably crop plants (e.g., corn, sorghum, wheat, sunflowers, tomatoes, crucifers, peppers, potatoes, cotton, rice, soybeans, sugar beets, sugarcane, tobacco, barley, rapeseed, etc.).

[0311] Any prokaryotic species can also be genetically modified using the methods presented herein.Non-limiting examples of prokaryotic species include archaea and bacteria (e.g., Bacillus species, Klebsiella species, Streptomyces species, Rhizobium species, Escherichia coli species, Pseudomonas species, Salmonella species, Shigella species, Vibrio species, Yersinia species, Mycoplasma species, Agrobacterium, and Lactobacillus species).

[0312] Any eukaryotic species or cells derived therefrom can be genetically modified using the methods presented herein.Non-limiting examples of eukaryotic species include animals (e.g. mammals, insects, fish, birds, and reptiles), fungi, amoebas, algae, and yeast.In some embodiments, the cells modified by the methods of the present disclosure include cells of hematopoietic origin, such as immune cells (i.e. cells of the innate or adaptive immune system), non-limiting examples of which include B cells, T cells, natural killer (NK) cells, pluripotent stem cells, induced pluripotent stem cells, chimeric antigen receptor T (CAR-T) cells, monocytes, macrophages, and dendritic cells.

[0313] The modified cells can be introduced into an organism. These cells are modified by an ex vivo approach and can be derived from the same organism (e.g., a human) in the case of autologous cell transplantation. In some embodiments, the cells are derived from another organism of the same species (e.g., another human) in the case of allogeneic cell transplantation.

[0314] XIII. Kit

[0315] Some aspects of the disclosure provide kits comprising the deaminase of the present invention. In some embodiments, the disclosure provides kits comprising the deaminase of the present invention and a fusion protein comprising a DNA-binding polypeptide (e.g., an RNA-guided DNA-binding polypeptide (such as an RGN polypeptide, e.g., a nuclease-inactive RGN or a nickase RGN)) and, optionally, a linker located between the DNA-binding polypeptide and the deaminase. In addition, in some embodiments, the kit includes appropriate reagents, buffers, and / or instructions for using the fusion protein for, e.g., in vitro or in vivo DNA or RNA editing. In some embodiments, the kit includes instructions for designing and using gRNA suitable for targeted nucleic acid sequence editing.

[0316] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "a polypeptide" means one or more polypeptides.

[0317] All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this disclosure pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0318] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent that certain changes and modifications can be practiced within the scope of the appended claims.

[0319] Non-limiting embodiments include the following:

[0320] 1.a) an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 2 and 7 to 12; b) an amino acid sequence having at least 95% identity with SEQ ID NO: 4 or 6 comprising an amino acid sequence selected from the group consisting of: A polypeptide with deaminase activity.

[0321] 2.a) an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 2 and 7 to 12; b) an amino acid sequence having at least 95% identity with SEQ ID NO: 4 or 6 comprising an amino acid sequence selected from the group consisting of: An isolated polypeptide having deaminase activity.

[0322] 3. A polypeptide according to embodiment 1 or 2, comprising an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 2 and 7 to 12.

[0323] 4. A polypeptide according to embodiment 1 or 2, comprising an amino acid sequence having 100% identity with any one of SEQ ID NOs: 2, 4, and 6 to 12.

[0324] 5. A polynucleotide encoding a deaminase polypeptide, the deaminase comprising: a) a nucleotide sequence having at least 80% identity to any one of SEQ ID NOs: 114 to 119; b) a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 109, 111, and 113; c) a nucleotide sequence encoding an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 2 and 7 to 12; and d) a nucleotide sequence encoding an amino acid sequence having at least 95% identity to SEQ ID NO: 4 or 6 A nucleic acid molecule encoded by a nucleotide sequence selected from the group consisting of:

[0325] 6. A polynucleotide encoding a deaminase polypeptide, the deaminase comprising: a) a nucleotide sequence having at least 80% identity to any one of SEQ ID NOs: 114 to 119; b) a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 109, 111, and 113; c) a nucleotide sequence encoding an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 2 and 7 to 12; and d) a nucleotide sequence encoding an amino acid sequence having at least 95% identity to SEQ ID NO: 4 or 6 An isolated nucleic acid molecule encoded by a nucleotide sequence selected from the group consisting of:

[0326] 7. The nucleic acid molecule of embodiment 5 or 6, wherein the deaminase is encoded by a nucleotide sequence having at least 90% identity to any one of SEQ ID NOs: 114 to 119.

[0327] 8. The nucleic acid molecule of embodiment 5 or 6, wherein the deaminase is encoded by a nucleotide sequence having at least 95% identity to any one of SEQ ID NOs: 114 to 119.

[0328] 9. The nucleic acid molecule of embodiment 5 or 6, wherein the deaminase is encoded by a nucleotide sequence having 100% identity to any one of SEQ ID NOs: 109, 111, and 113-119.

[0329] 10. The nucleic acid molecule of embodiment 5 or 6, wherein the deaminase polypeptide has an amino acid sequence that has at least 95% identity to any one of SEQ ID NOs: 2 and 7 to 12.

[0330] 11. The nucleic acid molecule of embodiment 5 or 6, wherein the deaminase polypeptide has an amino acid sequence that is 100% identical to any one of SEQ ID NOs: 2, 4, and 6-12.

[0331] 12. The nucleic acid molecule according to any one of embodiments 5 to 11, further comprising a heterologous promoter operably linked to said polypeptide.

[0332] 13. A vector comprising a nucleic acid molecule according to any one of embodiments 5 to 12.

[0333] 14. A cell comprising a nucleic acid molecule according to any one of embodiments 5 to 12 or a vector according to embodiment 13.

[0334] 15. The cell of embodiment 14, which is a prokaryotic cell.

[0335] 16. The cell of embodiment 14, which is a eukaryotic cell.

[0336] 17. The cell of embodiment 16, wherein the eukaryotic cell is a mammalian cell.

[0337] 18. The cell of embodiment 17, wherein the mammalian cell is a human cell.

[0338] 19. The cell of embodiment 18, wherein the human cell is an immune cell.

[0339] 20. The cell of embodiment 19, wherein the immune cell is a stem cell.

[0340] 21. The cell of embodiment 20, wherein the stem cell is an induced pluripotent stem cell.

[0341] 22. The cell of embodiment 16, wherein the eukaryotic cell is an insect cell or an avian cell.

[0342] 23. The cell of embodiment 16, wherein the eukaryotic cell is a fungal cell.

[0343] 24. The cell of embodiment 16, wherein the eukaryotic cell is a plant cell.

[0344] 25. A plant comprising a cell according to embodiment 24.

[0345] 26. A seed comprising a cell according to embodiment 24.

[0346] 27. A pharmaceutical composition comprising a polypeptide according to any one of embodiments 1 to 4, a nucleic acid molecule according to any one of embodiments 5 to 12, a vector according to embodiment 13, or a cell according to any one of embodiments 14 to 24, together with a pharma- ceutical acceptable carrier.

[0347] 28. The pharmaceutical composition according to embodiment 27, wherein the pharma- ceutically acceptable carrier is heterologous to the polypeptide or nucleic acid molecule.

[0348] 29. The pharmaceutical composition according to embodiment 27 or 28, wherein said pharma- ceutically acceptable carrier is non-naturally occurring.

[0349] 30. A method for producing a deaminase, comprising culturing a cell according to any one of embodiments 14 to 24 under conditions in which said deaminase is expressed.

[0350] 31. A method for producing a deaminase, comprising introducing a nucleic acid molecule according to any one of embodiments 5 to 12, or a vector according to embodiment 13, into a cell and culturing the cell under conditions in which the deaminase is expressed.

[0351] 32. The method of embodiment 30 or 31, further comprising purifying the deaminase.

[0352] 33. A DNA-binding polypeptide comprising: a) an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 2 and 7 to 12; b) an amino acid sequence having at least 95% identity with SEQ ID NO: 4 or 6 A deaminase having an amino acid sequence selected from the group consisting of A fusion protein comprising:

[0353] 34. The fusion protein of embodiment 33, wherein the deaminase has a sequence that is at least 95% identical to any one of SEQ ID NOs: 2 and 7 to 12.

[0354] 35. The fusion protein of embodiment 33, wherein the deaminase has an sequence that is 100% identical to any one of SEQ ID NOs: 2, 4, and 6 to 12.

[0355] 36. A fusion protein according to any one of embodiments 33 to 35, wherein the deaminase is a cytosine deaminase.

[0356] 37. A fusion protein according to any one of embodiments 33 to 36, wherein the DNA-binding polypeptide is a meganuclease, a zinc finger fusion protein, or a TALEN; or a variant of a meganuclease, a zinc finger fusion protein, or a TALEN, and wherein the nuclease activity is reduced or inhibited.

[0357] 38. The fusion protein of any one of embodiments 33 to 36, wherein the DNA-binding polypeptide is an RNA-guided DNA-binding polypeptide.

[0358] 39. The fusion protein of embodiment 38, wherein the RNA-guided DNA-binding polypeptide is an RNA-guided nuclease (RGN) polypeptide.

[0359] 40. The fusion protein of embodiment 39, wherein the RGN is a type II CRISPR-Cas polypeptide.

[0360] 41. The fusion protein of embodiment 39, wherein the RGN is a V-type CRISPR-Cas polypeptide.

[0361] 42. A fusion protein according to any one of embodiments 39 to 41, wherein the RGN is RGN nickase.

[0362] 43. The fusion protein of embodiment 42, wherein the RGN nickase has an inactive RuvC domain.

[0363] 44. The fusion protein of any one of embodiments 39 to 41, wherein the RGN is a nuclease-inactive RGN.

[0364] 45. The fusion protein of embodiment 39, wherein the RGN has an amino acid sequence that has at least 90% identity to any one of the RGN sequences in Table 1.

[0365] 46. ​​The fusion protein of embodiment 39, wherein the RGN has an amino acid sequence that has at least 95% identity to any one of the RGN sequences in Table 1.

[0366] 47. The fusion protein of embodiment 39, wherein the RGN has an amino acid sequence of any one of the RGN sequences in Table 1.

[0367] 48. The fusion protein of embodiment 39, wherein the RGN has an amino acid sequence that has at least 90% identity to any one of SEQ ID NOs: 74, 82, 87, 106, and 107.

[0368] 49. The fusion protein of embodiment 39, wherein the RGN has an amino acid sequence that has at least 95% identity to any one of SEQ ID NOs: 74, 82, 87, 106, and 107.

[0369] 50. The fusion protein of embodiment 39, wherein the RGN has an amino acid sequence of any one of SEQ ID NOs: 74, 82, 87, 106, and 107.

[0370] 51. The fusion protein of embodiment 42, wherein the RGN nickase has an amino acid sequence that has at least 90% identity to any one of SEQ ID NOs: 75 and 88 to 98.

[0371] 52. The fusion protein of embodiment 42, wherein the RGN nickase has an amino acid sequence that has at least 95% identity to any one of SEQ ID NOs: 75 and 88 to 98.

[0372] 53. The fusion protein of embodiment 42, wherein the RGN nickase has an amino acid sequence having any one of SEQ ID NOs: 75 and 88 to 98.

[0373] 54. A fusion protein according to any one of embodiments 33 to 53, further comprising at least one nuclear localization signal (NLS).

[0374] 55. A fusion protein according to any one of embodiments 33 to 54, wherein the deaminase is fused to the amino terminus of the DNA-binding polypeptide.

[0375] 56. A fusion protein according to any one of embodiments 33 to 54, wherein the deaminase is fused to the carboxyl terminus of the DNA-binding polypeptide.

[0376] 57. A fusion protein according to any one of embodiments 33 to 56, further comprising a linker sequence between the DNA-binding polypeptide and the deaminase.

[0377] 58. The fusion protein of embodiment 57, wherein the linker sequence has the amino acid sequence shown as SEQ ID NO: 78 or 79.

[0378] 59. The fusion protein of any one of embodiments 33 to 58, further comprising a uracil stabilizing protein (USP).

[0379] 60. The fusion protein of embodiment 59, wherein the USP has the sequence shown as SEQ ID NO: 81.

[0380] 61. The fusion protein of embodiment 59 or 60, further comprising a linker sequence between the USP and the deaminase or the DNA-binding polypeptide.

[0381] 62. The fusion protein of embodiment 61, wherein the linker sequence between the USP and the deaminase or the DNA-binding polypeptide has the amino acid sequence shown as SEQ ID NO: 120.

[0382] 63. The fusion protein of embodiment 33, having an amino acid sequence of any one of SEQ ID NOs: 67, 68, 146, and 147.

[0383] 64. A polynucleotide encoding a fusion protein comprising a DNA-binding polypeptide and a deaminase, wherein the deaminase is a) a nucleotide sequence having at least 80% identity to any one of SEQ ID NOs: 114 to 119; b) a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 109, 111, and 113; c) a nucleotide sequence encoding an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 2 and 7 to 12; and d) a nucleotide sequence encoding an amino acid sequence having at least 95% identity to SEQ ID NO: 4 or 6 A nucleic acid molecule encoded by a nucleotide sequence selected from the group consisting of:

[0384] 65. The nucleic acid molecule of embodiment 64, wherein the deaminase is encoded by a nucleotide sequence having at least 90% identity to any one of SEQ ID NOs: 114 to 119.

[0385] 66. The nucleic acid molecule of embodiment 64, wherein the deaminase is encoded by a nucleotide sequence having at least 95% identity to any one of SEQ ID NOs: 114 to 119.

[0386] 67. The nucleic acid molecule of embodiment 64, wherein the nucleotide sequence of the deaminase is 100% identical to any one of SEQ ID NOs: 109, 111, and 113 to 119.

[0387] 68. The nucleic acid molecule of embodiment 64, wherein the nucleotide sequence of the deaminase encodes an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 2 and 7 to 12.

[0388] 69. The nucleic acid molecule of embodiment 64, wherein the nucleotide sequence of the deaminase encodes an amino acid sequence having 100% identity to any one of SEQ ID NOs: 2, 4, 6 to 12.

[0389] 70. The nucleic acid molecule according to any one of embodiments 64 to 69, wherein the deaminase is a cytosine deaminase.

[0390] 71. The nucleic acid molecule of any one of embodiments 64 to 70, wherein the DNA-binding polypeptide is a meganuclease, a zinc finger fusion protein, or a TALEN; or a variant of a meganuclease, a zinc finger fusion protein, or a TALEN, and the nuclease activity is reduced or inhibited.

[0391] 72. The nucleic acid molecule according to any one of embodiments 64 to 70, wherein the DNA-binding polypeptide is an RNA-guided DNA-binding polypeptide.

[0392] 73. The nucleic acid molecule of embodiment 72, wherein the RNA-guided DNA-binding polypeptide is an RNA-guided nuclease (RGN) polypeptide.

[0393] 74. The nucleic acid molecule of embodiment 73, wherein the RGN is a type II CRISPR-Cas polypeptide.

[0394] 75. The nucleic acid molecule of embodiment 73, wherein the RGN is a type V CRISPR-Cas polypeptide.

[0395] 76. The nucleic acid molecule of any one of embodiments 73 to 75, wherein the RGN is RGN nickase.

[0396] 77. The nucleic acid molecule of embodiment 76, wherein the RGN nickase has an inactive RuvC domain.

[0397] 78. The nucleic acid molecule of any one of embodiments 73 to 75, wherein the RGN is a nuclease-inactive RGN.

[0398] 79. The nucleic acid molecule of embodiment 73, wherein the RGN has an amino acid sequence that has at least 90% identity to any one of the RGN sequences in Table 1.

[0399] 80. The nucleic acid molecule of embodiment 73, wherein the RGN has an amino acid sequence that has at least 95% sequence identity to any one of the RGN sequences in Table 1.

[0400] 81. The nucleic acid molecule of embodiment 73, wherein the RGN has an amino acid sequence of any one of the RGN sequences in Table 1.

[0401] 82. The nucleic acid molecule of embodiment 73, wherein the RGN has an amino acid sequence that has at least 90% identity to any one of SEQ ID NOs: 74, 82, 87, 106, and 107.

[0402] 83. The nucleic acid molecule of embodiment 73, wherein the RGN has an amino acid sequence that has at least 95% identity to any one of SEQ ID NOs: 74, 82, 87, 106, and 107.

[0403] 84. The nucleic acid molecule of embodiment 73, wherein the RGN has an amino acid sequence of any one of SEQ ID NOs: 74, 82, 87, 106, and 107.

[0404] 85. The nucleic acid molecule of embodiment 76, wherein the RGN nickase has an amino acid sequence that has at least 90% identity to any one of SEQ ID NOs: 75 and 88 to 98.

[0405] 86. The nucleic acid molecule of embodiment 76, wherein the RGN nickase has an amino acid sequence that has at least 95% identity to any one of SEQ ID NOs: 75 and 88 to 98.

[0406] 87. The nucleic acid molecule of embodiment 76, wherein the RGN nickase has an amino acid sequence having any one of SEQ ID NOs: 75 and 88 to 98.

[0407] 88. The nucleic acid molecule of any one of embodiments 64 to 87, wherein the polypeptide encoding the fusion protein is operably linked at its 5' end to a promoter.

[0408] 89. The nucleic acid molecule of any one of embodiments 64 to 88, wherein the polypeptide encoding the fusion protein is operably linked at its 3' end to a terminator.

[0409] 90. The nucleic acid molecule of any one of embodiments 64 to 89, wherein the fusion protein comprises one or more nuclear localization signals.

[0410] 91. The nucleic acid molecule of any one of embodiments 64 to 90, wherein the fusion protein is codon-optimized for expression in a eukaryotic cell.

[0411] 92. The nucleic acid molecule of any one of embodiments 64 to 90, wherein the fusion protein is codon-optimized for expression in a prokaryotic cell.

[0412] 93. The nucleic acid molecule of any one of embodiments 64 to 92, wherein the deaminase is fused to the amino terminus of the DNA-binding polypeptide.

[0413] 94. The nucleic acid molecule of any one of embodiments 64 to 92, wherein the deaminase is fused to the carboxyl terminus of the DNA-binding polypeptide.

[0414] 95. The nucleic acid molecule of any one of embodiments 64 to 94, wherein the fusion protein further comprises a linker sequence between the DNA-binding polypeptide and the deaminase.

[0415] 96. The nucleic acid molecule of embodiment 95, wherein the linker sequence has the amino acid sequence shown as SEQ ID NO: 78 or 79.

[0416] 97. The nucleic acid molecule of any one of embodiments 64 to 96, wherein the fusion protein further comprises a uracil stabilizing protein (USP).

[0417] 98. The nucleic acid molecule of embodiment 97, wherein the USP has the sequence shown as SEQ ID NO: 81.

[0418] 99. The nucleic acid molecule of embodiment 97 or 98, wherein the fusion protein further comprises a linker sequence between the USP and the deaminase or the DNA-binding polypeptide.

[0419] 100. The nucleic acid molecule of embodiment 99, wherein the linker sequence between the USP and the deaminase or the DNA-binding polypeptide has the amino acid sequence shown as SEQ ID NO: 120.

[0420] 101. The nucleic acid molecule of embodiment 64, wherein the fusion protein has an amino acid sequence of any one of SEQ ID NOs: 67, 68, 146, and 147.

[0421] 102. A vector comprising a nucleic acid molecule according to any one of embodiments 64 to 101.

[0422] 103. The vector of embodiment 102, further comprising at least one nucleotide sequence encoding a guide RNA (gRNA) capable of hybridizing to a target sequence.

[0423] 104. The vector of embodiment 103, wherein the gRNA is a single guide RNA.

[0424] 105. The vector of embodiment 102, wherein the gRNA is a dual guide RNA.

[0425] 106. A cell comprising a fusion protein according to any one of embodiments 33 to 63.

[0426] 107. The cell of embodiment 106, further comprising a guide RNA (gRNA).

[0427] 108. The cell of embodiment 107, wherein the gRNA is a single guide RNA.

[0428] 109. The cell of embodiment 107, wherein the gRNA is a dual guide RNA.

[0429] 110. A cell comprising a nucleic acid molecule according to any one of embodiments 64 to 101.

[0430] 111. A cell comprising a vector according to any one of embodiments 102 to 105.

[0431] 112. A cell according to any one of embodiments 106 to 111, which is a prokaryotic cell.

[0432] 113. A cell according to any one of embodiments 106 to 111, which is a eukaryotic cell.

[0433] 114. The cell according to embodiment 113, wherein the eukaryotic cell is a mammalian cell.

[0434] 115. The cell of embodiment 114, wherein the mammalian cell is a human cell.

[0435] 116. The cell of embodiment 115, wherein the human cell is an immune cell.

[0436] 117. The cell described in embodiment 116, wherein the immune cell is a stem cell.

[0437] 118. The cell described in embodiment 117, wherein the stem cell is an induced pluripotent stem cell.

[0438] 119. The cell according to embodiment 113, wherein the eukaryotic cell is an insect cell or an avian cell.

[0439] 120. The cell of embodiment 113, wherein the eukaryotic cell is a fungal cell.

[0440] 121. The cell of embodiment 113, wherein the eukaryotic cell is a plant cell.

[0441] 122. A plant comprising a cell according to embodiment 121.

[0442] 123. A seed comprising a cell according to embodiment 121.

[0443] 124. A pharmaceutical composition comprising a fusion protein according to any one of embodiments 33 to 63, a nucleic acid molecule according to any one of embodiments 64 to 101, a vector according to any one of embodiments 102 to 105, or a cell according to any one of embodiments 114 to 118, together with a pharma- ceutical acceptable carrier.

[0444] 125. A method for producing a fusion protein, comprising culturing a cell according to any one of embodiments 106 to 121 under conditions in which the fusion protein is expressed.

[0445] 126. A method for producing a fusion protein, comprising introducing into a cell a nucleic acid molecule according to any one of embodiments 64 to 101, or a vector according to any one of embodiments 102 to 105, and culturing the cell under conditions in which the fusion protein is expressed.

[0446] 127. The method of embodiment 125 or 126, further comprising purifying the fusion protein.

[0447] 128. A method for producing an RGN fusion ribonucleoprotein complex, comprising introducing into a cell a nucleic acid molecule according to any one of embodiments 72 to 87, and a nucleic acid molecule comprising an expression cassette encoding a guide RNA (gRNA), or a vector according to any one of embodiments 103 to 105, and culturing the cell under conditions in which the fusion protein and the gRNA are expressed to form an RGN fusion ribonucleoprotein complex.

[0448] 129. The method of embodiment 128, further comprising purifying the RGN fusion ribonucleoprotein complex.

[0449] 130. A system for modifying a target DNA molecule comprising a target DNA sequence, comprising: a) a fusion protein or a nucleotide sequence encoding said fusion protein, wherein said fusion protein comprises an RNA-guided nuclease polypeptide (RGN) and a deaminase, said deaminase comprising: i) an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 2 and 7 to 12; ii) an amino acid sequence having at least 95% identity with SEQ ID NO: 4 or 6 having an amino acid sequence selected from the group consisting of: b) one or more guide RNAs capable of hybridizing to said target DNA sequence, or one or more nucleotide sequences encoding said one or more guide RNAs. Including, A system in which the one or more guide RNAs form a complex with the fusion protein, enabling the fusion protein to bind to the target DNA sequence and modify the target DNA molecule.

[0450] 131. The system according to embodiment 130, wherein the deaminase has an amino acid sequence that has at least 95% identity to any one of SEQ ID NOs: 2 and 7 to 12.

[0451] 132. The system described in embodiment 130, wherein the deaminase has an amino acid sequence that has 100% identity to any one of SEQ ID NOs: 2, 4, and 6 to 12.

[0452] 133. The system according to any one of embodiments 130 to 132, wherein at least one of the nucleotide sequences encoding the one or more guide RNAs and the nucleotide sequence encoding the fusion protein is operably linked to a promoter.

[0453] 134. The system according to any one of embodiments 130 to 133, wherein the target DNA sequence is a eukaryotic target DNA sequence.

[0454] 135. A system according to any one of embodiments 130 to 134, wherein the target DNA sequence is located adjacent to a protospacer adjacent motif (PAM) recognised by the RGN.

[0455] 136. The system according to any one of embodiments 130 to 135, wherein the target DNA molecule is intracellular.

[0456] 137. The system according to embodiment 136, wherein the cell is a eukaryotic cell.

[0457] 138. The system according to embodiment 137, wherein the eukaryotic cell is a plant cell.

[0458] 139. The system according to embodiment 137, wherein the eukaryotic cell is a mammalian cell.

[0459] 140. The system according to embodiment 139, wherein the mammalian cells are human cells.

[0460] 141. The system according to embodiment 140, wherein the human cells are immune cells.

[0461] 142. The system of embodiment 141, wherein the immune cells are stem cells.

[0462] 143. The system according to embodiment 142, wherein the stem cells are induced pluripotent stem cells.

[0463] 144. The system according to embodiment 137, wherein the eukaryotic cell is an insect cell.

[0464] 145. The system according to embodiment 136, wherein the cells are prokaryotic cells.

[0465] 146. A system described in any one of embodiments 130 to 145, wherein the RGN of the fusion protein is a type II CRISPR-Cas polypeptide.

[0466] 147. A system described in any one of embodiments 130 to 145, wherein the RGN of the fusion protein is a V-type CRISPR-Cas polypeptide.

[0467] 148. The system according to any one of embodiments 130 to 145, wherein the RGN of the fusion protein has an amino acid sequence that has at least 90% identity to any one of the RGN sequences in Table 1.

[0468] 149. The system according to any one of embodiments 130 to 145, wherein the RGN of the fusion protein has an amino acid sequence that has at least 95% sequence identity with any one of the RGN sequences in Table 1.

[0469] 150. The system according to any one of embodiments 130 to 145, wherein the RGN of the fusion protein has an amino acid sequence of any one of the RGN sequences in Table 1.

[0470] 151. The system according to any one of embodiments 130 to 145, wherein the RGN of the fusion protein has an amino acid sequence that has at least 90% identity to any one of SEQ ID NOs: 74, 82, 87, 106, and 107.

[0471] 152. The system according to any one of embodiments 130 to 145, wherein the RGN of the fusion protein has an amino acid sequence that has at least 95% identity to any one of SEQ ID NOs: 74, 82, 87, 106, and 107.

[0472] 153. The system according to any one of embodiments 130 to 145, wherein the RGN of the fusion protein has an amino acid sequence according to any one of SEQ ID NOs: 74, 82, 87, 106, and 107.

[0473] 154. A system according to any one of embodiments 130 to 145, wherein the RGN of the fusion protein is RGN nickase.

[0474] 155. The system of embodiment 154, wherein the RGN nickase has an inactive RuvC domain.

[0475] 156. The system according to embodiment 154 or 155, wherein the RGN nickase has an amino acid sequence that has at least 90% identity to any one of SEQ ID NOs: 75 and 88 to 98.

[0476] 157. The system according to embodiment 154 or 155, wherein the RGN nickase has an amino acid sequence that has at least 95% identity to any one of SEQ ID NOs: 75 and 88 to 98.

[0477] 158. The system of embodiment 154 or 155, wherein the RGN nickase is any one of SEQ ID NOs: 75 and 88 to 98.

[0478] 159. A system according to any one of embodiments 130 to 145, wherein the RGN of the fusion protein is a nuclease-inactive RGN.

[0479] 160. The system according to any one of embodiments 130 to 159, wherein the fusion protein comprises one or more nuclear localization signals.

[0480] 161. The system according to any one of embodiments 130 to 160, wherein the deaminase is fused to the amino terminus of the DNA-binding polypeptide.

[0481] 162. The system according to any one of embodiments 130 to 160, wherein the deaminase is fused to the carboxyl terminus of the DNA-binding polypeptide.

[0482] 163. The system according to any one of embodiments 130 to 162, wherein the fusion protein further comprises a linker sequence between the DNA-binding polypeptide and the deaminase.

[0483] 164. The system according to embodiment 163, wherein the linker sequence has the amino acid sequence shown as SEQ ID NO: 78 or 79.

[0484] 165. The system according to any one of embodiments 130 to 164, wherein the fusion protein further comprises a uracil stabilizing protein (USP).

[0485] 166. The system described in embodiment 165, wherein the USP has the sequence shown as SEQ ID NO: 81.

[0486] 167. The system according to embodiment 165 or 166, wherein the fusion protein further comprises a linker sequence between the USP and the deaminase or the DNA-binding polypeptide.

[0487] 168. The system described in embodiment 167, wherein the linker sequence between the USP and the deaminase or the DNA-binding polypeptide has the amino acid sequence shown as SEQ ID NO: 120.

[0488] 169. The system described in embodiment 130, wherein the fusion protein has an amino acid sequence set forth as any one of SEQ ID NOs: 67, 68, 146, and 147.

[0489] 170. The system according to any one of embodiments 130 to 169, wherein the fusion protein is codon-optimized for expression in eukaryotic cells.

[0490] 171. The system according to any one of embodiments 130 to 170, wherein the nucleotide sequence encoding the one or more guide RNAs and the nucleotide sequence encoding the fusion protein are located on one vector.

[0491] 172. A ribonucleoprotein complex comprising at least one guide RNA and said fusion protein of a system according to any one of embodiments 130 to 171.

[0492] 173. A cell comprising a system according to any one of embodiments 130 to 171 or a ribonucleoprotein complex according to embodiment 172.

[0493] 174. The cell according to embodiment 173, which is a prokaryotic cell.

[0494] 175. The cell according to embodiment 173, which is a eukaryotic cell.

[0495] 176. The cell according to embodiment 175, wherein the eukaryotic cell is a mammalian cell.

[0496] 177. The cell of embodiment 176, wherein the mammalian cell is a human cell.

[0497] 178. The cell of embodiment 177, wherein the human cell is an immune cell.

[0498] 179. The cell described in embodiment 178, wherein the immune cell is a stem cell.

[0499] 180. The cell of embodiment 179, wherein the stem cell is an induced pluripotent stem cell.

[0500] 181. The cell according to embodiment 175, wherein the eukaryotic cell is an insect cell or an avian cell.

[0501] 182. The cell according to embodiment 175, wherein the eukaryotic cell is a fungal cell.

[0502] 183. The cell according to embodiment 175, wherein the eukaryotic cell is a plant cell.

[0503] 184. A plant comprising a cell according to embodiment 183.

[0504] 185. A seed comprising a cell according to embodiment 183.

[0505] 186. A pharmaceutical composition comprising a system according to any one of embodiments 130 to 171, a ribonucleoprotein complex according to embodiment 172, or a cell according to any one of embodiments 175 to 180, together with a pharma- ceutically acceptable carrier.

[0506] 187. A method for modifying a target DNA molecule comprising a target DNA sequence, comprising delivering a system according to any one of embodiments 130 to 171, or a ribonucleoprotein complex according to embodiment 172, to the target DNA molecule or to a cell comprising the target DNA molecule.

[0507] 188. The method of embodiment 187, wherein the modified target DNA molecule comprises a C>N mutation of at least one nucleotide within the target DNA molecule, where N is A, G, or T.

[0508] 189. The method of embodiment 188, wherein the modified target DNA molecule comprises a C>T mutation of at least one nucleotide in the target DNA molecule.

[0509] 190. The method of embodiment 188, wherein the modified target DNA molecule comprises a C>G mutation of at least one nucleotide within the target DNA molecule.

[0510] 191. A method for modifying a target DNA molecule containing a target sequence, comprising: a) Assembly of an RGN-deaminase complex under conditions suitable for the formation of the RGN-deaminase complex; i) one or more guide RNAs capable of hybridizing to said target DNA molecule; ii) a fusion protein comprising an RNA-guided nuclease polypeptide (RGN) and at least one deaminase, wherein the deaminase is I) an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 2 and 7 to 12; II) an amino acid sequence having at least 95% identity with SEQ ID NO: 4 or 6 (having an amino acid sequence selected from the group consisting of: b) contacting the target DNA molecule, or a cell containing the target DNA molecule, with the in vitro assembled RGN-deaminase complex; The one or more guide RNAs hybridize to the target DNA molecule, thereby allowing the fusion protein to bind to the target DNA molecule and cause modification of the target DNA molecule.

[0511] 192. The method of embodiment 191, wherein the deaminase has an amino acid sequence that has at least 95% identity to any one of SEQ ID NOs: 2 and 7 to 12.

[0512] 193. The method of embodiment 191, wherein the deaminase has an amino acid sequence that has 100% identity to any one of SEQ ID NOs: 2, 4, and 6-12.

[0513] 194. The method of any one of embodiments 191 to 193, wherein the modified target DNA molecule comprises a C>N mutation of at least one nucleotide within the target DNA molecule, where N is A, G, or T.

[0514] 195. The method of embodiment 194, wherein the modified target DNA molecule comprises a C>T mutation of at least one nucleotide in the target DNA molecule.

[0515] 196. The method of embodiment 194, wherein the modified target DNA molecule comprises a C>G mutation of at least one nucleotide in the target DNA molecule.

[0516] 197. The method of any one of embodiments 191 to 196, wherein the RGN of the fusion protein is a type II CRISPR-Cas polypeptide.

[0517] 198. The method of any one of embodiments 191 to 196, wherein the RGN of the fusion protein is a type V CRISPR-Cas polypeptide.

[0518] 199. The method of any one of embodiments 191 to 198, wherein the RGN of the fusion protein has an amino acid sequence that has at least 90% sequence identity to any one of the RGN sequences in Table 1.

[0519] 200. The method of any one of embodiments 191-198, wherein the RGN of the fusion protein has an amino acid sequence that has at least 95% sequence identity to any one of the RGN sequences in Table 1.

[0520] 201. The method of any one of embodiments 191-198, wherein the RGN of the fusion protein has an amino acid sequence of any one of the RGN sequences in Table 1.

[0521] 202. The method of any one of embodiments 191 to 198, wherein the RGN of the fusion protein has an amino acid sequence that has at least 90% identity to any one of SEQ ID NOs: 74, 82, 87, 106, and 107.

[0522] 203. The method of any one of embodiments 191 to 198, wherein the RGN of the fusion protein has an amino acid sequence that has at least 95% identity to any one of SEQ ID NOs: 74, 82, 87, 106, and 107.

[0523] 204. The method of any one of embodiments 191 to 198, wherein the RGN of the fusion protein has an amino acid sequence of any one of SEQ ID NOs: 74, 82, 87, 106, and 107.

[0524] 205. The method of any one of embodiments 191 to 198, wherein the RGN of the fusion protein is RGN nickase.

[0525] 206. The method of embodiment 205, wherein the RGN nickase has an inactive RuvC domain.

[0526] 207. The method of embodiment 205 or 206, wherein the RGN nickase has an amino acid sequence that has at least 90% identity to any one of SEQ ID NOs: 75 and 88 to 98.

[0527] 208. The method of embodiment 205 or 206, wherein the RGN nickase has an amino acid sequence that has at least 95% identity to any one of SEQ ID NOs: 75 and 88 to 98.

[0528] 209. The method of embodiment 205 or 206, wherein the RGN nickase is any one of SEQ ID NOs: 75 and 88 to 98.

[0529] 210. The method of any one of embodiments 191-198, wherein the RGN of the fusion protein is a nuclease-inactive RGN.

[0530] 211. The method of any one of embodiments 191 to 210, wherein the fusion protein comprises one or more nuclear localization signals.

[0531] 212. The method of any one of embodiments 191 to 211, wherein the deaminase is fused to the amino terminus of the DNA-binding polypeptide.

[0532] 213. The method of any one of embodiments 191 to 211, wherein the deaminase is fused to the carboxyl terminus of the DNA-binding polypeptide.

[0533] 214. The method of any one of embodiments 191 to 213, wherein the fusion protein further comprises a linker sequence between the DNA-binding polypeptide and the deaminase.

[0534] 215. The method of embodiment 214, wherein the linker sequence has the amino acid sequence shown as SEQ ID NO: 78 or 79.

[0535] 216. The method of any one of embodiments 191 to 215, wherein the fusion protein further comprises a uracil stabilizing protein (USP).

[0536] 217. The method of embodiment 216, wherein the USP has the sequence shown as SEQ ID NO: 81.

[0537] 218. The method of embodiment 216 or 217, wherein the fusion protein further comprises a linker sequence between the USP and the deaminase or the DNA-binding polypeptide.

[0538] 219. The method of embodiment 218, wherein the linker sequence between the USP and the deaminase or the DNA-binding polypeptide has the amino acid sequence shown as SEQ ID NO: 120.

[0539] 220. The method of embodiment 191, wherein the fusion protein has an amino acid sequence set forth as any one of SEQ ID NOs: 67, 68, 146, and 147.

[0540] 221. The method of any one of embodiments 191 to 220, wherein the target DNA sequence is a eukaryotic target DNA sequence.

[0541] 222. The method of any one of embodiments 191 to 221, wherein the target DNA sequence is located adjacent to a protospacer adjacent motif (PAM).

[0542] 223. The method of any one of embodiments 191 to 222, wherein the target DNA molecule is intracellular.

[0543] 224. The method of embodiment 223, wherein the cell is a eukaryotic cell.

[0544] 225. The method of embodiment 224, wherein the eukaryotic cell is a plant cell.

[0545] 226. The method of embodiment 224, wherein the eukaryotic cell is a mammalian cell.

[0546] 227. The method of embodiment 226, wherein the mammalian cell is a human cell.

[0547] 228. The method of embodiment 227, wherein the human cells are immune cells.

[0548] 229. The method of embodiment 228, wherein the immune cells are stem cells.

[0549] 230. The method of embodiment 229, wherein the stem cells are induced pluripotent stem cells.

[0550] 231. The method of embodiment 224, wherein the eukaryotic cell is an insect cell.

[0551] 232. The method of embodiment 223, wherein the cell is a prokaryotic cell.

[0552] 233. The method of any one of embodiments 223 to 232, further comprising selecting cells containing the modified DNA molecule.

[0553] 234. A cell comprising the modified target DNA according to the method of embodiment 233.

[0554] 235. The cell according to embodiment 234, which is a eukaryotic cell.

[0555] 236. The cell according to embodiment 235, wherein the eukaryotic cell is a plant cell.

[0556] 237. A plant comprising a cell according to embodiment 236.

[0557] 238. A seed comprising a cell according to embodiment 236.

[0558] 239. The cell according to embodiment 235, wherein the eukaryotic cell is a mammalian cell.

[0559] 240. The cell of embodiment 239, wherein the mammalian cell is a human cell.

[0560] 241. The cell of embodiment 240, wherein the human cell is an immune cell.

[0561] 242. The cell of embodiment 241, wherein the immune cell is a stem cell.

[0562] 243. The cell of embodiment 242, wherein the stem cell is an induced pluripotent stem cell.

[0563] 244. The cell according to embodiment 235, wherein the eukaryotic cell is an insect cell.

[0564] 245. The cell according to embodiment 234, which is a prokaryotic cell.

[0565] 246. A pharmaceutical composition comprising a cell according to any one of embodiments 293 to 243 and a pharma- ceutically acceptable carrier.

[0566] 247. A method for producing a genetically modified cell in which a causative mutation for a genetic disease has been corrected, comprising: a) a fusion protein or a polynucleotide encoding said fusion protein, wherein said fusion protein comprises an RNA-guided nuclease (RGN) and a deaminase, said deaminase comprising: i) an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 2 and 7 to 12; ii) an amino acid sequence having at least 95% identity with SEQ ID NO: 4 or 6 having an amino acid sequence selected from the group consisting of: b) one or more guide RNAs (gRNAs) capable of hybridizing to the target DNA sequence, or polynucleotides encoding said gRNAs; into said cell, This allows the fusion protein and gRNA to target the genomic locus of the causative mutation, thereby modifying the genomic sequence and eliminating the causative mutation.

[0567] 248. The method of embodiment 247, wherein the polynucleotide encoding the fusion protein is operably linked to a promoter active in the cell.

[0568] 249. The method of embodiment 247 or 248, wherein the polynucleotide encoding the gRNA is operably linked to a promoter active in the cell.

[0569] 250. The method of any one of embodiments 247 to 249, wherein the deaminase has an amino acid sequence that has at least 95% identity to any one of SEQ ID NOs: 2 and 7 to 12.

[0570] 251. The method according to any one of embodiments 247 to 249, wherein the deaminase has an amino acid sequence that has 100% identity to any one of SEQ ID NOs: 2, 4, and 6 to 12.

[0571] 252. The method of any one of embodiments 247 to 251, wherein the RGN of the fusion protein is a type II CRISPR-Cas polypeptide.

[0572] 253. The method of any one of embodiments 247 to 251, wherein the RGN of the fusion protein is a V-type CRISPR-Cas polypeptide.

[0573] 254. The method of any one of embodiments 247 to 253, wherein the RGN of the fusion protein has an amino acid sequence that has at least 90% sequence identity to any one of the RGN sequences in Table 1.

[0574] 255. The method of any one of embodiments 247 to 253, wherein the RGN of the fusion protein has an amino acid sequence that has at least 95% sequence identity to any one of the RGN sequences in Table 1.

[0575] 256. The method of any one of embodiments 247-253, wherein the RGN of the fusion protein has an amino acid sequence of any one of the RGN sequences in Table 1.

[0576] 257. The method of any one of embodiments 247 to 253, wherein the RGN of the fusion protein is RGN nickase.

[0577] 258. The method of embodiment 257, wherein the RGN nickase has an inactive RuvC domain.

[0578] 259. The method of embodiment 257 or 258, wherein the RGN nickase has an amino acid sequence that has at least 90% identity to any one of SEQ ID NOs: 75 and 88 to 98.

[0579] 260. The method of embodiment 257 or 258, wherein the RGN nickase has an amino acid sequence that has at least 95% identity to any one of SEQ ID NOs: 75 and 88 to 98.

[0580] 261. The method of embodiment 257 or 258, wherein the RGN nickase is any one of SEQ ID NOs: 75 and 88 to 98.

[0581] 262. The method of any one of embodiments 247 to 253, wherein the RGN of the fusion protein is a nuclease-inactive RGN.

[0582] 263. The method of any one of embodiments 247 to 262, wherein the fusion protein comprises one or more nuclear localization signals.

[0583] 264. The method of any one of embodiments 247 to 263, wherein the deaminase is fused to the amino terminus of the DNA-binding polypeptide.

[0584] 265. The method of any one of embodiments 247 to 263, wherein the deaminase is fused to the carboxyl terminus of the DNA-binding polypeptide.

[0585] 266. The method of any one of embodiments 247 to 265, wherein the fusion protein further comprises a linker sequence between the DNA-binding polypeptide and the deaminase.

[0586] 267. The method of embodiment 266, wherein the linker sequence has the amino acid sequence shown as SEQ ID NO: 78 or 79.

[0587] 268. The method of any one of embodiments 247 to 267, wherein the fusion protein further comprises a uracil stabilizing protein (USP).

[0588] 269. The method of embodiment 268, wherein the USP has the sequence shown as SEQ ID NO: 81.

[0589] 270. The method of embodiment 268 or 269, wherein the fusion protein further comprises a linker sequence between the USP and the deaminase or DNA-binding polypeptide.

[0590] 271. The method of embodiment 270, wherein the linker sequence between the USP and the deaminase or the DNA-binding polypeptide has the amino acid sequence shown as SEQ ID NO: 120.

[0591] 272. The method of any one of embodiments 247 to 249, wherein the fusion protein has an amino acid sequence set forth as any one of SEQ ID NOs: 67, 68, 146, and 147.

[0592] 273. The method of any one of embodiments 247 to 272, wherein the genomic modification comprises introducing a C>T mutation of at least one nucleotide into the target DNA sequence.

[0593] 274. The method of any one of embodiments 247 to 272, wherein the genomic modification comprises introducing a C>G mutation of at least one nucleotide into the target DNA sequence.

[0594] 275. The method of any one of embodiments 247 to 274, wherein the cell is an animal cell.

[0595] 276. The method of embodiment 275, wherein the animal cell is a mammalian cell.

[0596] 277. The method of embodiment 276, wherein the cells are derived from a dog, cat, mouse, rat, rabbit, horse, sheep, goat, cow, pig, or human.

[0597] 278. The method of any one of embodiments 247 to 277, wherein the correction of the causative mutation comprises correction of a nonsense mutation.

[0598] 279. The method of any one of embodiments 247-278, wherein the genetic disease is a disease listed in Table 23.

[0599] 280. The method of embodiment 279, wherein the gRNA further comprises a spacer sequence targeting any one of SEQ ID NOs: 122-144, or a complement thereof.

[0600] 281. a) a fusion protein comprising a DNA-binding polypeptide and a cytosine deaminase, or a nucleic acid molecule encoding said fusion protein; b) a second cytosine deaminase, or a nucleic acid molecule encoding said second deaminase, i) an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 2 and 7 to 12; ii) an amino acid sequence having at least 95% identity with SEQ ID NO: 4 or 6 having an amino acid sequence selected from the group consisting of A composition comprising:

[0601] 282. The composition of embodiment 281, wherein the second cytosine deaminase has a sequence that is at least 95% identical to any one of SEQ ID NOs: 2 and 7-12.

[0602] 283. The composition of embodiment 281, wherein the second cytosine deaminase has a sequence that is 100% identical to any one of SEQ ID NOs: 2, 4, and 6-12.

[0603] 284. The first cytosine deaminase is a) an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 2 and 7 to 12; b) an amino acid sequence having at least 95% identity with SEQ ID NO: 4 or 6 The composition of any one of embodiments 281 to 283, having an amino acid sequence selected from the group consisting of:

[0604] 285. The composition according to any one of embodiments 281 to 284, wherein the first cytosine deaminase has a sequence that is at least 95% identical to any one of SEQ ID NOs: 2 and 7 to 12.

[0605] 286. The composition according to any one of embodiments 281 to 284, wherein the first cytosine deaminase has a sequence that is 100% identical to any one of SEQ ID NOs: 2, 4, and 6 to 12.

[0606] 287. The composition of any one of embodiments 281 to 286, wherein the DNA-binding polypeptide is a meganuclease, a zinc finger fusion protein, or a TALEN; or a variant of a meganuclease, a zinc finger fusion protein, or a TALEN, and the nuclease activity is reduced or inhibited.

[0607] 288. The composition of any one of embodiments 281 to 286, wherein the DNA-binding polypeptide is an RNA-guided DNA-binding polypeptide.

[0608] 289. The composition of embodiment 288, wherein the RNA-guided DNA-binding polypeptide is an RNA-guided nuclease (RGN) polypeptide.

[0609] 290. The composition of embodiment 289, wherein the RGN is RGN nickase.

[0610] 291. The composition of embodiment 289, wherein the RGN is a nuclease-inactive RGN.

[0611] 292. A vector comprising a nucleic acid molecule encoding a fusion protein and a nucleic acid molecule encoding a second cytosine deaminase, wherein the fusion protein comprises a DNA-binding polypeptide and a first cytosine deaminase, and the second cytosine deaminase is a) an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 2 and 7 to 12; b) having an amino acid sequence selected from the group consisting of amino acid sequences having at least 95% identity to SEQ ID NO: 4 or 6).

[0612] 293. The vector of embodiment 292, wherein the second cytosine deaminase has a sequence that is at least 95% identical to any one of SEQ ID NOs: 2 and 7 to 12.

[0613] 294. The vector of embodiment 292, wherein the second cytosine deaminase has a sequence that is 100% identical to any one of SEQ ID NOs: 2, 4, and 6 to 12.

[0614] 295. The first cytosine deaminase is a) an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 2 and 7 to 12; b) an amino acid sequence having at least 95% identity with SEQ ID NO: 4 or 6 295. The vector of any one of embodiments 292 to 294, having an amino acid sequence selected from the group consisting of:

[0615] 296. The vector according to any one of embodiments 292 to 294, wherein the first cytosine deaminase has a sequence that is at least 95% identical to any one of SEQ ID NOs: 2 and 7 to 12.

[0616] 297. The vector according to any one of embodiments 292 to 294, wherein the first cytosine deaminase has a sequence that is 100% identical to any one of SEQ ID NOs: 2, 4, and 6 to 12.

[0617] 298. The vector of any one of embodiments 292 to 297, wherein the DNA-binding polypeptide is a meganuclease, a zinc finger fusion protein, or a TALEN; or a variant of a meganuclease, a zinc finger fusion protein, or a TALEN, and the nuclease activity is reduced or inhibited.

[0618] 299. The vector of any one of embodiments 292 to 297, wherein the DNA-binding polypeptide is an RNA-guided DNA-binding polypeptide.

[0619] 300. The vector of embodiment 299, wherein the RNA-guided DNA-binding polypeptide is an RNA-guided nuclease (RGN) polypeptide.

[0620] 301. The vector of embodiment 300, wherein the RGN is RGN nickase.

[0621] 302. The vector of embodiment 300, wherein the RGN is a nuclease-inactive RGN.

[0622] 303. A cell comprising a vector according to any one of embodiments 292 to 302.

[0623] 304. a) a fusion protein comprising a DNA-binding polypeptide and a first cytosine deaminase; or a nucleic acid molecule encoding said fusion protein; b) a second cytosine deaminase, or a nucleic acid molecule encoding said second deaminase, i) an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 2 and 7 to 12; ii) an amino acid sequence having at least 95% identity with SEQ ID NO: 4 or 6 having an amino acid sequence selected from the group consisting of Cells containing

[0624] 305. The cell of embodiment 304, wherein the second cytosine deaminase has a sequence that is at least 95% identical to any one of SEQ ID NOs: 2 and 7-12.

[0625] 306. The cell of embodiment 304, wherein the second cytosine deaminase has a sequence that is 100% identical to any one of SEQ ID NOs: 2, 4, and 6-12.

[0626] 307. The first cytosine deaminase is a) an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 2 and 7 to 12; b) an amino acid sequence having at least 95% identity with SEQ ID NO: 4 or 6 307. The cell of any one of embodiments 304 to 306, having an amino acid sequence selected from the group consisting of:

[0627] 308. The cell according to any one of embodiments 304 to 306, wherein the first cytosine deaminase has a sequence that is at least 95% identical to any one of SEQ ID NOs: 2 and 7 to 12.

[0628] 309. The cell according to any one of embodiments 304 to 306, wherein the first cytosine deaminase has a sequence that is 100% identical to any one of SEQ ID NOs: 2, 4, and 6 to 12.

[0629] 310. The cell of any one of embodiments 304 to 309, wherein the DNA-binding polypeptide is a meganuclease, a zinc finger fusion protein, or a TALEN; or a variant of a meganuclease, a zinc finger fusion protein, or a TALEN, and the nuclease activity is reduced or inhibited.

[0630] 311. The cell of any one of embodiments 304 to 309, wherein the DNA-binding polypeptide is an RNA-guided DNA-binding polypeptide.

[0631] 312. The cell of embodiment 311, wherein the RNA-guided DNA-binding polypeptide is an RNA-guided nuclease (RGN) polypeptide.

[0632] 313. The cell of embodiment 312, wherein the RGN is RGN nickase.

[0633] 314. The cell of embodiment 312, wherein the RGN is a nuclease-inactive RGN.

[0634] 315. A pharmaceutical composition comprising a composition according to any one of embodiments 281 to 291, a vector according to any one of embodiments 292 to 302, or a cell according to any one of embodiments 303 to 314, together with a pharma- ceutical acceptable carrier.

[0635] 316. A method for treating a disease, comprising administering to a subject in need thereof a fusion protein according to any one of embodiments 33 to 63, a nucleic acid molecule according to any one of embodiments 64 to 101, a vector according to any one of embodiments 102 to 105 and 292 to 302, a cell according to any one of embodiments 113 to 118, 239 to 243 and 303 to 314, a system according to any one of embodiments 130 to 171, a ribonucleoprotein complex according to embodiment 172, a composition according to any one of embodiments 281 to 291 or a pharmaceutical composition according to any one of embodiments 124, 186, 246 and 315.

[0636] 317. The method of embodiment 316, wherein the disease is associated with a causative mutation and the pharmaceutical composition corrects the causative mutation.

[0637] 318. The method of embodiment 316 or 317, wherein the disease is a disease listed in Table 23.

[0638] 319. Use of a fusion protein according to any one of embodiments 33 to 63, a nucleic acid molecule according to any one of embodiments 64 to 101, a vector according to any one of embodiments 102 to 105 and 292 to 302, a cell according to any one of embodiments 113 to 118, 239 to 243 and 303 to 314, a system according to any one of embodiments 130 to 171, a ribonucleoprotein complex according to embodiment 172 or a composition according to any one of embodiments 281 to 291 for treating a disease in a subject.

[0639] 320. The use according to embodiment 319, wherein the disease is associated with a causative mutation and the treatment comprises correcting the causative mutation.

[0640] 321. The use according to embodiment 319 or 320, wherein the disease is a disease listed in Table 23.

[0641] 322. Use of a fusion protein according to any one of embodiments 33 to 63, a nucleic acid molecule according to any one of embodiments 64 to 101, a vector according to any one of embodiments 102 to 105 and 292 to 302, a cell according to any one of embodiments 113 to 118, 239 to 243 and 303 to 314, a system according to any one of embodiments 130 to 171, a ribonucleoprotein complex according to embodiment 172 or a composition according to any one of embodiments 281 to 291 for the production of a drug useful for the treatment of a disease.

[0642] 323. The use according to embodiment 322, wherein the disease is associated with a causative mutation and an effective amount of the drug corrects the causative mutation.

[0643] 324. The use according to embodiment 322 or 323, wherein the disease is a disease listed in Table 23.

[0644] The following examples are offered by way of illustration and not by way of limitation.

[0645] experiment

[0646] Example 1: Demonstration of C base editing in mammalian cells

[0647] The deaminases shown as SEQ ID NOs: 1, 3, and 5 are truncated from both ends, and the truncated deaminases are shown as SEQ ID NOs: 2, 4, and 6. The bacterial cytosine deaminase RGN is shown as SEQ ID NOs: 7-12.

[0648] [Table 2]

[0649] To determine whether the deaminases in Table 2 can perform cytosine base editing in mammalian cells, each deaminase was functionally fused to RGN nickase to generate a fusion protein. Residues predicted to deactivate the RuvC domain of RGN APG07433.1 (SEQ ID NO: 74) (described in PCT Publication No. WO 2019 / 236566, incorporated herein by reference) were identified, and RGN was modified into a nickase variant (nAPG07433.1; SEQ ID NO: 75). The nickase variant of RGN is referred to herein as "nRGN". It should be understood that any nickase variant of RGN can be used to generate the fusion protein of the present invention.

[0650] The deaminase and the nRGN nucleotide sequence, codon-optimized for mammalian expression, were synthesized as fusion proteins with an N-terminal nuclear localization tag and cloned into the pTwist CMV (Twist Biosciences) expression plasmid. Starting from the amino terminus, each fusion protein contains an SV40 NLS (SEQ ID NO: 76), a 3×FLAG tag (SEQ ID NO: 77) operably linked to its C-terminus, a deaminase operably linked to its C-terminus, a peptide linker (L16 or L32; shown as SEQ ID NO: 78 or 79, respectively) operably linked to its C-terminus, an nRGN (e.g., nAPG07433.1, SEQ ID NO: 75) operably linked to its C-terminus, and finally a nucleoplasmin NLS (SEQ ID NO: 80) operably linked to its C-terminus. Table 3 shows the fusion proteins that were made and tested for activity. All fusion proteins contain at least one NLS and a 3×FLAG tag, as described above. The APG05840.1-nAPG07433.1-USP2 fusion protein in Table 3 further comprises a uracil stabilizing protein USP2 (shown as SEQ ID NO: 81) between the nRGN and the nucleoplasmin NLS. The fusion protein also comprises a peptide linker between nAPG07433.1 and USP2 having the sequence shown as SEQ ID NO: 120.

[0651] [Table 3]

[0652] Expression plasmids containing expression cassettes encoding sgRNAs were also generated. The human genomic target sequences and the sgRNA sequences directing the fusion proteins to the genomic targets are shown in Table 4.

[0653] [Table 4]

[0654] 500 ng of plasmids containing expression cassettes with the coding sequence of each fusion protein shown in Table 3 and 500 ng of plasmids containing expression cassettes encoding sgRNAs shown in Table 4 were simultaneously transfected into HEK293FT cells at 75–90% confluency in 24-well plates using Lipofectamine 2000 reagent (Life Technologies). The cells were then incubated at 37°C for 72 h. After incubation, genomic DNA was extracted using NucleoSpin 96 Tissue (Macherey-Nagel) according to the manufacturer's protocol. Genomic regions flanking the targeted genomic sites were amplified by PCR using primers from Table 4, and the products were purified using ZR-96 DNA Clean and Concentrator (Zymo Research) according to the manufacturer's protocol. The purified PCR products were then sent to an Illumina MiSeq (2 × 250) for next-generation sequencing. Results were analyzed for the formation of INDELs or the introduction of specific cytosine mutations.

[0655] Table 5 shows all cytosine base edits for each combination of fusion proteins from Table 3 and guide RNAs from Table 4. Tables 6-11 show specific nucleotide mutation profiles for selected representative samples. The position of each nucleotide in the target sequence was determined. For example, "C17" indicates a cytosine at position 17 of the target sequence. The position of each nucleotide in the target sequence was determined by placing the first nucleotide closest to the PAM in the target sequence at position 1, and the position number increases as the position number moves away from the PAM sequence in the 3' direction. Tables 6-11 also show the percentage of cytosines that were changed to which nucleotides. For example, Table 6 shows that for the APG30127-nAPG07433.1 fusion protein, cytosines at position 17 were mutated to thymines at a rate of 0.2%.

[0656] [Table 5-1] [Table 5-2]

[0657] [Table 6]

[0658] APG30127 showed predominantly C>G conversion at position C17.

[0659] [Table 7]

[0660] APG30127 exhibits C>T transversions at multiple positions, including C10, C12, and C17. There are also C>G and C>A transversions at position C17.

[0661] [Table 8]

[0662] APG30129 exhibits C>T transitions at positions C10, C12, and C17.

[0663] [Table 9]

[0664] APG30125 shows C>T transversions at positions C10, C12, and C17, with fewer C>G and C>A transversions at all positions than the APG30129 and APG30127 samples.

[0665] [Table 10]

[0666] A truncated APG05840 (APG05840.1) with a 16 amino acid linker (L16) and a uracil stabilizing protein (USP2) was examined. This construct showed high levels of specific C>T editing at several positions within the target SGN000169, including C9, C13, C15, C20, and C23, demonstrating that truncated deaminases and shorter linkers can still be used to generate site-specific single nucleotide edits.

[0667] [Table 11]

[0668] We investigated a truncated version of APG05840 (APG05840.1) with a 16 amino acid linker (L16) and a uracil stabilizing protein (USP2), which showed high levels of specific C>T editing at several positions within the target SGN000173, including C7, C8, C10, and C17.

[0669] Example 2: RGN-independent cytosine deaminase-driven off-target effect assay

[0670] To determine whether cytosine deaminase has any mutational effects on ssDNA, we performed an off-target mutation assay independent of RGN. We identified residues predicted to deactivate the RuvC and HNH domains of RGN APG09298 (SEQ ID NO: 82) (described in PCT Publication No. WO2021 / 217002, which is incorporated by reference in its entirety) and modified RGN to a dead variant (dAPG09298; SEQ ID NO: 83).

[0671] The dRGN nucleotide sequence, codon-optimized for expression, was synthesized as a fusion protein with an N-terminal nuclear localization tag and cloned into the pTwist CMV (Twist Biosciences) expression plasmid. Starting from the amino terminus, the dRGN fusion protein contains an SV40 NLS (SEQ ID NO: 76), a 3×FLAG tag (SEQ ID NO: 77) operably linked to its C-terminus, a dRGN (e.g., dAPG09298, SEQ ID NO: 83) operably linked to its C-terminus, and finally a nucleoplasmin NLS (SEQ ID NO: 80) operably linked to its C-terminus, constituting NLS-dAPG09298-NLS (SEQ ID NO: 84). This construct was used to create ssDNA in an R-loop at a position unrelated to the target sequence edited by the cytosine deaminase base editor.

[0672] An expression plasmid was generated containing an expression cassette encoding the sgRNA. The human genomic target sequence and the sgRNA sequence directing the fusion protein to the genomic target are shown in Table 4.

[0673] [Table 12]

[0674] 500 ng of plasmid containing an expression cassette containing the coding sequence of the fusion protein shown in Table 3, 500 ng of plasmid containing an expression cassette encoding the sgRNA shown in Table 4, 500 ng of plasmid containing an expression cassette containing the coding sequence of NLS-dAPG09298-NLS, and 500 ng of plasmid containing an expression cassette encoding the sgRNA for dAPG09298 shown in Table 12 were simultaneously transfected into HEK293FT cells at 75-90% confluency in 24-well plates using Lipofectamine 2000 reagent (Life Technologies). The cells were then incubated at 37 °C for 72 h. After incubation, genomic DNA was extracted using NucleoSpin 96 Tissue (Macherey-Nagel) according to the manufacturer's protocol. Genomic regions flanking the targeted genomic sites were amplified by PCR using primers in Table 4 or Table 12, and the products were purified using a ZR-96 DNA Clean and Concentrator (Zymo Research) according to the manufacturer's protocol. The purified PCR products were then sent to an Illumina MiSeq (2x250) for next generation sequencing. Results were analyzed for INDEL formation or specific cytosine mutations. On-target results were those identified by the amplicons in Table 4. Off-target results independent of RGN were those identified by the amplicons in Table 12.

[0675] [Table 13]

[0676] The putative on-target sites in each sample showed high levels of cytosine-specific mutations. The off-target site bound by dAPG09298 in SGN001165 showed no mutated reads in five of six samples. One target examined showed a low mutation rate at the off-target position, with 0.99% of reads carrying mutations. These could be RGN-independent, deaminase-driven mutational effects, but could also represent sequencing errors due to low sample representation.

[0677] Example 3: Demonstration of C>G base editing in mammalian cells

[0678] These studies assessed whether the orientation of the deaminase and RGN relative to each other in the fusion protein influenced the type of base editing caused by the resulting C base editor.

[0679] Residues predicted to deactivate the RuvC domain (SEQ ID NO: 74; PCT Publication No. WO2019 / 236566, incorporated herein by reference) were identified and RGN was modified into a nickase variant (nAPG07433.1; SEQ ID NO: 75).

[0680] The deaminases (APG09980 and APG05840; shown as SEQ ID NOs: 1 and 3, respectively) and the nAPG07433.1 nucleotide sequence, codon-optimized for expression, were synthesized as fusion proteins with an N-terminal nuclear localization tag and cloned into the pTwist CMV (Twist Biosciences) expression plasmid. Starting from the amino terminus, each fusion protein contains an SV40 NLS (SEQ ID NO: 76), a 3×FLAG tag (SEQ ID NO: 77) operably linked to its C-terminus, an nRGN-deaminase operably linked to its C-terminus, a deaminase-nRGN-USP2 or deaminase-nRGN fusion protein connected by a peptide linker (SEQ ID NO: 79), and finally a nucleoplasmin NLS (SEQ ID NO: 80) operably linked to its C-terminus. Table 14 shows the fusion proteins that were made and tested for activity. All fusion proteins contain at least one NLS and a 3×FLAG tag, as described above. The fusion proteins APG09980-nAPG07433.1-USP2 and APG05840.1-nAPG07433.1-USP2 in Table 14 further comprise a uracil stabilizing protein USP2 (shown as SEQ ID NO: 81) between nRGN and the nucleoplasmin NLS. The fusion proteins APG09980-nAPG07433.1-USP2 and APG05840.1-nAPG07433.1-USP2 also comprise a peptide linker having the sequence shown as SEQ ID NO: 120 between nAPG07433.1 and USP2.

[0681] [Table 14]

[0682] Expression plasmids containing expression cassettes encoding sgRNAs were also generated. The human genomic target sequences and the sgRNA sequences directing the fusion proteins to the genomic targets are shown in Table 15.

[0683] [Table 15]

[0684] 500 ng of plasmid containing an expression cassette with the coding sequence of the fusion protein shown in Table 14 and 500 ng of plasmid containing an expression cassette encoding the sgRNA shown in Table 15 were simultaneously transfected into HEK293FT cells at 75-90% confluency in 24-well plates using Lipofectamine 2000 reagent (Life Technologies). The cells were then incubated at 37°C for 72 hours. After incubation, genomic DNA was extracted using NucleoSpin 96 Tissue (Macherey-Nagel) according to the manufacturer's protocol. Genomic regions flanking the targeted genomic sites were amplified by PCR using the primers in Table 15, and the products were purified using ZR-96 DNA Clean and Concentrator (Zymo Research) according to the manufacturer's protocol. The purified PCR products were then sent to an Illumina MiSeq (2x250) for next-generation sequencing. Results were analyzed for INDEL formation or specific cytosine mutations.

[0685] Tables 16-20 show cytosine base editing for each combination of fusion protein from Table 14 and guide RNA from Table 15. The position of each nucleotide in the target sequence was determined. "C16" indicates, for example, a cytosine at position 16 of the target sequence. The position of each nucleotide in the target sequence was determined by placing the first nucleotide in the target sequence closest to the PAM at position 1, with the position number increasing the further away from the PAM sequence in the 3' direction. Tables 16-20 also show the percentage of cytosines that were changed to which nucleotide. For example, Table 16 shows that for the APG05840-nAPG07433.1-USP2 fusion protein, cytosines at position 16 were mutated to thymines at a rate of 11%.

[0686] [Table 16]

[0687] In the orientation of the deaminase N-terminally within the entire construct APG05840-nAPG07433.1-USP2, high levels of specific C>T conversions are evident at positions C16 and C21 within SGN000928.

[0688] [Table 17]

[0689] In the overall construct APG05840-nAPG07433.1-USP2, with the deaminase oriented N-terminally, high levels of specific C>T conversions are evident at positions C17 and C22 within SGN000930. Some C>G conversions are evident at position C17.

[0690] [Table 18]

[0691] When the orientation of the nickase-tethered deaminase is reversed and the deaminase is tethered to the C-terminus, the predominant editing outcome is a C>G conversion at position C16 within the target SGN000928. Very few C>T conversions are evident compared to the N-terminal construct.

[0692] [Table 19]

[0693] When the deaminase is tethered to the C-terminus of the nickase, the primary editing outcome is a C>G conversion at position C17 within the target SGN000930.

[0694] [Table 20]

[0695] With the second deaminase module, APG09980, the same trend is evident with the predominant mutational outcome being a C>G conversion at position C17 when tethered to the C-terminus of the nickase.

[0696] [Table 21]

[0697] When APG05840 is tethered to the N-terminus of nAPG07433.1, the primary mutational outcome is a C>G conversion at position C17 using guide SGN000930.

[0698] [Table 22]

[0699] Data in this table are averages from multiple editing experiments. The fraction of mutated reads is an estimate of the base editing rate in each sample. The fraction of reads with deletions is an estimate of the deletion rate in the sample. C-terminal fusions of APG09980 to nAPG07433.1 have a lower deletion rate than N-terminal fusions with and without USP.

[0700] APG05840-nAPG07433.1-USP2 showed predominantly C>T transitions at positions C17 in guide SGN000930 and C16 and C21 in SGN000928. nAPG07433.1-APG09980 and nAPG07433.1-APG05840 showed predominantly C>G transitions at these same positions. All constructs showed editing within the same window.

[0701] Example 4: Targeted base editing to correct disease-causing mutations

[0702] A database of clinical variants was obtained from the NCBI ClinVar database available at the NCBI ClinVar website through the world wide web. Pathogenic single nucleotide polymorphisms (SNPs) were identified from this list. Genomic locus information was used to identify CRISPR targets within the regions overlapping or surrounding each SNP. Table 23 lists a set of SNPs that can be corrected using base editing combined with RGN (e.g., APG07433.1 or its variants) to target causative mutations. Only one common name for each disease is listed in Table 23 below. "RS#" corresponds to the RS accession number in the SNP database at the NCBI website. The "Name" column contains the locus identifier, the gene name, the position of the mutation within the gene, and the change resulting from the mutation.

[0703] [Table 23]

[0704] Example 5: Demonstration of gene editing activity in plant cells

[0705] The base editing activity of the RGN-deaminase fusion protein of the present invention is demonstrated in plant cells using a protocol modified from Li et al., 2013 (Nat. Biotech. 31:688-691). Briefly, an expression vector containing an expression cassette capable of expressing an RGN-deaminase fusion protein operably linked to an SV40 nuclear localization signal (SEQ ID NO: 76) in plant cells and a second expression cassette encoding a guide RNA targeting one or more sites adjacent to a suitable PAM sequence in a plant PDS gene is introduced into Nicotiana benthamiana mesophyll protoplasts using PEG-mediated transformation. The transformed protoplasts are incubated in the dark for up to 36 hours. Genomic DNA is isolated from the protoplasts using the DNeasy Plant Mini Kit (Qiagen). Genomic regions adjacent to the RGN target sites are amplified by PCR, the products are purified, and the purified PCR products are analyzed by next generation sequencing on an Illumina MiSeq. Typically, 100,000 250bp paired-end reads (2 x 100,000 reads) are generated per amplicon. Reads are analyzed using CRISPResso (Pinello, et al. 2016 Nature Biotech, 34:695-697) and editing rates are calculated. Output alignments are analyzed for INDEL information or for the introduction of specific cytosine mutations.

Claims

1. a) an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 2 and 7 to 12; b) an amino acid sequence having at least 95% identity to SEQ ID NO: 4 or 6; and c) A polypeptide having deaminase activity, comprising an amino acid sequence selected from the group consisting of the amino acid sequences set forth as any one of SEQ ID NOs: 2, 4, and 6-12.

2. a polynucleotide encoding a deaminase polypeptide, the deaminase comprising: a) a nucleotide sequence having at least 80% identity to any one of SEQ ID NOs: 114-119; b) a nucleotide sequence having at least 95% identity to any one of SEQ ID NOs: 109, 111, and 113; c) a nucleotide sequence encoding an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 2 and 7 to 12; d) a nucleotide sequence encoding an amino acid sequence having at least 95% identity to SEQ ID NO: 4 or 6; e) a nucleotide sequence having at least 90% identity to any one of SEQ ID NOs: 114-119; f) a nucleotide sequence set forth as any one of SEQ ID NOs: 109, 111, and 113-119; and g) A nucleic acid molecule encoded by a nucleotide sequence selected from the group consisting of the nucleotide sequences encoding the amino acid sequence set forth as any one of SEQ ID NOs: 2, 4, and 6-12.

3. The nucleic acid molecule of claim 2 , further comprising a heterologous promoter operably linked to the polypeptide.

4. A vector comprising the nucleic acid molecule of claim 2.

5. a DNA binding polypeptide; a) an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 2 and 7 to 12; b) an amino acid sequence having at least 95% identity to SEQ ID NO: 4 or 6; and c) A fusion protein comprising a deaminase having an amino acid sequence selected from the group consisting of the amino acid sequences set forth as any one of SEQ ID NOs: 2, 4, and 6-12.

6. the DNA binding polypeptide a) a meganuclease, a zinc finger fusion protein, or a TALEN; or a variant of a meganuclease, a zinc finger fusion protein, or a TALEN, wherein said nuclease activity is reduced or inhibited; b) RNA-guided DNA-binding polypeptides; c) an RNA-guided nuclease (RGN) polypeptide; d) Type II CRISPR-Cas RGN polypeptide; e) a CRISPR-Cas RGN polypeptide of type V; f) RGN nickase; and g) a nuclease-inactive RGN.

7. The RGN nickase is a) an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 75 and 88-98; and b) the fusion protein of claim 6, having an amino acid sequence selected from the group consisting of the amino acid sequences set forth as any one of SEQ ID NOs: 75 and 88 to 98.

8. The fusion protein according to any one of claims 5 to 7, further comprising at least one nuclear localization signal (NLS).

9. The fusion protein of any one of claims 5 to 8, further comprising a uracil stabilizing protein (USP).

10. The fusion protein of claim 9, wherein the USP has the sequence shown as SEQ ID NO:

81.

11. The fusion protein of claim 5 having an amino acid sequence of any one of SEQ ID NOs: 67, 68, 146, and 147.

12. The polynucleotide encoding a fusion protein comprising a DNA-binding polypeptide and a deaminase, the deaminase comprising: a) a nucleotide sequence having at least 80% identity to any one of SEQ ID NOs: 114-119; b) a nucleotide sequence having at least 95% identity to any one of SEQ ID NOs: 109, 111, and 113; c) a nucleotide sequence encoding an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 2 and 7 to 12; and d) a nucleotide sequence encoding an amino acid sequence having at least 95% identity to SEQ ID NO: 4 or 6; e) a nucleotide sequence having at least 90% identity to any one of SEQ ID NOs: 114-119; f) a nucleotide sequence set forth as any one of SEQ ID NOs: 109, 111, and 113-119; and g) A nucleic acid molecule encoded by a nucleotide sequence selected from the group consisting of the nucleotide sequences encoding the amino acid sequence set forth as any one of SEQ ID NOs: 2, 4, and 6-12.

13. the DNA binding polypeptide a) a meganuclease, a zinc finger fusion protein, or a TALEN; or a variant of a meganuclease, a zinc finger fusion protein, or a TALEN, wherein said nuclease activity is reduced or inhibited; b) RNA-guided DNA-binding polypeptides; c) an RNA-guided nuclease (RGN) polypeptide; d) Type II CRISPR-Cas RGN polypeptide; e) a CRISPR-Cas RGN polypeptide of type V; f) RGN nickase; and g) nuclease-inactive RGN.

14. The RGN nickase is a) an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 75 and 88-98; and b) The nucleic acid molecule of claim 13, having an amino acid sequence selected from the group consisting of the amino acid sequences set forth as any one of SEQ ID NOs: 75 and 88 to 98.

15. The nucleic acid molecule according to any one of claims 12 to 14, wherein the polynucleotide encoding the fusion protein is operably linked at its 5' end to a promoter.

16. The nucleic acid molecule of any one of claims 12 to 15, wherein the fusion protein is codon-optimized for expression in a eukaryotic cell.

17. The nucleic acid molecule of claim 12, wherein the fusion protein has an amino acid sequence set forth as any one of SEQ ID NOs: 67, 68, 146, and 147.

18. A vector comprising the nucleic acid molecule according to any one of claims 12 to 17.

19. 20. The vector of claim 18, further comprising at least one nucleotide sequence encoding a guide RNA (gRNA) capable of hybridizing to a target sequence.

20. 1. A system for modifying a target DNA molecule comprising a target DNA sequence, comprising: a) a fusion protein or a nucleotide sequence encoding said fusion protein, wherein said fusion protein comprises an RNA-guided nuclease polypeptide (RGN) and a deaminase, said deaminase comprising: i) an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 2 and 7 to 12; ii) an amino acid sequence having at least 95% identity with SEQ ID NO: 4 or 6; ); and iii) having an amino acid sequence selected from the group consisting of the amino acid sequences set forth as any one of SEQ ID NOs: 2, 4, and 6-12; b) one or more guide RNAs capable of hybridizing to said target DNA sequence, or one or more nucleotide sequences encoding said one or more guide RNAs The system comprising:

21. 21. The system of claim 20, wherein the one or more guide RNAs form a complex with the fusion protein, enabling the fusion protein to bind to the target DNA sequence and modify the target DNA molecule.

22. The RGN of the fusion protein is a) a type II CRISPR-Cas polypeptide; b) CRISPR-Cas polypeptide of type V c) RGN nickase; and d) nuclease-inactive RGN; 22. The system according to claim 20 or 21, wherein

23. The RGN nickase is a) an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 75 and 88-98; and b) the system according to claim 22, having an amino acid sequence selected from the group consisting of the amino acid sequences set forth as any one of SEQ ID NOs: 75 and 88 to 98.

24. The system according to any one of claims 20 to 23, wherein the fusion protein comprises one or more nuclear localization signals.

25. The system according to any one of claims 20 to 24, wherein the fusion protein further comprises a uracil stabilising protein (USP).

26. 26. The system of claim 25, wherein the USP has the sequence shown as SEQ ID NO:

81.

27. The system of claim 20 , wherein the fusion protein has an amino acid sequence set forth as any one of SEQ ID NOs: 67, 68, 146, and 147.

28. A ribonucleoprotein complex comprising said at least one guide RNA and said fusion protein of the system according to any one of claims 20 to 27.

29. A cell comprising a nucleic acid molecule according to claim 2 or 3, a vector according to claim 4, a fusion protein according to any one of claims 5 to 11, a nucleic acid molecule according to any one of claims 12 to 17, a vector according to claim 18 or 19, a system according to any one of claims 20 to 27, or a ribonucleoprotein complex according to claim 28.

30. A pharmaceutical composition comprising a polypeptide according to claim 1, a nucleic acid molecule according to claim 2 or 3, a vector according to claim 4, a fusion protein according to any one of claims 5 to 11, a nucleic acid molecule according to any one of claims 12 to 17, a vector according to claim 18 or 19, a system according to any one of claims 20 to 27, or a ribonucleoprotein complex according to claim 28, or a cell according to claim 29, in combination with a pharma- ceutically acceptable carrier.

31. A method for modifying a target DNA molecule comprising a target DNA sequence, the method comprising delivering a system according to any one of claims 20 to 27 or a ribonucleoprotein complex according to claim 28 to said target DNA molecule or to a cell comprising said target DNA molecule.

32. 32. The method of claim 31 , wherein the modified target DNA molecule comprises a C>N mutation of at least one nucleotide within the target DNA molecule, where N is A, G, or T.

33. 1. An in vitro method for modifying a target DNA molecule comprising a target sequence, comprising: a) assembling an RGN-deaminase complex under conditions suitable for the formation of said RGN-deaminase complex; i) one or more guide RNAs capable of hybridizing to said target DNA molecule; ii) a fusion protein comprising an RNA-guided nuclease polypeptide (RGN) and at least one deaminase, wherein the deaminase is I) an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 2 and 7 to 12; II) an amino acid sequence having at least 95% identity to SEQ ID NO: 4 or 6; and III) having an amino acid sequence selected from the group consisting of the amino acid sequences set forth as any one of SEQ ID NOs: 2, 4, and 6-12; after performing in vitro combination; b) contacting the target DNA molecule, or a cell containing the target DNA molecule, with the in vitro assembled RGN-deaminase complex; The one or more guide RNAs hybridize to the target DNA molecule, thereby allowing the fusion protein to bind to the target DNA molecule and cause modification of the target DNA molecule.

34. 34. The method of claim 33, wherein the modified target DNA molecule comprises a C>N mutation of at least one nucleotide within the target DNA molecule, where N is A, G, or T.

35. The RGN of the fusion protein a) a type II CRISPR-Cas polypeptide; b) a CRISPR-Cas polypeptide of type V; c) RGN nickase; and d) nuclease-inactive RGN.

36. The RGN nickase is a) an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 75 and 88-98; and b) having an amino acid sequence selected from the group consisting of any one of SEQ ID NOs: 75 and 88 to 98.

37. The method of any one of claims 33 to 36, wherein the fusion protein further comprises a uracil stabilizing protein (USP).

38. 34. The method of claim 33, wherein the fusion protein has an amino acid sequence set forth as any one of SEQ ID NOs: 67, 68, 146, and 147.

39. 1. A method for producing a genetically modified cell having a corrected causative mutation for a genetic disease, comprising: a) a fusion protein or a polynucleotide encoding said fusion protein, wherein said fusion protein comprises an RNA-guided nuclease (RGN) and a deaminase, said deaminase comprising: i) an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 2 and 7 to 12; ii) an amino acid sequence having at least 95% identity to SEQ ID NO: 4 or 6; iii) having an amino acid sequence selected from the group consisting of amino acid sequences having 100% identity to any one of SEQ ID NOs: 2, 4, and 6-12; b) one or more guide RNAs (gRNAs) capable of hybridizing to the target DNA sequence, or a polynucleotide encoding said gRNAs; into said cell, This allows the fusion protein and gRNA to target the genomic locus of the causative mutation and modify the genomic sequence to correct the causative mutation.

40. The RGN of the fusion protein is a) Type II CRISPR-Cas polypeptide b) CRISPR-Cas polypeptide of type V c) RGN nickase; and d) nuclease inactive RGN.

41. The RGN nickase is a) an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 75 and 88-98; and b) having an amino acid sequence selected from the group consisting of any one of SEQ ID NOs: 75 and 88-98.

42. 42. The method of any one of claims 39 to 41, wherein the fusion protein further comprises a uracil stabilizing protein (USP).

43. 40. The method of claim 39, wherein the fusion protein has an amino acid sequence set forth as any one of SEQ ID NOs: 67, 68, 146, and 147.

44. The method of any one of claims 39 to 43, wherein the genomic modification comprises introducing a C>T or C>G mutation of at least one nucleotide in the target DNA sequence.

45. The method according to any one of claims 39 to 44, wherein the correction of the causative mutation comprises correction of a nonsense mutation.

46. 46. ​​The method of any one of claims 39 to 45, wherein the genetic disease is a disease listed in Table 23.

47. A fusion protein according to any one of claims 5 to 11, a nucleic acid molecule according to any one of claims 12 to 17, a vector according to claim 18 or 19, or a cell according to claim 29, a system according to any one of claims 20 to 27, or a ribonucleoprotein complex according to claim 28 for use in the treatment of a disease.

48. A fusion protein, a nucleic acid molecule, a vector, a cell, a system, or a ribonucleoprotein complex for use as claimed in claim 47, comprising: The disease is associated with a causative mutation, and an effective amount of the fusion protein, nucleic acid molecule, vector, cell, system, or ribonucleoprotein complex corrects the causative mutation.

49. 49. The fusion protein, nucleic acid molecule, vector, cell, system, or ribonucleoprotein complex for use according to claim 47 or 48, wherein the disease is a disease listed in Table 23.