Recombinant rabies viruses for gene therapy.

Abstract

Provided herein are recombinant rabies virus genomes and recombinant rabies viruses and methods of their use for delivering transgenes to target cells, as well as packaging systems and methods of using the packaging systems to produce recombinant rabies viruses.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 151,542, filed February 19, 2021, and U.S. Provisional Patent Application No. 63 / 241,989, filed September 8, 2021, the entire disclosures of which are incorporated herein by reference. [Background technology]

[0002] Gene therapy often uses viral gene delivery systems to transduce target cells and express transgenes. The viral systems widely used in gene therapy are derived from viruses and have significant drawbacks. Challenges in using current viral systems include limited packaging capacity, unintended consequences such as genome integration, limited tissue tropism, pre-existing immunity and / or immune responses in the target population, limited re-administration possibilities, and limited durability due to genome instability, immune clearance, and cytotoxicity. For example, adenoviral vector-mediated gene therapy exhibits high transduction efficiency and can be used to infect many different cell types, but it has several drawbacks, including lack of integration and high immunogenicity. Adeno-associated virus vector-mediated gene therapy has drawbacks such as high immunogenicity and limited packaging capacity. As another example, retroviral vector-mediated gene therapy suffers from low transduction efficiency and complement inactivation.

[0003] Therefore, there is a need for new viral gene delivery systems that are superior to current viral systems. Summary of the Invention [Means for solving the problem]

[0004] Provided herein are recombinant viral vectors and viruses derived from rabies virus that can be used to transduce target cells and express transgenes within those cells. The recombinant rabies virus vectors and viruses provided by the present disclosure find use as effective viral gene delivery systems. Also provided herein are viral packaging systems and methods for producing the recombinant viruses described herein.

[0005] In one aspect, a recombinant rabies virus genome is provided, comprising a nucleic acid encoding a therapeutic transgene, wherein the genome lacks a G gene encoding a rabies virus glycoprotein or a functional variant thereof, and / or the genome lacks an L gene encoding a rabies virus polymerase or a functional variant thereof.

[0006] In certain exemplary embodiments, the genome lacks a G gene encoding a rabies virus glycoprotein or a functional variant thereof. In certain exemplary embodiments, the genome lacks a G gene encoding a rabies virus glycoprotein or a functional variant thereof, and the genome lacks an L gene encoding a rabies virus polymerase or a functional variant thereof.

[0007] In certain exemplary embodiments, the genome comprises an N gene encoding a rabies virus nucleoprotein or a functional variant thereof, a P gene encoding a rabies virus phosphoprotein or a functional variant thereof, and an M gene encoding a rabies virus matrix protein or a functional variant thereof.

[0008] In another aspect, a recombinant rabies virus particle is provided comprising a rabies virus glycoprotein and a recombinant rabies virus genome described herein.

[0009] In another aspect, there is provided a recombinant rabies virus particle comprising a rabies virus glycoprotein and a recombinant rabies virus genome comprising a nucleic acid encoding a therapeutic transgene, wherein the genome lacks a G gene encoding the rabies virus glycoprotein or a functional variant thereof, and / or the genome lacks an L gene encoding the rabies virus polymerase or a functional variant thereof.

[0010] In certain exemplary embodiments, the genome lacks a G gene encoding a rabies virus glycoprotein or a functional variant thereof. In certain exemplary embodiments, the genome lacks a G gene encoding a rabies virus glycoprotein or a functional variant thereof, and the genome lacks an L gene encoding a rabies virus polymerase or a functional variant thereof.

[0011] In certain exemplary embodiments, the genome comprises: the G gene encoding the rabies virus glycoprotein or a functional variant thereof; the L gene encoding the rabies virus polymerase or a functional variant thereof; and It lacks the M gene, which encodes the rabies virus matrix protein or a functional variant thereof.

[0012] In certain exemplary embodiments, the genome comprises: the G gene encoding the rabies virus glycoprotein or a functional variant thereof; the L gene encoding the rabies virus polymerase or a functional variant thereof; the M gene encoding the rabies virus matrix protein or a functional variant thereof; and It lacks the P gene, which encodes the rabies virus phosphoprotein or a functional variant thereof.

[0013] In certain exemplary embodiments, the genome comprises: the G gene encoding the rabies virus glycoprotein or a functional variant thereof; the L gene encoding the rabies virus polymerase or a functional variant thereof; the M gene encoding the rabies virus matrix protein or a functional variant thereof; a P gene encoding the rabies virus phosphoprotein or a functional variant thereof; and It lacks the N gene, which encodes the rabies virus nucleoprotein or a functional variant thereof.

[0014] In certain exemplary embodiments, the genome encodes only the therapeutic transgene.

[0015] In certain exemplary embodiments, the genome comprises an N gene encoding a rabies virus nucleoprotein or a functional variant thereof, a P gene encoding a rabies virus phosphoprotein or a functional variant thereof, and an M gene encoding a rabies virus matrix protein or a functional variant thereof.

[0016] In other exemplary embodiments, the genome lacks the N gene, which encodes the rabies virus nucleoprotein or a functional variant thereof, the P gene, which encodes the rabies virus phosphoprotein or a functional variant thereof, and / or the M gene, which encodes the rabies virus matrix protein or a functional variant thereof.

[0017] In certain exemplary embodiments, the recombinant rabies virus is replication-incompetent. In other exemplary embodiments, the recombinant rabies virus is replication-deficient.

[0018] In certain exemplary embodiments, each of the genes is operably linked to a transcriptional regulatory element. In certain exemplary embodiments, the transcriptional regulatory element comprises a transcription initiation signal. In certain exemplary embodiments, the transcription initiation signal is exogenous to the rabies virus. In certain exemplary embodiments, the transcription initiation signal is endogenous to the rabies virus.

[0019] In certain exemplary embodiments, each of the genes is operably linked to a transcription termination and polyadenylation signal.

[0020] In certain exemplary embodiments, the therapeutic transgene comprises a sequence encoding a nucleic acid editing system or a component thereof. In certain exemplary embodiments, the nucleic acid editing system comprises a Clustered Regulatory Interspaced Short Palindromic Repeat (CRISPR) system, a zinc finger nuclease (ZFN), a meganuclease, and a transcription activator-like effector nuclease (TALEN). In certain exemplary embodiments, the nucleic acid editing system comprises a CRISPR system. In certain exemplary embodiments, the CRISPR system comprises a nucleobase editor comprising a polynucleotide programmable nucleotide binding domain and a nucleobase editing domain.

[0021] In certain exemplary embodiments, the nucleobase editing domain is adenosine deaminase, cytidine deaminase, or a functional variant thereof. In certain exemplary embodiments, the nucleobase editing domain is adenosine deaminase. In certain exemplary embodiments, the adenosine deaminase is TadA deaminase derived from any of the adenosine base editors listed in Table 10, Table 11, Table 12, Table 13, Table 14, or Table 15.

[0022] The beauty of the beauty of the painting is presented in the picture below. BE0.1、BE0.2、BE1.1、BE1.2、BE2 .1、HE2.2、HE2.3、HE2.4、HE2.5、HE2.6、HE2.7、HE2.8、HE2.9、HE 2.10、ABE2.11、ABE2.12、ABE3.1、ABE3.2、ABE3.3、ABE3.4、ABE3.5、ABE3.6 wood. 、ABE5.5、ABE5.6、ABE5.7、ABE5.8、ABE5.9、ABE5.10、ABE5.11、ABE5.12、AB E5.13、AB5.14、FI6.1、FI6.2、FI6.3、FI6.4、FI6.5、FIBE6.6、FIBE7.1、 ABE7.2、ABE7.3、ABE7.4、ABE7.5、ABE7.6、ABE7.7、ABE7.8、ABE7.9、ABE7.1 0、ABE8.1-m、ABE8.2-m、ABE8.3-m、ABE8.4-m、ABE8.5-m、ABE8.6-m、ABE8.7- m、ABE8.8-m、ABE8.9-m、ABE8.10-m、ABE8.11-m、ABE8.12-m、ABE8.13-m、AB E8.14-m、ABE8.15-m、ABE8.16-m、ABE8.17-m、ABE8.18-m、ABE8.19-m、ABE8. 20-m、ABE8.21-m、ABE8.22-m、ABE8.23-m、ABE8.24-m、ABE8.1-d、ABE8.2-d 、ABE8.3-d、ABE8.4-d、ABE8.5-d、ABE8.6-d、ABE8.7-d、ABE8.8-d、ABE8.9-d 、ABE8.10-d、ABE8.11-d、ABE8.12-d、ABE8.13-d、ABE8.14-d、ABE8.15-d、A BE8.16-d、ABE8.17-d、ABE8.18-d、ABE8.19-d、ABE8.20-d、ABE8.21-d、ABE8 .22-d、ABE8.23-d、ABE8.24-d、ABE8a-m、ABE8b-m、ABE8c-m、ABE8d-m、ABE8 e-m、ABE8a-d、ABE8b-d、ABE8c-d、ABE8d-d、ABE8e-d、ABE9.1、ABE9.2、ABE9.3, ABE9.4, ABE9.5, ABE9.6, ABE9.7, ABE9.8, ABE9.9, ABE9.10, ABE9.11, ABE9.12, ABE9.13, ABE9.14, ABE9.15, ABE9.16, ABE9.17, ABE9.18, ABE 9.19, ABE9.2, ABE9.21, ABE9.22, ABE9.23, ABE9.24, ABE9.25, ABE9.26, ABE9.27, ABE9.28, ABE9.29, ABE9.30, ABE9.31, ABE9.32, ABE9.33, ABE and adenosine deaminases derived from any one of the adenosine base editors ABE9.9.34, ABE9.35, ABE9.36, ABE9.37, ABE9.38, ABE9.39, ABE9.40, ABE9.41, ABE9.42, ABE9.43, ABE9.44, ABE9.45, ABE9.46, ABE9.47, ABE9.48, ABE9.49, ABE9.50, ABE9.51, ABE9.52, ABE9.53, ABE9.54, ABE9.55, ABE9.56, ABE9.57, and ABE9.58.

[0023] The beauty of the beauty of the painting is presented in the picture below. BE0.1、BE0.2、BE1.1、BE1.2、BE2 .1、HE2.2、HE2.3、HE2.4、HE2.5、HE2.6、HE2.7、HE2.8、HE2.9、HE 2.10、ABE2.11、ABE2.12、ABE3.1、ABE3.2、ABE3.3、ABE3.4、ABE3.5、ABE3.6 wood. 、ABE5.5、ABE5.6、ABE5.7、ABE5.8、ABE5.9、ABE5.10、ABE5.11、ABE5.12、AB E5.13、AB5.14、FI6.1、FI6.2、FI6.3、FI6.4、FI6.5、FIBE6.6、FIBE7.1、 ABE7.2、ABE7.3、ABE7.4、ABE7.5、ABE7.6、ABE7.7、ABE7.8、ABE7.9、ABE7.1 0、ABE8.1-m、ABE8.2-m、ABE8.3-m、ABE8.4-m、ABE8.5-m、ABE8.6-m、ABE8.7- m、ABE8.8-m、ABE8.9-m、ABE8.10-m、ABE8.11-m、ABE8.12-m、ABE8.13-m、AB E8.14-m、ABE8.15-m、ABE8.16-m、ABE8.17-m、ABE8.18-m、ABE8.19-m、ABE8. 20-m、ABE8.21-m、ABE8.22-m、ABE8.23-m、ABE8.24-m、ABE8.1-d、ABE8.2-d 、ABE8.3-d、ABE8.4-d、ABE8.5-d、ABE8.6-d、ABE8.7-d、ABE8.8-d、ABE8.9-d 、ABE8.10-d、ABE8.11-d、ABE8.12-d、ABE8.13-d、ABE8.14-d、ABE8.15-d、A BE8.16-d、ABE8.17-d、ABE8.18-d、ABE8.19-d、ABE8.20-d、ABE8.21-d、ABE8 .22-d、ABE8.23-d、ABE8.24-d、ABE8a-m、ABE8b-m、ABE8c-m、ABE8d-m、ABE8 e-m、ABE8a-d、ABE8b-d、ABE8c-d、ABE8d-d、ABE8e-d、ABE9.1、ABE9.2、ABE9.3, ABE9.4, ABE9.5, ABE9.6, ABE9.7, ABE9.8, ABE9.9, ABE9.10, ABE9.11, ABE9.12, ABE9.13, ABE9.14, ABE9.15, ABE9.16, ABE9.17, ABE9.18, ABE9.19, ABE9.2, ABE9.21, ABE9.22, ABE9.23, ABE9.24, ABE9.25, ABE9.26, ABE9.27, ABE9.28, ABE9.29, ABE9.30, ABE9.31, ABE9.32, ABE9. In certain exemplary embodiments, the adenosine base editor is selected from the group consisting of ABE9.33, ABE9.34, ABE9.35, ABE9.36, ABE9.37, ABE9.38, ABE9.39, ABE9.40, ABE9.41, ABE9.42, ABE9.43, ABE9.44, ABE9.45, ABE9.46, ABE9.47, ABE9.48, ABE9.49, ABE9.50, ABE9.51, ABE9.52, ABE9.53, ABE9.54, ABE9.55, ABE9.56, ABE9.57, and ABE9.58. In certain exemplary embodiments, the adenosine deaminase is ABE7.10 or ABE8.20.

[0024] In certain exemplary embodiments, the nucleobase editing domain comprises a cytidine deaminase, hi certain exemplary embodiments, the cytidine deaminase is selected from the group consisting of Petromyzon marinus cytosine deaminase 1 (PmCDA1), activation-induced cytidine deaminase (AID), and APOBEC.

[0025] In certain exemplary embodiments, the polynucleotide-programmable nucleotide binding domain comprises a Cas9 polypeptide, a Cas12 polypeptide, or a functional variant thereof.

[0026] In certain exemplary embodiments, the CRISPR system further comprises a guide RNA (gRNA) or a nucleic acid sequence encoding the gRNA.

[0027] In certain exemplary embodiments, the therapeutic transgene comprises a sequence encoding a uracil glycosylase inhibitor (UGI).

[0028] In certain exemplary embodiments, the therapeutic transgene comprises a sequence encoding a nuclear localization signal (NLS).

[0029] In certain exemplary embodiments, the polynucleotide programmable nucleotide binding domain and / or the nucleobase editing domain further comprises a nuclear localization signal (NLS).

[0030] In certain exemplary embodiments, the cytidine deaminase further comprises a uracil glycosylase inhibitor (UGI).

[0031] In certain exemplary embodiments, the therapeutic transgene comprises a therapeutic polypeptide and / or a therapeutic nucleic acid, hi certain exemplary embodiments, the therapeutic polypeptide and / or the therapeutic nucleic acid is secreted from the cell.

[0032] In certain exemplary embodiments, the nucleic acid encoding a therapeutic transgene is greater than about 300 bp, about 400 bp, about 500 bp, about 600 bp, about 700 bp, about 800 bp, about 900 bp, about 1,000 bp, about 1,100 bp, about 1,200 bp, about 1,300 bp, about 1,400 bp, about 1,500 bp, about 1,600 bp, about 1,700 bp, about 1,800 bp, about 1,900 bp, about 2,000 bp, about 2,100 bp, about 2,200 bp, about 2,300 bp, about 2,400 bp, about 2,500 bp, about 2,600 bp, about 2,700 bp, about 2,800 bp, about 2,900 bp, or about 3,000 bp. In certain exemplary embodiments, the nucleic acid encoding the therapeutic transgene is larger than about 300 bp. In certain exemplary embodiments, the nucleic acid encoding the therapeutic transgene is larger than about 650 bp. In certain exemplary embodiments, the nucleic acid encoding the therapeutic transgene is larger than about 1,000 bp. In certain exemplary embodiments, the nucleic acid encoding the therapeutic transgene is larger than about 3,000 bp. In certain exemplary embodiments, the nucleic acid encoding the therapeutic transgene is larger than about 4,500 bp. In certain exemplary embodiments, the nucleic acid encoding the therapeutic transgene is larger than about 8,500 bp. In certain exemplary embodiments, the nucleic acid encoding the therapeutic transgene is larger than about 10,000 bp.

[0033] In certain exemplary embodiments, the therapeutic transgene is operably linked to a transcriptional regulatory element. In certain exemplary embodiments, the transcriptional regulatory element comprises a transcription initiation signal. In certain exemplary embodiments, the transcription initiation signal is exogenous to the rabies virus. In certain exemplary embodiments, the transcription initiation signal is endogenous to the rabies virus.

[0034] In certain exemplary embodiments, the therapeutic transgene is operably linked to a transcription termination polyadenylation signal.

[0035] In another aspect, a pharmaceutical composition is provided that includes the recombinant viral particles described herein.

[0036] In another aspect, a method for expressing a therapeutic transgene in a target cell is provided, the method comprising transducing the target cell with a recombinant viral particle described herein.

[0037] In another aspect, a method for expressing a nucleobase editor in a target cell is provided, comprising transducing the target cell with a recombinant rabies virus particle, wherein the recombinant virus particle comprises a rabies virus glycoprotein and a recombinant rabies virus genome comprising a nucleic acid encoding a nucleobase editor comprising a polynucleotide-programmable nucleotide-binding domain and a nucleobase editing domain, wherein the genome lacks a G gene encoding the rabies virus glycoprotein or a functional variant thereof, and / or the genome lacks an L gene encoding the rabies virus polymerase or a functional variant thereof.

[0038] In certain exemplary embodiments, the genome further lacks the M gene, which encodes the rabies virus matrix protein or a functional variant thereof.

[0039] In certain exemplary embodiments, the genome further lacks the M gene, which encodes the rabies virus matrix protein or a functional variant thereof, and the P gene, which encodes the rabies virus phosphoprotein or a functional variant thereof.

[0040] In certain exemplary embodiments, the genome further lacks the M gene, which encodes a rabies virus matrix protein or a functional variant thereof, the P gene, which encodes a rabies virus phosphoprotein or a functional variant thereof, and the N gene, which encodes a rabies virus nucleoprotein or a functional variant thereof.

[0041] In certain exemplary embodiments, the genome encodes only nucleobase editors.

[0042] In certain exemplary embodiments, the genome comprises an N gene encoding a rabies virus nucleoprotein or a functional variant thereof, a P gene encoding a rabies virus phosphoprotein or a functional variant thereof, and an M gene encoding a rabies virus matrix protein or a functional variant thereof.

[0043] In certain exemplary embodiments, each of the genes is operably linked to a transcriptional regulatory element. In certain exemplary embodiments, the transcriptional regulatory element comprises a transcription initiation signal. In certain exemplary embodiments, the transcription initiation signal is exogenous to the rabies virus. In certain exemplary embodiments, the transcription initiation signal is endogenous to the rabies virus.

[0044] In certain exemplary embodiments, each of the genes is operably linked to a transcription termination and polyadenylation signal.

[0045] In certain exemplary embodiments, the nucleobase-editing domain comprises an adenosine deaminase, a cytidine deaminase, or a functional variant thereof.

[0046] In certain exemplary embodiments, the nucleobase editing domain comprises an adenosine deaminase, hi certain exemplary embodiments, the adenosine deaminase is a TadA deaminase derived from any of the adenosine base editors listed in Table 10, Table 11, Table 12, Table 13, Table 14, or Table 15.

[0047] The beauty of the beauty of the house is also updated. Pages Publications HEB0.1、HEB0.2、HE1.1、HE1.2、HE2.1、HE2.2、HE2.3、HE2 .4、HEB2.5、HE2.6、HE2.7、HE2.8、HE2.9、HE2.10、HE2.11、HE2.12 wood. .1、HE4.2、HE4.3、HE5.1、HE5.2、HE5.3、HE5.4、HE5.5、HE5.6、AB E5.7. E6.1、ABS6.2、ABS6.3、ABS6.4、ABS6.5、ABS. ABE7.4、ABE7.5、ABE7.6、ABE7.7、ABE7.8、ABE7.9、ABE7.10、ABE8.1-m、AB E8.2-m、ABE8.3-m、ABE8.4-m、ABE8.5-m、ABE8.6-m、ABE8.7-m、ABE8.8-m、 ABE8.9-m、ABE8.10-m、ABE8.11-m、ABE8.12-m、ABE8.13-m、ABE8.14-m、AB E8.15-m、ABE8.16-m、ABE8.17-m、ABE8.18-m、ABE8.19-m、ABE8.20-m、ABE 8.21-m、ABE8.22-m、ABE8.23-m、ABE8.24-m、ABE8.1-d、ABE8.2-d、ABE8.3 -d、ABE8.4-d、ABE8.5-d、ABE8.6-d、ABE8.7-d、ABE8.8-d、ABE8.9-d、ABE8 .10-d、ABE8.11-d、ABE8.12-d、ABE8.13-d、ABE8.14-d、ABE8.15-d、ABE8. 16-d、ABE8.17-d、ABE8.18-d、ABE8.19-d、ABE8.20-d、ABE8.21-d、ABE8.2 2-d、ABE8.23-d、ABE8.24-d、ABE8a-m、ABE8b-m、ABE8c-m、ABE8d-m、ABE8e -m、ABE8a-d、ABE8b-d、ABE8c-d、ABE8d-d、ABE8e-d、ABE9.1、ABE9.2、ABE9.3, ABE9.4, ABE9.5, ABE9.6, ABE9.7, ABE9.8, ABE9.9, ABE9.10, ABE9.11, ABE9.12, ABE9.13, ABE9.14, ABE9.15, ABE9.16, ABE9.17, ABE9.18, ABE 9.19, ABE9.2, ABE9.21, ABE9.22, ABE9.23, ABE9.24, ABE9.25, ABE9.26, ABE9.27, ABE9.28, ABE9.29, ABE9.30, ABE9.31, ABE9.32, ABE9.33, ABE9 and adenosine deaminases derived from any one of the following adenosine base editors: ABE9.34, ABE9.35, ABE9.36, ABE9.37, ABE9.38, ABE9.39, ABE9.40, ABE9.41, ABE9.42, ABE9.43, ABE9.44, ABE9.45, ABE9.46, ABE9.47, ABE9.48, ABE9.49, ABE9.50, ABE9.51, ABE9.52, ABE9.53, ABE9.54, ABE9.55, ABE9.56, ABE9.57, and ABE9.58.

[0048] The beauty of the beauty of the painting is presented in the picture below. BE0.1、BE0.2、BE1.1、BE1.2、BE2 .1、HE2.2、HE2.3、HE2.4、HE2.5、HE2.6、HE2.7、HE2.8、HE2.9、HE 2.10、ABE2.11、ABE2.12、ABE3.1、ABE3.2、ABE3.3、ABE3.4、ABE3.5、ABE3.6 wood. 、ABE5.5、ABE5.6、ABE5.7、ABE5.8、ABE5.9、ABE5.10、ABE5.11、ABE5.12、AB E5.13、AB5.14、FI6.1、FI6.2、FI6.3、FI6.4、FI6.5、FIBE6.6、FIBE7.1、 ABE7.2、ABE7.3、ABE7.4、ABE7.5、ABE7.6、ABE7.7、ABE7.8、ABE7.9、ABE7.1 0、ABE8.1-m、ABE8.2-m、ABE8.3-m、ABE8.4-m、ABE8.5-m、ABE8.6-m、ABE8.7- m、ABE8.8-m、ABE8.9-m、ABE8.10-m、ABE8.11-m、ABE8.12-m、ABE8.13-m、AB E8.14-m、ABE8.15-m、ABE8.16-m、ABE8.17-m、ABE8.18-m、ABE8.19-m、ABE8. 20-m、ABE8.21-m、ABE8.22-m、ABE8.23-m、ABE8.24-m、ABE8.1-d、ABE8.2-d 、ABE8.3-d、ABE8.4-d、ABE8.5-d、ABE8.6-d、ABE8.7-d、ABE8.8-d、ABE8.9-d 、ABE8.10-d、ABE8.11-d、ABE8.12-d、ABE8.13-d、ABE8.14-d、ABE8.15-d、A BE8.16-d、ABE8.17-d、ABE8.18-d、ABE8.19-d、ABE8.20-d、ABE8.21-d、ABE8 .22-d、ABE8.23-d、ABE8.24-d、ABE8a-m、ABE8b-m、ABE8c-m、ABE8d-m、ABE8 e-m、ABE8a-d、ABE8b-d、ABE8c-d、ABE8d-d、ABE8e-d、ABE9.1、ABE9.2、ABE9.3, ABE9.4, ABE9.5, ABE9.6, ABE9.7, ABE9.8, ABE9.9, ABE9.10, ABE9.11, ABE9.12, ABE9.13, ABE9.14, ABE9.15, ABE9.16, ABE9.17, ABE9.18, ABE9.19, ABE9.2, ABE9.21, ABE9.22, ABE9.23, ABE9.24, ABE9.25, ABE9.26, ABE9.27, ABE9.28, ABE9.29, ABE9.30, ABE9.31, ABE9.32, ABE9. and ABE9.50, ABE9.51, ABE9.52, ABE9.53, ABE9.54, ABE9.55, ABE9.56, ABE9.57, and ABE9.58.

[0049] In certain exemplary embodiments, the nucleobase editing domain comprises an ABE7.10 or ABE8.20 adenosine base editor.

[0050] In certain exemplary embodiments, the nucleobase editing domain is a cytidine deaminase, hi certain exemplary embodiments, the cytidine deaminase is selected from the group consisting of Petromyzon marinus cytosine deaminase 1 (PmCDA1), activation-induced cytidine deaminase (AID), and APOBEC.

[0051] In certain exemplary embodiments, the polynucleotide-programmable nucleotide binding domain comprises a Cas9 polypeptide, a Cas12 polypeptide, or a functional variant thereof.

[0052] In certain exemplary embodiments, the recombinant genome further comprises a guide RNA (gRNA). In certain exemplary embodiments, the guide RNA (gRNA) is exogenously provided to the target cell. In certain exemplary embodiments, the gRNA is capable of targeting a genomic locus in the target cell.

[0053] In certain exemplary embodiments, the target cells are transduced ex vivo. In certain exemplary embodiments, the target cells are human cells. In certain exemplary embodiments, the target cells are obtained from a human. In certain exemplary embodiments, the target cells are autologous to the human. In certain exemplary embodiments, the target cells are allogeneic to the human.

[0054] In certain exemplary embodiments, the target cell is transduced in vivo. In certain exemplary embodiments, the target cell is a human cell. In certain exemplary embodiments, the target cell is a neuronal cell, an epithelial cell, or a hepatic cell. In certain exemplary embodiments, the target cell is a cell within a human.

[0055] In another aspect, a method is provided for delivering a therapeutic transgene to a subject, the method comprising administering to the subject a recombinant viral particle described herein, or a pharmaceutical composition described herein.

[0056] In another aspect, provided is a method for delivering a nucleobase editor to a subject, comprising administering to the subject a recombinant rabies virus particle, wherein the recombinant virus particle comprises a rabies virus glycoprotein and a recombinant rabies virus genome comprising a nucleic acid encoding a nucleobase editor comprising a polynucleotide-programmable nucleotide-binding domain and a nucleobase editing domain, wherein the genome lacks a G gene encoding the rabies virus glycoprotein or a functional variant thereof, and / or the genome lacks an L gene encoding the rabies virus polymerase or a functional variant thereof.

[0057] In certain exemplary embodiments, the genome comprises an N gene encoding a rabies virus nucleoprotein or a functional variant thereof, a P gene encoding a rabies virus phosphoprotein or a functional variant thereof, and an M gene encoding a rabies virus matrix protein or a functional variant thereof.

[0058] In certain exemplary embodiments, the nucleobase editing domain is adenosine deaminase, cytidine deaminase, or a functional variant thereof. In certain exemplary embodiments, the base editor is adenosine deaminase. In certain exemplary embodiments, the adenosine deaminase is ABE7.10.

[0059] In certain exemplary embodiments, the polynucleotide-programmable nucleotide binding domain comprises a Cas9 polypeptide, a Cas12 polypeptide, or a functional variant thereof.

[0060] In certain exemplary embodiments, the recombinant genome further comprises a nucleic acid sequence encoding a guide RNA (gRNA). In certain exemplary embodiments, the guide RNA (gRNA) is exogenously provided to the target cell. In certain exemplary embodiments, the gRNA is capable of targeting a genomic locus in the target cell.

[0061] In certain exemplary embodiments, the subject is a mammal, hi certain exemplary embodiments, the subject is a human.

[0062] In another aspect, there is provided a packaging system for the recombinant preparation of rabies virus particles, the packaging system comprising an N gene encoding a rabies virus nucleoprotein or a functional variant thereof, a P gene encoding a rabies virus phosphoprotein or a functional variant thereof, an L gene encoding a rabies virus polymerase or a functional variant thereof, and a recombinant rabies virus genome, wherein the genome lacks the G gene encoding the rabies virus glycoprotein or a functional variant thereof and / or the genome lacks the L gene encoding the rabies virus polymerase or a functional variant thereof.

[0063] In certain exemplary embodiments, the recombinant rabies virus genome comprises a nucleic acid encoding a transgene or therapeutic transgene.

[0064] In certain exemplary embodiments, the recombinant rabies virus genome is contained within a viral genome vector.

[0065] In certain exemplary embodiments, the N, P, and L genes are each contained within a separate vector. In certain exemplary embodiments, each of the N, P, and L genes is operably linked to a transcriptional regulator. In certain exemplary embodiments, the transcriptional regulator comprises a promoter and / or an enhancer. In certain exemplary embodiments, the promoter is a constitutive promoter. In certain exemplary embodiments, the promoter is an elongation factor 1 alpha promoter. In certain exemplary embodiments, the separate vectors are each contained within a separate transfection plasmid.

[0066] In certain exemplary embodiments, the N, P, and L genes are contained within a single vector. In certain exemplary embodiments, the single vector comprises a first expression cassette comprising the N gene and the P gene and a second expression cassette comprising the L gene. In certain exemplary embodiments, the first expression cassette comprises, from 5' to 3', a transcriptional regulator, the P gene, and the N gene. In certain exemplary embodiments, the first expression cassette comprises, from 5' to 3', a transcriptional regulator, the P gene, a ribosome skipping element, and the N gene. In certain exemplary embodiments, the ribosome skipping element is an IRES element. In certain exemplary embodiments, the ribosome skipping element is a 2A element. In certain exemplary embodiments, the second expression cassette comprises, from 5' to 3', a transcriptional regulator and the L gene. In certain exemplary embodiments, the transcriptional regulator comprises a promoter and / or an enhancer. In certain exemplary embodiments, the promoter is a constitutive promoter. In certain exemplary embodiments, the promoter is an elongation factor 1α promoter. In certain exemplary embodiments, the first and second expression cassettes are in opposite orientations on the vector, hi certain exemplary embodiments, the single vector is contained within a single transfection plasmid.

[0067] In certain exemplary embodiments, the packaging system further comprises an M gene encoding a rabies virus matrix protein or a functional variant thereof. In certain exemplary embodiments, the M gene is contained within a vector. In certain exemplary embodiments, the M gene is operably linked to a transcriptional regulator. In certain exemplary embodiments, the transcriptional regulator comprises a promoter and / or an enhancer. In certain exemplary embodiments, the vector comprising the M gene is contained within a transfection plasmid.

[0068] In certain exemplary embodiments, the packaging system further comprises a G gene encoding a rabies virus glycoprotein or a functional variant thereof. In certain exemplary embodiments, the G gene is contained within a vector. In certain exemplary embodiments, the G gene is operably linked to a transcriptional regulator. In certain exemplary embodiments, the transcriptional regulator comprises a promoter and / or an enhancer. In certain exemplary embodiments, the vector comprising the G gene is contained within a transfection plasmid.

[0069] In another aspect, a method for producing recombinant rabies virus particles is provided, comprising introducing the packaging system described herein into a cell under conditions operative to package a recombinant rabies virus genome to form a recombinant rabies virus particle.

[0070] In certain exemplary embodiments, the introducing is mediated by electroporation, nucleofection, or lipofection.

[0071] In another aspect, a recombinant rabies virus particle packaging cell is provided that comprises the packaging system described herein.

[0072] In another aspect, a recombinant rabies virus particle packaging cell is provided, comprising an N gene encoding a rabies virus nucleoprotein or a functional variant thereof, a P gene encoding a rabies virus phosphoprotein or a functional variant thereof, and an L gene encoding a rabies virus polymerase or a functional variant thereof.

[0073] In certain exemplary embodiments, the first expression cassette comprises an N gene and a P gene. In certain exemplary embodiments, the first expression cassette comprises, from 5' to 3', a transcriptional regulator, a P gene, and an N gene. In certain exemplary embodiments, the first expression cassette comprises, from 5' to 3', a transcriptional regulator, a P gene, a ribosome skipping element, and an N gene. In certain exemplary embodiments, the ribosome skipping element is an IRES element. In certain exemplary embodiments, the ribosome skipping element is a 2A element.

[0074] In certain exemplary embodiments, the second expression cassette comprises the L gene. In certain exemplary embodiments, the second expression cassette comprises, from 5' to 3', a transcriptional regulator and the L gene. In certain exemplary embodiments, the transcriptional regulator comprises a promoter and / or an enhancer. In certain exemplary embodiments, the promoter is a constitutive promoter. In certain exemplary embodiments, the promoter is an elongation factor 1 alpha promoter.

[0075] In certain exemplary embodiments, the first and second expression cassettes are in opposite orientations on the vector.

[0076] In certain exemplary embodiments, the recombinant rabies virus particle packaging cell further comprises an M gene encoding a rabies virus matrix protein and / or a G gene encoding a rabies virus glycoprotein. In certain exemplary embodiments, the M gene and / or the G gene are operably linked to a transcriptional regulator. In certain exemplary embodiments, the transcriptional regulator comprises a promoter and / or an enhancer. In certain exemplary embodiments, the third expression cassette and / or the fourth expression cassette comprises the rabies virus G gene and / or the rabies virus M gene.

[0077] In certain exemplary embodiments, the packaging cells are of mammalian, bacterial, or insect origin, hi certain exemplary embodiments, the packaging cells are selected from the group consisting of HEK293 cells, VERO cells, BHK cells, and BSR cells.

[0078] In another aspect, there is provided a method for producing a recombinant rabies virus, comprising introducing into a packaging cell described herein a nucleic acid comprising a recombinant rabies virus genome, the nucleic acid comprising a therapeutic transgene, under conditions operative to package the recombinant rabies virus genome into a glycoprotein to form a recombinant rabies virus, wherein the genome lacks an endogenous G gene encoding the rabies virus glycoprotein and the genome lacks an endogenous L gene encoding the rabies virus polymerase.

[0079] In certain exemplary embodiments, the recombinant genome comprises an N gene encoding a rabies virus nucleoprotein or a functional variant thereof, a P gene encoding a rabies virus phosphoprotein or a functional variant thereof, and an M gene encoding a rabies virus matrix protein or a functional variant thereof.

[0080] In certain exemplary embodiments, each of the genes is operably linked to a transcriptional regulatory element. In certain exemplary embodiments, the transcriptional regulatory element comprises a transcription initiation signal. In certain exemplary embodiments, the transcription initiation signal is exogenous to the rabies virus. In certain exemplary embodiments, the transcription initiation signal is endogenous to the rabies virus.

[0081] In certain exemplary embodiments, each of the genes is operably linked to a transcription termination and polyadenylation signal.

[0082] In certain exemplary embodiments, the titer of the recombinant rabies virus is greater than about 1E8 TU / mL, hi certain exemplary embodiments, the titer of the recombinant rabies virus is between about 1E8 TU / mL and about 1E9 TU / mL.

[0083] In another aspect, a method of treating a disease or disorder in a subject is provided, comprising administering to the subject a recombinant rabies virus particle described herein, or a pharmaceutical composition described herein.

[0084] In certain exemplary embodiments, the disease or disorder is a nervous system disease or disorder. In certain exemplary embodiments, the disease or disorder is an ophthalmological disease or disorder.

[0085] In another aspect, there is provided the use of a recombinant rabies virus described herein, or a pharmaceutical composition described herein, in the manufacture of a medicament for treating a disease or disorder in a subject. [Brief explanation of the drawings]

[0086] [Figure 1] FIG. 1 is a graph showing the relative infectivity for 293T cells obtained from equal volumes of virus-containing supernatants harvested on the indicated days from various stable cell lines. [Figure 2-1] Figure 2A is a schematic diagram showing the VIR218 replicon. Figure 2B is a schematic diagram showing the production and infection scheme for recombinant rabies virus particle-mediated gene delivery. Figure 2C is a graph showing that recombinant rabies virus particles containing a recombinant rabies virus genome encoding a nucleobase editor can perform gene editing of a target sequence. [Figure 2-2] Same as above. [Figure 2-3] Same as above. [Figure 3-1]Figure 3A is a schematic diagram showing the structure of a recombinant rabies virus genome containing a gRNA, a polynucleotide-programmable nucleotide-binding domain, and a nucleobase editor. Figure 3B is a schematic diagram showing a gRNA-tRNA expression cassette encoding a gRNA between two tRNA sequences, with the arrow indicating the RNA cleavage site. Figure 3C is a schematic diagram showing a gRNA-tRNA expression cassette encoding gRNAs (a first gRNA and a second gRNA), where the first gRNA is between the first tRNA and the second tRNA, followed by the second gRNA. Figure 3D is a schematic diagram showing a gRNA-tRNA expression cassette encoding gRNAs (a first gRNA and a second gRNA), where the first gRNA is between the first tRNA and the second tRNA, and the second gRNA is between the second tRNA and the third tRNA. Figure 3E is a graph showing % infection and % A>G base editing in HEK cells transduced with recombinant rabies virus particles containing a recombinant rabies virus genome encoding a nucleobase editor and a gRNA encoded between multiple tRNAs. % base editing was measured at the Hek2 and IEDG sites targeted by the Hek2-targeting gRNA and IEDG-targeting gRNA. [Figure 3-2] Same as above. [Figure 4-1] Figure 4A is a schematic diagram showing the ΔG, ΔGL, and ΔALL (i.e., ΔGLNPM) rabies virus replicons encoding the Cre Recombinase-2A-GFP reporter gene. Figure 4B shows fluorescence images of cells transfected with rabies virus vectors in the ΔG, ΔGL, or ΔALL (i.e., ΔGLNPM) background. Each vector encodes the Cre Recombinase-2A-GFP reporter gene. Images were taken 6 days post-transfection for standard and optimized transfections, as shown in Figure 2B. Figure 4C shows a bar graph depicting percent viral entry into reporter cells as measured by GFP fluorescence. [Figure 4-2] Same as above. [Figure 4-3] Same as above. [Figure 5] Figure 5 is a graph showing the percentage of A>G base editing in 293T cells transduced with RABV containing a RABV genome encoding the ABE8.20 base editor. A codon-optimized version of the RABV SAD B19 ΔG virus, designated SynV, was used as the RABV genome. The G gene in the RABV genome was replaced with a base editor gene. U6-driven guide RNAs (gRNAs) targeting the HEK2 and ABCA4 loci were transfected into target cells at the time of infection. The multiplicity of infection (MOI) was indicated based on functional virus titers. [Figure 6] Figure 6 shows two graphs showing the percent C>T base editing in 293T cells transduced with RABV containing RABV genomes encoding one of several different base editors. SynV RABV genomes were used. The G gene in the RABV genome was replaced with a base editor gene. U6-driven gRNAs targeting the HEK2 (top graph) and ABCA4 (bottom graph) loci were transfected into target cells at the time of infection. MOIs are shown based on functional virus titers. [Figure 7] Figure 7 shows two graphs showing the percentage of A>G base editing in 293T cells transduced with RABV containing a RABV genome encoding ABE7.10 bases. The SAD B19 ΔGL RABV genome was used to encode the base editor, replacing the G protein in the RABV genome with the base editor. One set of cells was additionally transfected with a plasmid encoding the RABV L protein (virus + L). A U6-driven gRNA targeting the HEK2 and ABCA4 loci was transfected into target cells in trans at the time of infection. The MOI is indicated based on functional virus titer. [Figure 8]Figure 8 shows fluorescence images of cells transfected with rabies virus vectors in the ΔN, ΔP, ΔM, and ΔL backgrounds. Each vector encodes a Cre recombinase-2A-GFP reporter gene. Either 293T or CE1.30 cells were transfected with or without complementation of the defective gene. Images were taken 4 days postinfection. [Figure 9] Figure 9 shows a bar graph representing the percentage of virus entry into reporter cells (293T or CE1.30) as measured by GFP fluorescence. Supernatants from transfected cells in Figure 8 were used. [Figure 10-1] Figure 10 shows a graph depicting % viral entry into CE1.30 cells as measured by GFP and mScarlet fluorescence. ΔGL, ΔMGL, ΔPMGL, and ΔALL rabies virus replicons encoding Cre-2a-GFP as the transgene were grown in CE1.30 cells and titrated in reporter cells, demonstrating functional delivery of both Cre and GFP mRNA. [Figure 10-2] Same as above. [Figure 11] Figure 11 shows fluorescence images of cells transfected with rabies virus vectors in the ΔGL, ΔMGL, ΔPMGL, and ΔALL backgrounds. Each vector encodes a Cre recombinase-2A-GFP reporter gene. They were transfected into either 293T or CE1.30 cells. Images were taken 3 days postinfection. [Figure 12]Figure 12 shows a schematic diagram of the expression vectors used to express RABV genes. Vector VIR069 contains, from 5' to 3', a first expression cassette consisting of the EF1α promoter, the RABV N gene, a 2a ribosome skipping element, the RABV P gene, a 2a ribosome skipping element, and the RABV M gene. VIR069 contains, from 5' to 3', a second expression cassette consisting of the RPBSA promoter and the BFP-Zeocin selectable marker gene, in the reverse orientation to the first expression cassette. Vector VIR071 contains, from 5' to 3', a first expression cassette consisting of the EF1α promoter, the RABV N gene, an IRES, the RABV P gene, a 2a ribosome skipping element, and the RABV M gene. VIR071 contains, from 5' to 3', a second expression cassette consisting of the RPBSA promoter, the RABV L gene, a 2a ribosome skipping element, and the Zeocin selectable marker gene, in the reverse orientation to the first expression cassette. Vector VIR112 contains, from 5' to 3', a first expression cassette of the EF1α promoter, the RABV N gene, an IRES, the RABV P gene, an IRES, and the RABV M gene. VIR071 contains, in the opposite orientation to the first expression cassette, a second expression cassette of the RPBSA promoter, the RABV L gene, the 2a ribosomal skipping element, and the Zeocin selectable marker gene. DETAILED DESCRIPTION OF THE INVENTION

[0087] Provided herein is a recombinant rabies virus genome comprising a nucleic acid that includes a transgene (e.g., a therapeutic transgene). In certain embodiments, the recombinant rabies virus genome lacks the endogenous G gene that encodes the rabies virus glycoprotein and lacks the endogenous L gene that encodes the rabies virus polymerase. Also provided are methods for producing recombinant rabies virus particles. Such methods generally involve introducing a packaging system described herein into a suitable packaging cell.

[0088] It is understood that the methods described herein are not limited to the particular methods and experimental conditions disclosed herein, as such methods and conditions may vary. It is also understood that the terminology used herein is used for the purpose of describing particular embodiments only and is not intended to be limiting. The methods described herein employ conventional molecular and cell biology and immunological techniques and are well within the skill of those of ordinary skill in the art. Such techniques are well known to those skilled in the art and are described in the scientific literature.

[0089] A.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The following references provide those skilled in the art with general definitions of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings set forth below unless otherwise specified.

[0090] "Adenosine deaminase" refers to a polypeptide or fragment thereof capable of catalyzing the hydrolytic deamination of adenine or adenosine. In some embodiments, the deaminase or deaminase domain is an adenosine deaminase capable of catalyzing the hydrolytic deamination of adenosine to inosine or deoxyadenosine to deoxyinosine. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenine or adenosine in deoxyribonucleic acid (DNA). The adenosine deaminases provided herein (e.g., engineered adenosine deaminases, evolved adenosine deaminases) can be derived from any organism, such as bacteria.

[0091] By "Adenosine deaminase base editor 8 (ABE8) polypeptide" or "ABE8" is meant a base editor as defined herein, including an adenosine deaminase variant comprising an alteration at amino acid position 82 and / or 166 of the following reference sequence: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 8).

[0092] In some embodiments, ABE8 further comprises modifications described herein relative to the reference sequence.

[0093] By "adenosine deaminase base editor 8 (ABE8) polynucleotide" is meant a polynucleotide that encodes ABE8.

[0094] "Administering," as used herein, refers to providing one or more compositions described herein to a patient or subject.

[0095] By "agent" is meant any small molecule compound, antibody, nucleic acid molecule, or polypeptide, or fragment thereof.

[0096] "Alteration" refers to a change (increase or decrease) in the level, structure, or activity of an analyte, gene, or polypeptide, as detected by standard methods known in the art, such as those described herein. As used herein, alteration includes a 10% change in expression levels, a 25% change, a 40% change, and a 50% or greater change in expression levels. In some embodiments, alteration includes an insertion, deletion, or substitution of a nucleic acid base or amino acid.

[0097] By "ameliorate" is meant to reduce, suppress, attenuate, decrease, arrest, or stabilize the occurrence or progression of a disease.

[0098] "Analog" refers to a molecule that is not identical but has similar functional or structural characteristics. For example, a polypeptide analog retains the biological activity of the corresponding native polypeptide while possessing certain biochemical modifications that enhance the function of the analog compared to the native polypeptide. Such biochemical modifications can increase the analog's protease resistance, membrane permeability, or half-life, for example, without altering ligand binding. Analogs can include unnatural amino acids.

[0099] "Base editor (BE)" or "nucleobase editor polypeptide (NBE)" refers to an agent that binds to a polynucleotide and has nucleobase-modifying activity. In various embodiments, a base editor comprises a nucleobase-modifying polypeptide (e.g., a deaminase) and a polynucleotide-programmable nucleotide-binding domain (e.g., Cas9 or Cpf1) that interfaces with a guide polynucleotide (e.g., a guide RNA (gRNA)). Representative nucleic acid and protein sequences of base editors are provided in the Sequence Listing as SEQ ID NOs: 274-283.

[0100] "Base editing activity" refers to acting to chemically modify a base within a polynucleotide. In one embodiment, a first base is converted to a second base. In one embodiment, the base editing activity is cytidine deaminase activity, e.g., converting a target C·G to T·A. In another embodiment, the base editing activity is adenosine or adenine deaminase activity, e.g., converting A·T to G·C.

[0101] The term "base editor system" refers to an intermolecular complex that edits nucleobases of a target nucleotide sequence. In various embodiments, a base editor (BE) system comprises: (1) a polynucleotide-programmable nucleotide-binding domain, a deaminase domain (e.g., cytidine deaminase or adenosine deaminase) for deaminating nucleobases in a target nucleotide sequence, and (2) one or more guide polynucleotides (e.g., guide RNAs) that interface with the polynucleotide-programmable nucleotide-binding domain. In various embodiments, the base editor (BE) system comprises a nucleobase editor domain selected from adenosine deaminase or cytidine deaminase, and a domain with nucleic acid sequence-specific binding activity. In some embodiments, the base editor system comprises: (1) a base editor (BE) comprising a polynucleotide-programmable DNA-binding domain and a deaminase domain for deaminating one or more nucleobases in a target nucleotide sequence, and (2) one or more guide RNAs that interface with the polynucleotide-programmable DNA-binding domain. In some embodiments, the polynucleotide-programmable nucleotide-binding domain is a polynucleotide-programmable DNA-binding domain. In some embodiments, the base editor is a cytidine base editor (CBE). In some embodiments, the base editor is an adenine or adenosine base editor (ABE). In some embodiments, the base editor is an adenine or adenosine base editor (ABE) or a cytidine base editor (CBE).

[0102] "Base editing activity" refers to acting to chemically modify a base within a polynucleotide. In one embodiment, a first base is converted to a second base. In one embodiment, the base editing activity is cytidine deaminase activity, e.g., converting a target C·G to T·A. In another embodiment, the base editing activity is adenosine deaminase activity, e.g., converting A·T to G·C.

[0103] The term "Cas9" or "Cas9 domain" refers to an RNA-guided nuclease comprising a Cas9 protein or a fragment thereof (e.g., a protein comprising an active, inactive, or partially active Cas9 DNA cleavage domain and / or a Cas9 gRNA binding domain). Cas9 nucleases are also sometimes referred to as casnl nucleases or CRISPR (clustered regularly interspaced short palindromic repeat)-associated nucleases.

[0104] The term "conservative amino acid substitution" or "conservative mutation" refers to the replacement of one amino acid with another amino acid that shares common properties. A functional method for defining common properties between individual amino acids is to analyze the normalized frequency of amino acid changes between corresponding proteins of the same organism (Schulz, GE and Schirmer, RH, Principles of Protein Structure, Springer-Verlag, New York (1979)). According to such an analysis, amino acids within a group are preferentially exchanged with each other, thereby defining groups of amino acids whose effects on the overall structure of the protein are most similar to each other (Schulz, GE and Schirmer, RH, supra). Non-limiting examples of conservative mutations include amino acid substitutions, such as the substitution of lysine with arginine, or vice versa, to maintain a positive charge; the substitution of glutamic acid with aspartic acid, or vice versa, to maintain a negative charge; the substitution of serine with threonine, or vice versa, to maintain a free -OH; and the substitution of glutamine with asparagine, or vice versa, to maintain a free -NH2.

[0105] The terms "coding sequence" or "protein-coding sequence," as used interchangeably herein, refer to a segment of a polynucleotide that encodes a protein. A coding sequence is sometimes referred to as an open reading frame. The region or sequence is bounded by a start codon near the 5' end and a stop codon near the 3' end. Stop codons useful in the base editors described herein include the following: Glutamine CAG → TAG stop codon CAA → TAA Arginine CGA → TGA Tryptophan TGG → TGA TGG → TAG TGG→TAA

[0106] "Cytidine deaminase" refers to a polypeptide or fragment thereof capable of catalyzing a deamination reaction that converts an amino group to a carbonyl group. In one embodiment, the cytidine deaminase converts cytosine to uracil or 5-methylcytosine to thymine. PmCDA1 (SEQ ID NOS: 41-42) from Petromyzon marinus (Petromyzon marinus cytosine deaminase 1, "PmCDA1"), AID (activation-induced cytidine deaminase; AICDA) from mammals (e.g., humans, pigs, cows, horses, monkeys, etc.) (exemplary AID polypeptide sequences are provided in the Sequence Listing as SEQ ID NOS: 43-44, 1372, and 1374-1377), and APOBEC are exemplary cytidine deaminases (exemplary APOBEC polypeptide sequences are provided in the Sequence Listing as SEQ ID NOS: 1378-1416, 1421, and 1422). Further exemplary cytidine deaminase (CDA) sequences are provided in the Sequence Listing as SEQ ID NOs: 1373, 1417-1420. Additional exemplary cytidine deaminase sequences, including APOBEC polypeptide sequences, are provided in the Sequence Listing as SEQ ID NOs: 1378-1422. Some aspects of the present disclosure provide base editor proteins and base editor systems capable of deaminating cytosines in nucleic acid molecules (e.g., DNA or RNA). In some embodiments, the base editor protein or base editor system comprises a cytidine deaminase or cytosine deaminase capable of deaminating cytidine to uridine in a nucleic acid molecule (e.g., DNA or RNA). It is understood that any of the cytidine or cytosine deaminases provided herein include variants of naturally occurring cytidine and cytosine deaminases. Such variants can be engineered to increase the efficiency of on-target deaminase activity of the base editor protein or system in a nucleic acid molecule (e.g., DNA or RNA).

[0107] The term "deaminase" or "deaminase domain," as used herein, refers to a protein or enzyme that catalyzes a deamination reaction.

[0108] "Detecting" refers to determining the presence, absence, or amount of an analyte being detected. In one embodiment, a sequence variation in a polynucleotide or polypeptide is detected. In another embodiment, the presence of an indel is detected.

[0109] "Detectable label" refers to a composition that can be attached to a molecule of interest to enable the molecule to be detected by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioisotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (e.g., those commonly used in enzyme-linked immunosorbent assays (ELISAs)), biotin, digoxigenin, or haptens.

[0110] "Disease" means any condition or disorder in which the normal function of a cell, tissue, or organ is impaired or disrupted. Exemplary diseases include neurological diseases and ophthalmologic diseases.

[0111] An "effective amount" refers to the amount of an agent or active compound, e.g., a base editor described herein, required to ameliorate the symptoms of a disease compared to an untreated patient or disease-free individual, i.e., a healthy individual, or is the amount of an agent or active compound sufficient to elicit a desired biological response. The effective amount of the active compound(s) used in practicing the present invention for the therapeutic treatment of a disease will vary depending on the mode of administration, the age, weight, and general health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amount and dosing regimen. Such an amount is also referred to as an "effective" amount. In one embodiment, an effective amount is the amount of a base editor of the present invention sufficient to introduce a modification into a gene of interest in a cell (e.g., a cell in vitro or in vivo). In one embodiment, an effective amount is the amount of a base editor required to achieve a therapeutic effect. Such a therapeutic effect need not be sufficient to modify pathogenic genes in all cells of a subject, tissue, or organ, but need only be sufficient to modify pathogenic genes in about 1%, 5%, 10%, 25%, 50%, 75%, or more of the cells present in the subject, tissue, or organ. In one embodiment, the effective amount is sufficient to ameliorate one or more symptoms of the disease.

[0112] The term "exonuclease" refers to a protein or polypeptide capable of digesting nucleic acids (eg, RNA or DNA) from their free ends.

[0113] The term "endonuclease" refers to a protein or polypeptide that is capable of catalyzing (eg, cleaving) an internal region in a nucleic acid (eg, DNA or RNA).

[0114] By "fragment" is meant a portion of a polypeptide or nucleic acid molecule, which portion contains at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment can contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

[0115] "Guide RNA" or "gRNA" refers to a polynucleotide or complex of polynucleotides that is specific to a target sequence and can form a complex with a polynucleotide-programmable nucleotide-binding domain protein (e.g., Cas9 or Cpf1). In one embodiment, the guide polynucleotide is a guide RNA (gRNA). The gRNA can exist as a complex of two or more RNAs, or as a single RNA molecule.

[0116] By "tRNA" or "transfer RNA" is meant an RNA molecule that contains a secondary and / or tertiary structure that can be cleaved in the cell. Cleavage can occur, for example, by an RNase, such as RNase P or RNase Z.

[0117] "Hybridization" refers to hydrogen bonding between complementary nucleobases, which can be Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.

[0118] By "increase" is meant a positive change of at least 10%, 25%, 50%, 75%, or 100%.

[0119] The term "inhibitor of base repair," "base repair inhibitor," "IBR," or grammatical equivalents thereof, refers to a protein capable of inhibiting the activity of a nucleic acid repair enzyme, e.g., a base excision repair enzyme.

[0120] The terms "isolated," "purified," or "biologically pure" refer to material that has been separated, to varying degrees, from components that normally accompany it as found in its native state. "Isolated" refers to some degree of separation from the original source or environment. "Purified" refers to a greater degree of separation than isolation. A "purified" or "biologically pure" protein is one that has been sufficiently removed from other substances so that any impurities do not significantly affect the biological properties of the protein or cause other adverse effects. That is, a nucleic acid or peptide of the invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term "purified" can indicate that the nucleic acid or protein appears as essentially a single band on an electrophoretic gel. In the case of proteins that can be modified, eg, phosphorylated or glycosylated, different modifications will result in different isolated proteins, which can be individually purified.

[0121] An "isolated polynucleotide" refers to a nucleic acid (e.g., DNA) that is free of the genes that flank it in the naturally occurring genome of the organism from which the nucleic acid molecule of the invention is derived. Thus, the term includes, for example, recombinant DNA incorporated into a vector, a self-replicating plasmid or virus, or the genomic DNA of a prokaryote or eukaryote, or recombinant DNA that exists as a separate molecule independent of other sequences (e.g., cDNA or genomic or cDNA fragments produced by PCR or restriction endonuclease digestion). Additionally, the term includes RNA molecules transcribed from DNA molecules and recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequences.

[0122] By "isolated polypeptide" is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, a polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, by weight, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention. Isolated polypeptides of the invention can be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide, or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0123] The term "mutation," as used herein, refers to the substitution of a residue in a sequence, e.g., a nucleic acid or amino acid sequence, with another residue, or the deletion or insertion of one or more residues in a sequence. Mutations are typically described herein by identifying the original residue, followed by the position of the residue in the sequence and the newly substituted residue. Various methods for making the amino acid substitutions (mutations) provided herein are well known in the art and are described, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4 th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012).

[0124] The terms "nucleic acid" and "nucleic acid molecule," as used herein, refer to a compound comprising a nucleobase and an acid moiety, e.g., a nucleoside, a nucleotide, or a polymer of nucleotides. Typically, polymeric nucleic acids, e.g., nucleic acid molecules comprising three or more nucleotides, are linear molecules in which adjacent nucleotides are linked to each other via phosphodiester linkages. In some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides). In some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising three or more individual nucleotide residues. As used herein, the terms "oligonucleotide" and "polynucleotide" can be used interchangeably to refer to a polymer of nucleotides (e.g., a string of at least three nucleotides). In some embodiments, "nucleic acid" encompasses RNA and single- and / or double-stranded DNA. Nucleic acids can occur naturally, for example, in the context of a genome, transcript, mRNA, tRNA, rRNA, siRNA, snRNA, plasmid, cosmid, chromosome, chromatid, or other naturally occurring nucleic acid molecule. Alternatively, a nucleic acid molecule may be a non-natural molecule, e.g., recombinant DNA or RNA, an artificial chromosome, an engineered genome or fragment thereof, or synthetic DNA, RNA, DNA / RNA hybrid, or contain non-natural nucleotides or nucleosides. Furthermore, the terms "nucleic acid," "DNA," "RNA," and / or similar terms include nucleic acid analogs, e.g., analogs having other than a phosphodiester backbone. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, or chemically synthesized. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids can contain nucleoside analogs, such as analogs with chemically modified bases or sugars and backbone modifications. Nucleic acid sequences are presented in the 5' to 3' direction unless otherwise specified.In some embodiments, nucleic acids are selected from the group consisting of natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, The bases may be or contain: 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine; chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite linkages).

[0125] The term "nuclear localization sequence," "nuclear localization signal," or "NLS" refers to an amino acid sequence that promotes the import of a protein into the cell nucleus. Nuclear localization sequences are known in the art and are described, for example, in International PCT Application PCT / EP2000 / 011690 to Plank et al., filed November 23, 2000, and published May 31, 2001 as WO / 2001 / 038547, the contents of which are incorporated herein by reference for their disclosure of exemplary nuclear localization sequences. In other embodiments, the NLS is an optimized NLS, for example, as described in Koblan et al., Nature Biotech. 2018 doi:10.1038 / nbt.4172. In some embodiments, the NLS comprises the amino acid sequence KRTADGSEFESPKKKRKV (SEQ ID NO: 84), KRPAATKKAGQAKKKK (SEQ ID NO: 85), KKTELQTTNAENKTKKL (SEQ ID NO: 86), KRGINDRNFWRGENGRKTR (SEQ ID NO: 87), RKSGKIAAIVVKRPRK (SEQ ID NO: 88), PKKKRKV (SEQ ID NO: 89), or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 90).

[0126] The terms "nucleobase," "nitrogenous base," or "base," as used interchangeably herein, refer to nitrogen-containing biological compounds that form nucleosides, which are components of nucleotides. The ability of nucleobases to base pair and stack one on top of the other directly leads to the helical structure of long chains such as ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). The five nucleobases, adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U), are referred to as the major or standard nucleobases. Adenine and guanine are derived from purines, while cytosine, uracil, and thymine are derived from pyrimidines. DNA and RNA may also contain other (minor) bases that have been modified. Non-limiting exemplary modified nucleobases include hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine (m5C), and 5-hydromethylcytosine. Hypoxanthine and xanthine can be formed in the presence of mutagens, both resulting from deamination (replacement of an amine group with a carbonyl group). Hypoxanthine can be modified from adenine. Xanthine can be modified from guanine. Uracil can be obtained from the deamination of cytosine. A "nucleoside" consists of a nucleobase and a five-carbon sugar (either ribose or deoxyribose). Examples of nucleosides include adenosine, guanosine, uridine, cytidine, 5-methyluridine (m5U), deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine, and deoxycytidine. Examples of nucleosides containing modified nucleobases include inosine (I), xanthosine (X), 7-methylguanosine (m7G), dihydrouridine (D), 5-methylcytidine (m5C), and pseudouridine (Ψ). A "nucleotide" consists of a nucleobase, a five-carbon sugar (either ribose or deoxyribose), and at least one phosphate group.

[0127] The terms "nucleic acid" and "nucleic acid molecule," as used herein, refer to a compound comprising a nucleobase and an acid moiety, e.g., a nucleoside, nucleotide, or polymer of nucleotides.

[0128] As used herein, the terms "oligonucleotide" and "polynucleotide" can be used interchangeably to refer to a polymer of nucleotides.

[0129] The term "nucleic acid programmable DNA binding protein" or "napDNAbp" can be used interchangeably with "polynucleotide programmable nucleotide binding domain" and refers to a protein that associates with a nucleic acid (e.g., DNA or RNA), such as a guide nucleic acid or guide polynucleotide (e.g., gRNA), to guide the napDNAbp to a specific nucleic acid sequence. In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable DNA binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable RNA binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain is a Cas9 protein. The Cas9 protein can associate with a guide RNA, and the guide RNA guides the Cas9 protein to a specific DNA sequence complementary to the guide RNA. In some embodiments, the napDNAbp is a Cas9 domain, such as a nuclease-active Cas9, Cas9 nickase (nCas9), or nuclease-inactive Cas9 (dCas9). Non-limiting examples of nucleic acid programmable DNA binding proteins include Cas9 (e.g., dCas9 and nCas9), Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, and Cas12j / CasΦ (Cas12j / Casphi).Non-limiting examples of Cas enzymes include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (also known as Csn1 or Csx12), Cas10, Cas10d, Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, Cas12j / CasΦ, Cpf1, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, and Cs a5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, type II Cas effector proteins, type V Cas effector proteins, type VI Cas effector proteins, CARF, DinG, homologs thereof, or modified or engineered forms thereof. Although not specifically listed in this disclosure, other nucleic acid programmable DNA binding proteins are also within the scope of this disclosure. For example, see Makarova et al. "Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?" CRISPR J. 2018 Oct; 1: 325-336. doi: 10.1089 / crispr.2018.0033; Yan et al., "Functionally diverse type V CRISPR-Cas systems" Science. 2019 Jan 4; 363(6422): 88-91. doi: 10.1126 / science.aav7271, the entire contents of each of which are incorporated herein by reference.Exemplary nucleic acid programmable DNA binding proteins and nucleic acid sequences encoding nucleic acid programmable DNA binding proteins are provided in the Sequence Listing as SEQ ID NOs: 223, 230-232, 235-242, 246-256, and 285-294.

[0130] The terms "nucleobase editing domain" or "nucleobase editing protein," as used herein, refer to a protein or enzyme that can catalyze nucleobase modifications in RNA or DNA, such as the deamination of cytosine (or cytidine) to uracil (or uridine) or thymine (or thymidine) and the deamination of adenine (or adenosine) to hypoxanthine (or inosine), as well as non-templated nucleotide addition and insertion. In some embodiments, the nucleobase editing domain is a deaminase domain (e.g., an adenine deaminase or adenosine deaminase, or a cytidine deaminase or cytosine deaminase).

[0131] As used herein, "obtaining" in "obtaining an agent" includes synthesizing, purchasing, or otherwise obtaining the agent.

[0132] As used herein, "patient" or "subject" refers to a mammalian subject or individual who has been diagnosed with, is at risk of, or is suspected of having or developing a disease or disorder. In some embodiments, the term "patient" refers to a mammalian subject who has a higher-than-average likelihood of developing a disease or disorder. Exemplary patients can be humans, non-human primates, cats, dogs, pigs, cows, felines, horses, camels, llamas, goats, sheep, rodents (e.g., mice, rabbits, rats, or guinea pigs), and other mammals that can benefit from the therapies disclosed herein. Exemplary human patients can be male and / or female.

[0133] A "patient in need thereof" or "subject in need thereof," as used herein, refers to a patient who has been diagnosed with, is at risk of, is predetermined to have, or is suspected of having a disease or disorder.

[0134] The terms "pathogenic mutation," "pathogenic variant," "disease-causing mutation," "disease-causing variant," "deleterious mutation," or "predisposing mutation" refer to an alteration or mutation in a gene that increases an individual's susceptibility or predisposition to a particular disease or disorder. In some embodiments, a pathogenic mutation comprises at least one wild-type amino acid substituted with at least one pathogenic amino acid in the protein encoded by the gene.

[0135] The terms "protein," "peptide," "polypeptide," and their grammatical equivalents are used interchangeably herein to refer to a polymer of amino acid residues joined together by peptide (amide) bonds. A protein, peptide, or polypeptide may be natural, recombinant, or synthetic, or any combination thereof.

[0136] As used herein, the term "fusion protein" refers to a hybrid polypeptide comprising protein domains derived from at least two different proteins.

[0137] The term "recombinant" as used herein with respect to a protein or nucleic acid refers to a protein or nucleic acid that does not occur in nature and is a man-made product. For example, in some embodiments, a recombinant protein or nucleic acid molecule comprises an amino acid or nucleotide sequence that contains at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations compared to any naturally occurring sequence.

[0138] By "reduction" is meant a negative change of at least 10%, 25%, 50%, 75%, or 100%.

[0139] A "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence can be a subset or the entirety of a specified sequence, for example, a segment of a full-length cDNA or gene sequence, or a complete cDNA or gene sequence. For polypeptides, the length of a reference polypeptide sequence is generally at least about 16 amino acids, at least about 20 amino acids, at least about 25 amino acids, at least about 35 amino acids, at least about 50 amino acids, or at least about 100 amino acids. For nucleic acids, the length of a reference nucleic acid sequence is generally at least about 50 nucleotides, at least about 60 nucleotides, at least about 75 nucleotides, at least about 100 nucleotides, or at least about 300 nucleotides, or any integer therebetween. In some embodiments, the reference sequence is the wild-type sequence of a protein of interest. In other embodiments, the reference sequence is a polynucleotide sequence encoding a wild-type protein.

[0140] The terms "RNA programmable nuclease" and "RNA-guided nuclease" are used in conjunction with (e.g., bound to or associated with) one or more RNA(s) that are not the target of cleavage. In some embodiments, when an RNA programmable nuclease is complexed with an RNA, it can be referred to as a nuclease:RNA complex. Typically, the bound RNA(s) are referred to as guide RNAs (gRNAs). In some embodiments, the RNA programmable nuclease is a (CRISPR-associated system) Cas9 endonuclease, e.g., Cas9 (Csnl) from Streptococcus pyogenes.

[0141] The term "single nucleotide polymorphism (SNP)" refers to a single nucleotide variation occurring at a particular location in the genome, each variation present to some appreciable extent in a population (eg, greater than 1%).

[0142] By "specifically binds" is meant a nucleic acid molecule, polypeptide, polypeptide / polynucleotide complex, compound, or molecule that recognizes and binds to a polypeptide and / or nucleic acid molecule of the invention, but does not substantially recognize or bind to other molecules in a sample, e.g., a biological sample.

[0143] "Substantially identical" refers to a polypeptide or nucleic acid molecule that exhibits at least 50% identity to a reference amino acid sequence. In one embodiment, the reference sequence is a wild-type amino acid or nucleic acid sequence. In another embodiment, the reference sequence is any one of the amino acid or nucleic acid sequences described herein. In one embodiment, such a sequence is at least 60%, 80%, 85%, 90%, 95%, or even 99% identical at the amino acid or nucleic acid level to the sequence used for comparison.

[0144] Sequence identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary method for determining the degree of identity, the BLAST program can be used, e.g., -3 ~e -100 A probability score between 0 and 1 is used to indicate closely related sequences.

[0145] COBALT is used, for example, with the following parameters: a) Alignment parameters: gap penalty -11, -1 and end gap penalty -5, -1; b) CDD parameters: Use RPS BLAST on; Blast E-value 0.003; Search and recalculate conservative columns on; and c) Query clustering parameters: Use query cluster on; word size 4; max cluster distance 0.8; alphabet regular.

[0146] The EMBOSS Needle is used, for example, with the following parameters: a) Matrix: BLOSUM62; b) Gap start: 10; c) Gap extension: 0.5; d) Output format: Pair; e) End gap penalty: none; f) End gap start: 10; and g) End gap extension: 0.5.

[0147] Nucleic acid molecules useful in the methods of the present invention include any nucleic acid molecule encoding a polypeptide of the present invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but typically exhibit substantial identity. A polynucleotide having "substantial identity" to an endogenous sequence is typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the present invention include any nucleic acid molecule encoding a polypeptide of the present invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but typically exhibit substantial identity. A polynucleotide having "substantial identity" to an endogenous sequence is typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. "Hybridize" refers to pairing between complementary polynucleotide sequences (e.g., genes described herein) or portions thereof to form a double-stranded molecule under various stringency conditions. (See, e.g., Wahl, GM and SL Berger (1987) Methods Enzymol. 152:399; Kimmel, AR (1987) Methods Enzymol. 152:507.)

[0148] For example, stringent salt concentrations are typically less than about 750 mM NaCl and less than 75 mM trisodium citrate, preferably less than about 500 mM NaCl and less than 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and less than 25 mM trisodium citrate. Low stringency hybridization can be achieved in the absence of organic solvents, such as formamide, while high stringency hybridization can be achieved in the presence of at least about 35% formamide, more preferably at least about 50% formamide. Stringent temperature conditions typically include a temperature of at least about 30°C, more preferably at least about 37°C, and most preferably at least about 42°C. Various additional parameters, such as hybridization time, concentration of detergents, e.g., sodium dodecyl sulfate (SDS), and inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency can be achieved by combining these various conditions as needed. In a preferred embodiment, hybridization is performed at 30°C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization is performed at 37°C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization is performed at 42°C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA. Useful variations of these conditions will be readily apparent to those of skill in the art.

[0149] For most applications, the wash step following hybridization can also vary in stringency. Wash stringency conditions can be defined by salt concentration and temperature. As noted above, wash stringency can be increased by decreasing salt concentration or increasing temperature. For example, stringent salt concentrations for wash steps are preferably less than about 30 mM NaCl and less than 3 mM trisodium citrate, most preferably less than about 15 mM NaCl and less than 1.5 mM trisodium citrate. Stringent temperature conditions for wash steps typically include temperatures of at least about 25°C, more preferably at least about 42°C, and even more preferably at least about 68°C. In one embodiment, wash steps are performed at 25°C, 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In another embodiment, wash steps are performed at 42°C, 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the wash steps are performed at 68°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0150] "Split" means to divide into two or more pieces.

[0151] "Split Cas9 protein" or "split Cas9" refers to a Cas9 protein that is provided as an N-terminal fragment and a C-terminal fragment that are encoded by two separate nucleotide sequences. Polypeptides corresponding to the N-terminal and C-terminal portions of the Cas9 protein can be spliced ​​to form a "reconstituted" Cas9 protein.

[0152] The term "target site" refers to a sequence within a nucleic acid molecule that is deaminated by a deaminase (e.g., a cytidine or adenine deaminase) or a fusion protein comprising a deaminase (e.g., a dCas9-adenosine deaminase fusion protein or a base editor disclosed herein).

[0153] As used herein, the terms "treat," "treating," "treatment," and the like refer to reducing or ameliorating a disorder and / or its associated symptoms, or achieving a desired pharmacological and / or physiological effect. It will be understood that treating a disorder or condition does not require, but does not exclude, complete elimination of the disorder, condition, or its associated symptoms. In some embodiments, the effect is therapeutic, i.e., without limitation, the effect is a partial or complete reduction, decrease, neutralization, alleviation, mitigation, lessening in intensity, or cure of the disease and / or adverse symptoms resulting from the disease. In some embodiments, the effect is prophylactic, i.e., the effect inhibits or prevents the onset or recurrence of the disease or condition. To this end, the methods of the present disclosure comprise administering a therapeutically effective amount of a composition described herein.

[0154] "Uracil glycosylase inhibitor" or "UGI" refers to an agent that inhibits the uracil excision repair system. Base editors that contain cytidine deaminase convert cytosine to uracil, which is then converted to thymine by DNA replication or repair. Inclusion of an inhibitor of uracil DNA glycosylase (UGI) in a base editor prevents base excision repair of U back to C. An exemplary UGI comprises the following amino acid sequence: >splP14739IUNGI_BPPB2 Uracil-DNA glycosylase inhibitor MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSD APEYKPWALVIQDSNGENKIKML (SEQ ID NO: 106).

[0155] It is understood that ranges provided herein are shorthand notations for all values ​​within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or subrange in the group consisting of 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, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0156] The recitation of a list of chemical groups in any definition of a variable herein includes definitions of that variable of any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0157] All terms are intended to be understood as understood by one of ordinary skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0158] In this application, the use of the singular includes the plural unless expressly stated otherwise. It should be noted that as used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. In this application, the use of "or" means "and / or" unless expressly stated otherwise. Furthermore, the use of the term "including," as well as other forms such as "include," "includes," and "included," is not limiting.

[0159] As used in the specification and claim(s), the words "comprising" (and all forms of comprising, such as "comprise" and "comprises"), "having" (and all forms of having, such as "have" and "has"), "including" (and all forms of including, such as "includes" and "include"), or "containing" (and all forms of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the disclosure, and vice versa. Furthermore, the compositions of the disclosure can be used to achieve the methods of the disclosure.

[0160] The term "about" or "approximately" means within an acceptable range of error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation, in accordance with practice in the art. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within 10-fold, e.g., within 5-fold, within 2-fold of a value. Where a particular value is described in this application and claims, unless otherwise indicated, the term "about" shall mean within an acceptable range of error for that particular value.

[0161] References herein to "certain embodiments," "some embodiments," "an embodiment," "one embodiment," or "other embodiments" mean that the particular feature, structure, or characteristic described in connection with an embodiment is included in at least some embodiments of the present disclosure, but need not be included in all embodiments.

[0162] B. Recombinant rabies virus Provided herein are recombinant rabies viruses useful for transducing target cells. In one aspect, the recombinant rabies viruses of the present disclosure comprise a rabies virus glycoprotein and a recombinant rabies virus genome. In certain embodiments, the recombinant rabies virus genome encodes a nucleic acid comprising a transgene. In certain embodiments, the recombinant rabies virus genome encodes a nucleic acid comprising a therapeutic transgene. Thus, the recombinant rabies viruses of the present disclosure can be employed in methods for transducing target cells, wherein the recombinant rabies virus comprises a recombinant rabies virus genome comprising a rabies virus glycoprotein and a nucleic acid comprising a transgene (e.g., a therapeutic transgene). Upon transduction of the target cell, the transgene contained within the recombinant rabies virus genome is expressed, producing a transgene product.

[0163] Rabies virus, also known as Rabies lyssavirus, is a single-stranded, negative-strand RNA virus in the genus Lyssavirus, family Rhabdoviridae. Rabies virus has a cylindrical morphology and its structure includes a lipoprotein envelope composed of glycoprotein G, surrounding a helical ribonucleoprotein core. The rabies virus genome contains five genes encoding proteins that facilitate genome transcription and replication and proteins that form the structural elements of the virus. These five genes are the N gene encoding the rabies virus nucleoprotein, the P gene encoding the rabies virus phosphoprotein, the M gene encoding the rabies virus matrix protein, the G gene encoding the rabies virus glycoprotein, and the L gene encoding the rabies virus polymerase. The viral genomic RNA and nucleoprotein combine to form a ribonucleoprotein, which serves as a template for replication and transcription by the rabies virus polymerase (RNA-dependent RNA polymerase).

[0164] In certain embodiments, the recombinant rabies virus genome of the present disclosure has one or more rabies virus genes deleted. For example, the N gene, P gene, M gene, L gene, and / or G gene may be absent from the recombinant rabies virus genome. In certain embodiments, the recombinant rabies virus genome lacks the G gene, which encodes the rabies virus glycoprotein or a functional variant thereof. A recombinant rabies virus genome lacking the G gene, which encodes the rabies virus glycoprotein, prevents the virus from endogenously producing the glycoprotein. Because the glycoprotein is required only for the final stages of the viral life cycle, this deletion prevents the virus from spreading beyond the initially infected cell and does not prevent the virus from completing all of its replication cycles up to that point. In certain embodiments, the recombinant rabies virus genome lacks the L gene, which encodes the rabies virus polymerase or a functional variant thereof. The L gene product is required for both viral gene transcription and viral genome replication, and deletion of the L gene may reduce cytotoxicity in transduced target cells. See, e.g., Chatterjee et al., Nat. Neurosci. (2018) 21(4):638-646, the disclosure of which is incorporated by reference in its entirety. In certain embodiments, the recombinant rabies virus genome lacks the G gene encoding the rabies virus glycoprotein or a functional variant thereof, and lacks the L gene encoding the rabies virus polymerase or a functional variant thereof.

[0165] It will be readily understood by those skilled in the art that the recombinant rabies virus genome lacking a rabies virus gene described herein refers to a rabies virus genome lacking all or a portion of a rabies virus gene. For example, a recombinant rabies virus genome lacking the G gene may lack all or a portion of the G gene, where the portion of the G gene is necessary for the function of the G gene product. In certain embodiments, a truncated, non-functional glycoprotein may be produced by deleting a portion of the G gene necessary for the function of the G gene product. In certain embodiments, a recombinant rabies virus genome lacking the L gene may lack all or a portion of the L gene, where the portion of the L gene is necessary for the function of the L gene product. In certain embodiments, a truncated, non-functional RNA-dependent RNA polymerase may be produced by deleting a portion of the L gene necessary for the function of the L gene product.

[0166] In certain embodiments, the recombinant rabies virus genome of the present disclosure encodes a nucleic acid comprising a transgene. In certain embodiments, the nucleic acid comprising a transgene replaces one or more deleted rabies virus genes described herein. For example, the nucleic acid comprising a transgene can replace all or part of a rabies virus gene. In certain embodiments, the nucleic acid comprising a transgene replaces all or part of the G gene, where the part of the G gene is required for the function of the G gene product. In certain embodiments, the nucleic acid comprising a transgene replaces all or part of the L gene, where the part of the L gene is required for the function of the L gene product. In certain embodiments, the nucleic acid comprising a transgene replaces all or part of the L gene, where the part of the L gene is required for the function of the L gene product, and also replaces all or part of the G gene, where the part of the G gene is required for the function of the G gene product.

[0167] In certain embodiments, a recombinant rabies virus genome of the present disclosure encodes a nucleic acid comprising a transgene, where the transgene replaces one or more of the deleted rabies virus genes described herein. In certain embodiments, the recombinant rabies virus genome comprises an N gene encoding a rabies virus nucleoprotein or a functional variant thereof, a P gene encoding a rabies virus phosphoprotein or a functional variant thereof, and / or an M gene encoding a rabies virus matrix protein or a functional variant thereof.

[0168] Exemplary nucleic acid sequences for the N, P, M, L, and G genes, as well as the amino acid sequences of their gene products, are provided in Table 1. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8]

[0169] In certain embodiments, the recombinant rabies virus genome comprises an N gene having a nucleic acid sequence that is about 60%, about 65%, 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%, or about 99% identical to the nucleic acid sequence set forth in SEQ ID NO:4001. In certain embodiments, the recombinant rabies virus genome comprises an N gene having a nucleic acid sequence that is at least 60%, at least 65%, about 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 nucleic acid sequence set forth in SEQ ID NO: 4001. In certain embodiments, the recombinant rabies virus genome comprises an N gene comprising the nucleic acid sequence set forth in SEQ ID NO: 4001. In certain embodiments, the recombinant rabies virus genome comprises an N gene consisting of the nucleic acid sequence set forth in SEQ ID NO: 4001. In certain embodiments, the N gene encodes an amino acid sequence that is 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%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO:4002. In certain embodiments, the N gene encodes an amino acid sequence that is 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 set forth in SEQ ID NO:4002.In certain embodiments, the N gene encodes an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 4002. In certain embodiments, the N gene encodes an amino acid sequence consisting of the amino acid sequence set forth in SEQ ID NO: 4002.

[0170] In certain embodiments, the recombinant rabies virus genome comprises an L gene having a nucleic acid sequence that is about 60%, about 65%, 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%, or about 99% identical to the nucleic acid sequence set forth in SEQ ID NO:4003. In certain embodiments, the recombinant rabies virus genome comprises an L gene having a nucleic acid sequence that is at least 60%, at least 65%, about 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 nucleic acid sequence set forth in SEQ ID NO: 4003. In certain embodiments, the recombinant rabies virus genome comprises an L gene comprising the nucleic acid sequence set forth in SEQ ID NO: 4003. In certain embodiments, the recombinant rabies virus genome comprises an L gene consisting of the nucleic acid sequence set forth in SEQ ID NO: 4003. In certain embodiments, the L gene encodes an amino acid sequence that is 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%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO:4004. In certain embodiments, the L gene encodes an amino acid sequence that is 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 set forth in SEQ ID NO:4004.In certain embodiments, the L gene encodes an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 4004. In certain embodiments, the L gene encodes an amino acid sequence consisting of the amino acid sequence set forth in SEQ ID NO: 4004.

[0171] In certain embodiments, the recombinant rabies virus genome comprises an M gene having a nucleic acid sequence that is about 60%, about 65%, 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%, or about 99% identical to the nucleic acid sequence set forth in SEQ ID NO:4005. In certain embodiments, the recombinant rabies virus genome comprises an M gene having a nucleic acid sequence that is at least 60%, at least 65%, about 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 nucleic acid sequence set forth in SEQ ID NO: 4005. In certain embodiments, the recombinant rabies virus genome comprises an M gene comprising the nucleic acid sequence set forth in SEQ ID NO: 4005. In certain embodiments, the recombinant rabies virus genome comprises an M gene consisting of the nucleic acid sequence set forth in SEQ ID NO: 4005. In certain embodiments, the M gene encodes an amino acid sequence that is 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%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO:4006. In certain embodiments, the M gene encodes an amino acid sequence that is 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 set forth in SEQ ID NO:4006.In certain embodiments, the M gene encodes an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 4006. In certain embodiments, the M gene encodes an amino acid sequence consisting of the amino acid sequence set forth in SEQ ID NO: 4006.

[0172] In certain embodiments, the recombinant rabies virus genome comprises a P gene having a nucleic acid sequence that is about 60%, about 65%, 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%, or about 99% identical to the nucleic acid sequence set forth in SEQ ID NO:4007. In certain embodiments, the recombinant rabies virus genome comprises a P gene having a nucleic acid sequence that is at least 60%, at least 65%, about 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 nucleic acid sequence set forth in SEQ ID NO: 4007. In certain embodiments, the recombinant rabies virus genome comprises a P gene comprising the nucleic acid sequence set forth in SEQ ID NO: 4007. In certain embodiments, the recombinant rabies virus genome comprises a P gene consisting of the nucleic acid sequence set forth in SEQ ID NO: 4007. In certain embodiments, the P gene encodes an amino acid sequence that is 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%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO:4008. In certain embodiments, the P gene encodes an amino acid sequence that is 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 set forth in SEQ ID NO:4008.In certain embodiments, the P gene encodes an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 4008. In certain embodiments, the P gene encodes an amino acid sequence consisting of the amino acid sequence set forth in SEQ ID NO: 4008.

[0173] In certain embodiments, the recombinant rabies virus genome comprises a G gene having a nucleic acid sequence that is about 60%, about 65%, 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%, or about 99% identical to the nucleic acid sequence set forth in SEQ ID NO:4009. In certain embodiments, the recombinant rabies virus genome comprises a G gene having a nucleic acid sequence that is at least 60%, at least 65%, about 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 nucleic acid sequence set forth in SEQ ID NO: 4009. In certain embodiments, the recombinant rabies virus genome comprises a G gene comprising the nucleic acid sequence set forth in SEQ ID NO: 4009. In certain embodiments, the recombinant rabies virus genome comprises a G gene consisting of the nucleic acid sequence set forth in SEQ ID NO: 4009. In certain embodiments, the G gene encodes an amino acid sequence that is 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%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO:4010. In certain embodiments, the G gene encodes an amino acid sequence that is 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 set forth in SEQ ID NO:4010.In certain embodiments, the G gene encodes an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 4010. In certain embodiments, the G gene encodes an amino acid sequence consisting of the amino acid sequence set forth in SEQ ID NO: 4010.

[0174] Each of the genes contained within the recombinant rabies virus genome of the present disclosure may be operably linked to a transcriptional regulator. In certain embodiments, when genes are linked to a single expression cassette, it may be possible for a single transcriptional regulator to control the expression of the genes. In certain embodiments, each gene is operably linked to a separate transcriptional regulator. In certain embodiments, the transcriptional regulator for each gene may be the same. In certain embodiments, the transcriptional regulator for each gene may be different.

[0175] In certain embodiments, each of the genes is operably linked to a transcriptional regulatory element, and the transcriptional regulatory element is capable of controlling the expression of the gene operably linked to that element. In certain embodiments, the transcriptional regulatory element comprises a transcription initiation signal. The transcription initiation signal can be endogenous or exogenous to the rabies virus. In certain embodiments, the transcription initiation signal is a synthetic transcription initiation signal. In certain embodiments, the nucleic acid encoding the transgene is further operably linked to a transcription termination polyadenylation signal. The transcription termination polyadenylation signal can be endogenous or exogenous to the rabies virus. In certain embodiments, the transcription termination polyadenylation signal is a synthetic transcription termination polyadenylation signal. Examples of suitable transcription initiation signals and transcription termination polyadenylation signals are known to those of skill in the art and are described, for example, in Albertini et al., Adv. Virus. Res. (2011) 79:1-22; Ogino and Green, Viruses (2019) 11(6):504; Ogino et al., Nucl. Acids. Res. (2019) 47(1):299-309; and Ogino and Green, Front. Microbiol. (2019) 10:1490, the disclosures of which are incorporated herein by reference in their entireties.

[0176] In certain embodiments, the recombinant rabies virus is replication-incompetent. As used herein, the term "replication-incompetent" refers to a virus that is unable to complete a full replication cycle in a host cell after infection. Generally, a virus can be made replication-incompetent by the deletion or inactivation of viral genes necessary for viral replication. In certain embodiments, the recombinant rabies virus is made replication-incompetent by the deletion or inactivation of any one or more of the P gene, L gene, M gene, N gene, and G gene in the recombinant rabies virus genome. In other embodiments, the recombinant rabies virus is replication-deficient. As used herein, the term "replication-deficient" refers to a modified virus that is less efficient at completing a full replication cycle in a host cell after infection compared to a wild-type or unmodified virus.

[0177] C. Guide RNA and the recombinant rabies virus genome encoding it In one aspect, the present disclosure provides a recombinant rabies virus genome comprising a nucleic acid encoding a first guide RNA (gRNA) comprising a 5' end and a 3' end, and a nucleic acid encoding a first transfer RNA (tRNA) located either upstream of the 5' end of the nucleic acid encoding the first gRNA or downstream of the 3' end of the nucleic acid encoding the first gRNA.

[0178] In certain embodiments, the recombinant rabies virus genome comprises a nucleic acid encoding a second tRNA. In certain embodiments, the recombinant rabies virus genome comprises a nucleic acid encoding a third tRNA. In certain embodiments, the recombinant rabies virus genome comprises a nucleic acid encoding a fourth tRNA. In certain embodiments, the recombinant rabies virus genome comprises a nucleic acid encoding a fifth tRNA.

[0179] In certain embodiments, the nucleic acid encoding the first tRNA is located upstream of the 5' end of the nucleic acid encoding the first gRNA, and the nucleic acid encoding the second tRNA is located downstream of the 3' end of the nucleic acid encoding the first gRNA.

[0180] In certain embodiments, the nucleotide sequence of the first tRNA and the nucleotide sequence of the second tRNA, the third tRNA, the fourth tRNA, and / or the fifth tRNA are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical.

[0181] In certain embodiments, the first tRNA and the second tRNA, the third tRNA, the fourth tRNA, and / or the fifth tRNA specify the same amino acid, e.g., the first tRNA and the second tRNA have different anticodon loop sequences, each of which corresponds to the same amino acid (e.g., a first tRNA with an anticodon loop sequence comprising 5'GGC3' that specifies Ala, and a second tRNA with an anticodon loop sequence comprising 5'AGC3' that also specifies Ala).

[0182] In certain embodiments, the first tRNA and the second tRNA, the third tRNA, the fourth tRNA, and / or the fifth tRNA specify different amino acids, e.g., the first tRNA and the second tRNA have different anticodon loop sequences, each corresponding to a different amino acid (e.g., a first tRNA with an anticodon loop sequence comprising 5'GGC3' that specifies Ala and a second tRNA with an anticodon loop sequence comprising 5'AAA3' that specifies Phe).

[0183] In certain embodiments, the recombinant rabies virus genome comprises two or more nucleic acids encoding a first tRNA, a second tRNA, a third tRNA, a fourth tRNA, and / or a fifth tRNA. In certain embodiments, the recombinant rabies virus genome comprises two nucleic acids encoding a first tRNA, a second tRNA, a third tRNA, a fourth tRNA, and / or a fifth tRNA. In certain embodiments, the recombinant rabies virus genome comprises three nucleic acids encoding a first tRNA, a second tRNA, a third tRNA, a fourth tRNA, and / or a fifth tRNA. In certain embodiments, the recombinant rabies virus genome comprises four nucleic acids encoding a first tRNA, a second tRNA, a third tRNA, a fourth tRNA, and / or a fifth tRNA. In certain embodiments, the recombinant rabies virus genome comprises five nucleic acids encoding a first tRNA, a second tRNA, a third tRNA, a fourth tRNA, and / or a fifth tRNA.

[0184] In certain embodiments, the recombinant rabies virus genome comprises nucleic acid encoding a second gRNA, a third gRNA, a fourth gRNA, and / or a fifth gRNA.

[0185] In certain embodiments, the two or more nucleic acids encode the same gRNA. In certain embodiments, the two or more nucleic acids encode at least one different gRNA. In certain embodiments, the nucleotide sequence of the first gRNA and the nucleotide sequence of the second gRNA, the third gRNA, the fourth gRNA, and / or the fifth gRNA are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical.

[0186] In certain embodiments, the first gRNA and the second gRNA, the third gRNA, the fourth gRNA, and / or the fifth gRNA specifically hybridize to the same target nucleic acid sequence, while in certain embodiments, the first gRNA and the second gRNA, the third gRNA, the fourth gRNA, and / or the fifth gRNA specifically hybridize to different target nucleic acid sequences.

[0187] In certain embodiments, the first tRNA, the second tRNA, the third tRNA, the fourth tRNA, and / or the fifth tRNA are each selected from the group consisting of tRNA-ala, tRNA-arg, tRNA-asn, tRNA-asp, tRNA-cys, tRNA-gln, tRNA-gly, tRNA-his, tRNA-ile, tRNA-leu, tRNA-lys, tRNA-met, tRNA-phe, tRNA-pro, tRNA-pyl, tRNA-sec, tRNA-ser, tRNA-thr, tRNA-trp, tRNA-tyr, and tRNA-val.

[0188] In certain embodiments, the recombinant rabies virus genome comprises a nucleic acid encoding a negative-strand RNA viral gene.

[0189] In certain embodiments, the recombinant rabies virus genome comprises a nucleic acid encoding a transgene (e.g., a nucleobase editor).

[0190] In certain embodiments, the nucleic acid encoding the first gRNA and the nucleic acid encoding the first tRNA are located between two nucleic acids that respectively encode negative-strand RNA viral genes.

[0191] In certain embodiments, the nucleic acid encoding the first gRNA and the nucleic acid encoding the first tRNA are located between two nucleic acids that each encode a transgene.

[0192] In certain embodiments, the nucleic acid encoding the first gRNA and the nucleic acid encoding the first tRNA are located between the nucleic acid encoding the negative-strand RNA viral gene and the nucleic acid encoding the transgene.

[0193] In certain embodiments, the recombinant rabies virus genome comprises a gRNA expression cassette comprising, from 5' to 3', a negative-strand RNA viral transcription initiation signal, a nucleic acid encoding a tRNA, a nucleic acid encoding a gRNA, and a transcription termination polyadenylation signal.

[0194] In certain embodiments, the recombinant rabies virus genome comprises a gRNA expression cassette comprising, from 5' to 3', a negative-strand RNA viral transcription initiation signal, a nucleic acid encoding a first tRNA, a nucleic acid encoding a first gRNA, a nucleic acid encoding a second tRNA, and a transcription termination polyadenylation signal.

[0195] In certain embodiments, the recombinant rabies virus genome comprises a gRNA expression cassette comprising, from 5' to 3', a negative-strand RNA viral transcription initiation signal, a nucleic acid encoding a first tRNA, a nucleic acid encoding a first gRNA, a nucleic acid encoding a second tRNA, a nucleic acid encoding a second gRNA, and a transcription termination polyadenylation signal.

[0196] In certain embodiments, the recombinant rabies virus genome comprises a gRNA expression cassette comprising, from 5' to 3', a negative-strand RNA viral transcription initiation signal, a nucleic acid encoding a first tRNA, a nucleic acid encoding a first gRNA, a nucleic acid encoding a second tRNA, a nucleic acid encoding a second gRNA, and a transcription termination polyadenylation signal.

[0197] In certain embodiments, the recombinant rabies virus genome comprises a gRNA expression cassette comprising, from 5' to 3', a negative-strand RNA viral transcription initiation signal, a nucleic acid encoding a first tRNA, a nucleic acid encoding a first gRNA, a nucleic acid encoding a second tRNA, a nucleic acid encoding a second gRNA, a nucleic acid encoding a third tRNA, and a transcription termination polyadenylation signal.

[0198] In certain embodiments of gRNA expression cassettes, the nucleic acids encoding the first tRNA, the second tRNA, and / or the third tRNA are identical. In certain embodiments of gRNA expression cassettes, the nucleic acids encoding the first tRNA, the second tRNA, and / or the third tRNA are different. In certain embodiments of gRNA expression cassettes, the nucleotide sequence of the first tRNA and the nucleotide sequence of the second tRNA are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In certain embodiments of gRNA expression cassettes, the first tRNA and the second tRNA specify the same amino acid. In certain embodiments of gRNA expression cassettes, the first tRNA and the second tRNA specify different amino acids. In certain embodiments of gRNA expression cassettes, the nucleic acids encoding the first gRNA and / or the second gRNA are identical. In certain embodiments of gRNA expression cassettes, the nucleic acids encoding the first gRNA and / or the second gRNA are different. In certain embodiments of gRNA expression cassettes, the nucleotide sequence of the first gRNA and the nucleotide sequence of the second gRNA are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In certain embodiments of gRNA expression cassettes, the first gRNA and the second gRNA specifically hybridize to the same target nucleic acid sequence. In certain embodiments of gRNA expression cassettes, the first gRNA and the second gRNA specifically hybridize to different target nucleic acid sequences. In certain embodiments of gRNA expression cassettes, the transcription termination polyadenylation signal comprises an endogenous transcription termination polyadenylation signal. In certain embodiments of gRNA expression cassettes, the transcription termination polyadenylation signal comprises a heterologous transcription termination polyadenylation signal.

[0199] D. Therapeutic Transgenes In certain embodiments, the recombinant rabies virus genome of the present disclosure encodes a nucleic acid comprising a therapeutic transgene. As used herein, the term "therapeutic" refers to treatment and / or prevention. As used herein, the term "therapeutic transgene" refers to a transgene encoding a transgene product capable of providing treatment and / or prevention to a subject in need thereof. In certain embodiments, the therapeutic effect is achieved by suppression, amelioration, or eradication of a disease state from which the subject suffers. A therapeutic transgene may encode any therapeutic agent capable of providing treatment and / or prevention to a subject in need thereof, resulting in the suppression, amelioration, or eradication of a disease state from which the subject suffers. As used herein, the term "therapeutic transgene" refers to a transgene encoding a transgene product that, when processed, e.g., intracellularly, is capable of providing treatment and / or prevention to a subject in need thereof.

[0200] In certain embodiments, the nucleic acid encoding a therapeutic transgene is greater than about 300 bp, about 400 bp, about 500 bp, about 600 bp, about 700 bp, about 800 bp, about 900 bp, about 1,000 bp, about 1,100 bp, about 1,200 bp, about 1,300 bp, about 1,400 bp, about 1,500 bp, about 1,600 bp, about 1,700 bp, about 1,800 bp, about 1,900 bp, about 2,000 bp, about 2,100 bp, about 2,200 bp, about 2,300 bp, about 2,400 bp, about 2,500 bp, about 2,600 bp, about 2,700 bp, about 2,800 bp, about 2,900 bp, or about 3,000 bp.

[0201] In certain embodiments, the nucleic acid encoding the therapeutic transgene is larger than about 300 bp (e.g., the therapeutic transgene is about 350 bp, about 400 bp, about 450 bp, about 500 bp, about 550 bp, about 600 bp, or about 650 bp). In certain embodiments, the nucleic acid encoding the therapeutic transgene is larger than about 650 bp (e.g., the therapeutic transgene is about 700 bp, about 750 bp, about 800 bp, about 850 bp, about 900 bp, about 950 bp, or about 1,000 bp). In certain embodiments, the nucleic acid encoding the therapeutic transgene is larger than about 1,000 bp (e.g., the therapeutic transgene is about 1,500 bp, about 2,000 bp, about 2,500 bp, or about 3,000 bp). In certain embodiments, the nucleic acid encoding the therapeutic transgene is larger than about 3,000 bp (eg, the therapeutic transgene is about 3,500 bp, about 4,000 bp, or about 4,500 bp).

[0202] In certain embodiments, the nucleic acid encoding the therapeutic transgene is larger than about 4,500 bp (e.g., the therapeutic transgene is about 5,000 bp, about 5,500 bp, about 6,000 bp, about 6,500 bp, about 7,000 bp, about 7,500 bp, about 8,000 bp, or about 8,500 bp).

[0203] In certain embodiments, the nucleic acid encoding the therapeutic transgene is larger than about 8,500 bp (eg, the therapeutic transgene is about 9,000 bp, about 9,500 bp, or about 10,000 bp).

[0204] In certain embodiments, the nucleic acid encoding the therapeutic transgene is larger than about 10,000 bp (e.g., the therapeutic transgene is about 10,500 bp, about 11,000 bp, about 11,500 bp, about 12,000 bp, about 12,500 bp, about 13,000 bp, about 13,500 bp, about 14,000 bp, about 14,500 bp, or about 15,000 bp).

[0205] In certain embodiments, the nucleic acid encoding the therapeutic transgene is between about 4,000 bp and about 6,000 bp (eg, the therapeutic transgene is about 4,000 bp, about 4,500 bp, about 5,000 bp, about 5,500 bp, or about 6,000 bp).

[0206] In certain embodiments, the therapeutic transgene encodes a therapeutic nucleic acid. The therapeutic transgene may encode any therapeutic nucleic acid known in the art, including, but not limited to, any antisense RNA (single-stranded RNA), any small interfering RNA (double-stranded RNA), any RNA aptamer, and / or any messenger RNA (mRNA). For example, the therapeutic transgene may encode, but is not limited to, miRNA, miRNA mimic, siRNA, shRNA, gRNA, long non-coding RNA, enhancer RNA, RNA aptazyme, RNA aptamer, antagomiR, and / or synthetic RNA. In certain embodiments, the therapeutic nucleic acid may be an RNA-binding site, such as an miRNA-binding site. Various other types of therapeutic nucleic acids are known to those skilled in the art.

[0207] In certain embodiments, the therapeutic transgene encodes a therapeutic polypeptide. The therapeutic transgene may encode any therapeutic polypeptide known in the art, including, but not limited to, a therapeutic polypeptide capable of replacing a missing or abnormal protein, a therapeutic polypeptide capable of enhancing an existing pathway, a therapeutic polypeptide capable of providing a new function or activity (e.g., a new function or activity beneficial to a subject suffering from the deficiency), a therapeutic polypeptide that interferes with a molecule or organism (e.g., an organism other than the one hosting the target cell), and / or a therapeutic polypeptide that delivers other compounds or proteins (e.g., radionuclides, cytotoxic drugs, and / or effector proteins). For example, the therapeutic transgene may encode, but is not limited to, a nucleic acid-modifying protein (e.g., an adenine or cytidine base editor) or system, an antibody or antibody-based drug, an anticoagulant, a blood factor, a bone morphogenetic protein, an artificial protein scaffold, an enzyme, an Fc fusion protein, a growth factor, a hormone, an interferon, an interleukin, and / or a thrombolytic agent. Various other types of therapeutic polypeptides are known to those of skill in the art.

[0208] In certain embodiments, the therapeutic transgene encodes a nucleic acid editing system or a component thereof. In some embodiments, the therapeutic transgene encodes a protein comprising a nucleic acid binding protein (e.g., a zinc finger, TALE, or nucleic acid-programmable nucleic acid binding protein such as Cas9). In some embodiments, the component of the nucleic acid editing system is a nucleic acid-programmable nucleic acid binding protein (e.g., Cas9). In some embodiments, the component of the nucleic acid editing system is a guide RNA (gRNA).

[0209] In some embodiments, the therapeutic transgene encodes a CRISPR system. In some embodiments, the CRISPR system comprises a nucleobase editor comprising a polynucleotide-programmable nucleotide-binding domain and a nucleobase-editing domain. In some embodiments, the nucleobase-editing domain is adenosine deaminase, cytidine deaminase, cytosine deaminase, or a functional variant thereof (e.g., a functional variant capable of deaminating a nucleobase in a nucleic acid molecule such as DNA or RNA). In some embodiments, the nucleobase-editing domain is adenosine deaminase. In some embodiments, the adenosine deaminase is ABE7.10. In some embodiments, the polynucleotide-programmable nucleotide-binding domain is a Cas9 polypeptide, a Cas12 polypeptide, or a functional variant thereof. In some embodiments, the CRISPR system further comprises a guide RNA (gRNA) or a nucleic acid encoding a gRNA.

[0210] In some embodiments, the therapeutic transgene encodes a nucleobase-modifying protein (e.g., a base editor protein). In some embodiments, the therapeutic transgene encodes an adenosine base editor (e.g., ABE7.10). In some embodiments, the therapeutic transgene encodes a cytidine base editor. In some embodiments, the therapeutic transgene encodes a cytosine base editor capable of deaminating cytosines in DNA or RNA.

[0211] In certain embodiments, the therapeutic transgene encodes a nucleic acid editing system, e.g., a base editor system as further described herein.

[0212] It will be readily apparent to one of skill in the art that the recombinant rabies virus genome of the present disclosure described herein encodes a nucleic acid comprising a therapeutic transgene, wherein the therapeutic transgene encodes a therapeutic polypeptide and / or a therapeutic nucleic acid; for example, in certain embodiments, the therapeutic transgene encodes a combination of a therapeutic polypeptide and a therapeutic nucleic acid. In certain embodiments, the therapeutic transgene encodes one or more therapeutic polypeptides. In certain embodiments, the therapeutic transgene encodes one or more therapeutic nucleic acids. In certain embodiments, the therapeutic transgene encodes a combination of one or more therapeutic polypeptides and one or more therapeutic nucleic acids. Delivery of a combination of a therapeutic polypeptide and a therapeutic nucleic acid to a target cell can serve various purposes known to those of skill in the art. In certain embodiments, a therapeutic polypeptide can be delivered to a target cell, with delivery being detargeted to a certain other cell type. For example, a therapeutic transgene encodes a therapeutic polypeptide and / or a therapeutic nucleic acid and can also include an miRNA binding site. The miRNA binding site can function to detarget the cell type. For example, miRNA122a, which is exclusively expressed in the liver, can be employed for hepatocyte detargeting. See, e.g., Dhungel et al., Molecules (2018) 23(7):1500.

[0213] In certain embodiments, the therapeutic transgene further encodes one or more reporter sequences. When expressed in target cells, the reporter sequence generates a detectable signal, either directly or indirectly. Examples of suitable reporter sequences include, but are not limited to, sequences encoding fluorescent proteins (e.g., GFP, RFP, YFP), alkaline phosphatase, thymidine kinase, chloramphenicol acetyltransferase (CAT), luciferase, β-galactosidase (LacZ), and β-lactamase. Sequences encoding cell surface membrane-associated proteins may also be suitable as reporter sequences, including, for example, membrane-associated proteins bound by high-affinity antibodies, such as influenza hemagglutinin protein (HA), CD2, CD4, CD8, and others known to those skilled in the art, such as membrane-associated proteins tagged with antigenic domains (e.g., HA tag, FLAG tag, Myc tag, polyhistidine tag).

[0214] In certain embodiments, the therapeutic transgene does not encode a reporter gene and / or a selectable marker. In certain embodiments, the therapeutic transgene does not encode a fluorescent reporter protein (e.g., GFP, YFP, RFP, tdTomato). In certain embodiments, the therapeutic transgene does not encode β-galactosidase (LacZ). In certain embodiments, the therapeutic transgene does not encode chloramphenicol acetyltransferase (CAT).

[0215] In certain embodiments, the therapeutic transgene does not encode a polymerase (eg, a DNA polymerase, a DNA-dependent RNA polymerase, an RNA-dependent DNA polymerase (RT), a telomerase).

[0216] In certain embodiments, the therapeutic transgene does not encode a site-specific recombinase (e.g., Cre, FLP, Hin, or Tre recombinase).

[0217] In certain embodiments, the therapeutic transgene does not encode a viral antigen.

[0218] In certain embodiments, the therapeutic transgene does not encode a pro-apoptotic protein (eg, cytochrome c).

[0219] In certain embodiments, the therapeutic transgene does not encode an immunoglobulin (e.g., an immunoglobulin heavy and / or light chain).

[0220] In certain embodiments, the therapeutic transgene does not encode a neurotransmitter, neuropeptide, receptor, nerve growth factor, or neurogene.

[0221] In certain embodiments, the therapeutic transgene encodes a therapeutic polypeptide and / or therapeutic nucleic acid, and the therapeutic polypeptide and / or therapeutic nucleic acid is secreted (e.g., secreted from the cell). For example, a recombinant rabies virus genome of the present disclosure described herein can be introduced into a target cell, where the recombinant rabies virus genome encodes a nucleic acid comprising a therapeutic transgene, and the therapeutic transgene encodes a secreted therapeutic polypeptide and / or therapeutic nucleic acid (e.g., a secretable therapeutic transgene and / or a secretable therapeutic nucleic acid). The therapeutic polypeptide and / or nucleic acid, upon expression, can be secreted outside the target cell. In certain embodiments, the therapeutic polypeptide and / or nucleic acid, upon expression, is secreted by endogenous elements present on the therapeutic polypeptide and / or nucleic acid (e.g., an endogenous signal peptide that directs extracellular secretion). In certain embodiments, the therapeutic polypeptide and / or nucleic acid, upon expression, is secreted by exogenous elements present on the therapeutic polypeptide and / or nucleic acid (e.g., an exogenous signal peptide that directs extracellular secretion). Delivery of secretable therapeutic polypeptides and / or nucleic acids is useful for treating certain diseases. For example, lysosomal storage diseases (LSDs), which result from metabolic dysfunction of the lysosome, have unique cross-collection properties that allow specific extracellular LSD enzymes to be internalized and targeted to the lysosomes of enzyme-deficient or enzyme-abnormal cells. The cross-collection properties of certain enzymes are the basis of an approved treatment known as enzyme replacement therapy. See, e.g., Rastall and Amalfitano, Appl. Clin. Genet. (2015) 8:157-169.

[0222] In certain embodiments, a recombinant rabies virus genome of the present disclosure comprises a transcriptional control element operably linked to a nucleic acid encoding a transgene. The transcriptional control element is capable of controlling expression of the transgene (e.g., expression of an encoded therapeutic polypeptide and / or nucleic acid) operably linked to the element. In certain embodiments, the transcriptional control element comprises a transcription initiation signal. The transcription initiation signal may be endogenous or exogenous to the rabies virus. In certain embodiments, the transcription initiation signal is a synthetic transcription initiation signal. In certain embodiments, the nucleic acid encoding the transgene is further operably linked to a transcription termination polyadenylation signal. The transcription termination polyadenylation signal may be endogenous or exogenous to the rabies virus. In certain embodiments, the transcription termination polyadenylation signal is a synthetic transcription termination polyadenylation signal. Examples of suitable transcription initiation signals and transcription termination polyadenylation signals are known to those of skill in the art and are described, for example, in Albertini et al., Adv. Virus. Res. (2011) 79:1-22; Ogino and Green, Viruses (2019) 11(6):504; and Ogino and Green, Front. Microbiol. (2019) 10:1490, the disclosures of which are incorporated herein by reference in their entireties.

[0223] The recombinant rabies virus genome of the present disclosure, which comprises a nucleic acid comprising a therapeutic transgene, may further comprise any element known to those of skill in the art that supports and / or enhances expression of the therapeutic transgene.

[0224] The recombinant rabies virus genome of the present disclosure is incorporated into a recombinant rabies virus particle by the methods described herein. In certain embodiments, the recombinant rabies virus particle of the present disclosure comprises a recombinant rabies virus genome comprising a rabies virus glycoprotein and a nucleic acid comprising a therapeutic transgene described herein. In certain embodiments, the recombinant rabies virus particle comprises a rabies virus glycoprotein and a recombinant rabies virus genome comprising a nucleic acid comprising a therapeutic transgene, wherein the genome lacks an endogenous G gene encoding the rabies virus glycoprotein. In certain embodiments, the recombinant rabies virus particle comprises a rabies virus glycoprotein and a recombinant rabies virus genome comprising a nucleic acid comprising a therapeutic transgene, wherein the genome lacks an endogenous G gene encoding the rabies virus glycoprotein and the genome lacks an endogenous L gene encoding the rabies virus polymerase.

[0225] E. Nucleobase Editor In certain exemplary embodiments, a therapeutic transgene useful in the methods and compositions described herein is a nucleobase editor that edits, modifies, or alters a target nucleotide sequence of a polynucleotide. The nucleobase editors described herein typically include a polynucleotide-programmable nucleotide-binding domain and a nucleobase-editing domain (e.g., adenosine deaminase or cytidine deaminase). The polynucleotide-programmable nucleotide-binding domain, in conjunction with an associated guide polynucleotide (e.g., gRNA), can specifically bind to the target polynucleotide sequence, thereby positioning the base editor at the target nucleic acid sequence where editing is desired.

[0226] Polynucleotide-programmable nucleotide-binding domains The polynucleotide-programmable nucleotide binding domain binds to a polynucleotide (e.g., RNA, DNA). The base editor polynucleotide-programmable nucleotide binding domain may itself comprise one or more domains (e.g., one or more nuclease domains). In some embodiments, the nuclease domain of the polynucleotide-programmable nucleotide binding domain may comprise an endonuclease or an exonuclease. An endonuclease can cleave one strand of a double-stranded nucleic acid or both strands of a double-stranded nucleic acid molecule. In some embodiments, the nuclease domain of the polynucleotide-programmable nucleotide binding domain can cleave zero, one, or two strands of a target polynucleotide.

[0227] Non-limiting examples of polynucleotide-programmable nucleotide-binding domains that can be incorporated into base editors include domains derived from CRISPR proteins, restriction nucleases, meganucleases, TAL nucleases (TALENs), and zinc finger nucleases (ZFNs). In some embodiments, the base editor comprises a polynucleotide-programmable nucleotide-binding domain comprising a naturally occurring or modified protein or portion thereof capable of binding to a nucleic acid sequence via a bound guide nucleic acid during CRISPR (i.e., Clustered Regularly Interspaced Short Palindromic Repeats)-mediated nucleic acid modification. Such proteins are referred to herein as "CRISPR proteins." Accordingly, disclosed herein are base editors comprising a polynucleotide-programmable nucleotide-binding domain comprising all or a portion of a CRISPR protein (i.e., a base editor comprising all or a portion of a CRISPR protein as a domain, also referred to as the "CRISPR protein-derived domain" of the base editor). The CRISPR protein-derived domain incorporated into the base editor may be modified compared to a wild-type or naturally occurring CRISPR protein. For example, as described below, a domain derived from a CRISPR protein can contain one or more mutations, insertions, deletions, rearrangements, and / or recombinations compared to a wild-type or naturally occurring CRISPR protein.

[0228] Cas proteins that can be used herein include class 1 and class 2. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 or Csx12), Cas10, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, and Csb3. , Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, Cas12a / Cpf1, Cas12b / C2c1 (e.g., SEQ ID NO: 258), Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, and Cas12j / CasΦ, CARF, DinG, homologs thereof, or modified forms thereof. CRISPR enzymes can induce cleavage of one or both strands of a target sequence, such as within the target sequence and / or within the complement of the target sequence. For example, CRISPR enzymes can induce cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500 or more base pairs from the first or last nucleotide of the target sequence.

[0229] A vector can be used that encodes a CRISPR enzyme that is mutated relative to the corresponding wild-type enzyme, such that the mutant CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence. A Cas protein (e.g., Cas9, Cas12) or Cas domain (e.g., Cas9, Cas12) can refer to a polypeptide or domain that contains at least or at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or sequence homology to a wild-type exemplary Cas polypeptide or Cas domain. Cas (e.g., Cas9, Cas12) can refer to a wild-type or modified Cas protein that can contain amino acid changes such as deletions, insertions, substitutions, variants, mutations, fusions, chimeras, or any combination thereof.

[0230] In some embodiments, the base editor CRISPR protein-derived domains are derived from Corynebacterium ulcerans (NCBI Refs:NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs:NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref:NC_021284.1); Prevotella intermedia (NCBI Ref:NC_017861.1); Spiroplasma taiwanense (NCBI Ref:NC_021846.1); Streptococcus iniae (NCBI Ref:NC_021314.1); Belliella baltica (NCBI Ref:NC_018010.1); Psychroflexus torquis (NCBI Ref:NC_018721.1); Streptococcus thermophilus (NCBI Ref:YP_820832.1); Listeria innocua (NCBI Ref:NP_472073.1); Campylobacter jejuni (NCBI Ref:YP_002344900.1); Neisseria meningitidis (NCBI Ref:YP_002342100.1), Streptococcus pyogenes, or Staphylococcus aureus.

[0231] The sequence and structure of Cas9 nuclease are well known to those of skill in the art (see, e.g., "Complete genome sequence of an M1 strain of Streptococcus pyogenes," Ferretti et al., Proc. Natl. Acad. Sci. USA 98:4658-4663 (2001); "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III," Deltcheva E., et al., Nature 471:602-607 (2011); and "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity," Jinek M., et al., Science 337:816-821 (2012), the entire contents of each of which are incorporated herein by reference). Cas9 orthologs have been described in various species, including, but not limited to, S. pyogenes and S. thermophilus. Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on the present disclosure, including Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier, "The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems" (2013) RNA Biology 10:5, 726-737, the entire contents of which are incorporated herein by reference.

[0232] High-fidelity Cas9 domain Some aspects of the present disclosure provide high-fidelity Cas9 domains. High-fidelity Cas9 domains are known in the art and are described, for example, in Kleinstiver, B.P., et al., "High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects," Nature 529, 490-495 (2016); and Slaymaker, I.M., et al., "Rationally engineered Cas9 nucleases with improved specificity," Science 351, 84-88 (2015), the entire contents of each of which are incorporated herein by reference. An exemplary high-fidelity Cas9 domain is provided in the Sequence Listing as SEQ ID NO: 1423. In some embodiments, the high-fidelity Cas9 domain is an engineered Cas9 domain containing one or more mutations that reduce the electrostatic interaction between the Cas9 domain and the sugar-phosphate backbone of DNA compared to the corresponding wild-type Cas9 domain. High-fidelity Cas9 domains with reduced electrostatic interactions with the sugar-phosphate backbone of DNA have fewer off-target effects. In some embodiments, the Cas9 domain (e.g., a wild-type Cas9 domain (SEQ ID NOs: 223 and 233)) comprises one or more mutations that reduce the association between the Cas9 domain and the sugar-phosphate backbone of DNA. In some embodiments, the Cas9 domain comprises one or more mutations that reduce the association between the Cas9 domain and the sugar-phosphate backbone of DNA by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70%.

[0233] In some embodiments, any of the Cas9 fusion proteins provided herein contain one or more of the following mutations: D10A, N497X, R661X, Q695X, and / or Q926X, or the corresponding mutations in any of the amino acid sequences provided herein, where X is any amino acid. In some embodiments, the high-fidelity Cas9 enzyme is SpCas9(K855A), eSpCas9(1.1), SpCas9-HF1, or a high-precision Cas9 variant (HypaCas9). In some embodiments, the modified Cas9 eSpCas9(1.1) contains an alanine substitution that weakens the interaction between the HNH / RuvC groove and the non-target DNA strand, thereby preventing strand separation and cleavage at off-target sites. Similarly, SpCas9-HF1 reduces off-target editing due to an alanine substitution that prevents Cas9 from interacting with the DNA phosphate backbone. HypaCas9 contains mutations in the REC3 domain (SpCas9 N692A / M694A / Q695A / H698A) that increase Cas9 proofreading and target discrimination. All three high-fidelity enzymes produce fewer off-target edits than wild-type Cas9.

[0234] Cas9 domains with reduced exclusivity Typically, Cas9 proteins, such as Cas9 from S. pyogenes (spCas9), require a "protospacer adjacent motif (PAM)" or PAM-like motif, which is a 2-6 base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system. The presence of the NGG PAM sequence is required for binding to specific nucleic acid regions, where "N" in "NGG" is adenosine (A), thymidine (T), or cytosine (C), and G is guanosine. This can limit the ability to edit desired bases in the genome. In some embodiments, the base editing fusion proteins provided herein may need to be placed at a precise location, e.g., in the region containing the target base upstream of the PAM. See, e.g., Komor, AC, et al., "Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage," Nature 533, 420-424 (2016), the entire contents of which are incorporated herein by reference. Exemplary polypeptide sequences of spCas9 proteins capable of binding to PAM sequences are provided in the Sequence Listing as SEQ ID NOS: 223, 234, and 1304-1307. Thus, in some embodiments, any of the fusion proteins provided herein can contain a Cas9 domain capable of binding to a nucleotide sequence that does not contain a canonical (e.g., NGG) PAM sequence. Cas9 domains that bind to non-canonical PAM sequences have been described in the art and would be apparent to one of skill in the art.For example, Cas9 domains that bind to non-canonical PAM sequences are described in Kleinstiver, B.P., et al., "Engineered CRISPR-Cas9 nucleases with altered PAM specificities" Nature 523, 481-485 (2015); and Kleinstiver, B.P., et al., "Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition" Nature Biotechnology 33, 1293-1298 (2015), the entire contents of each of which are incorporated herein by reference.

[0235] Nickase In some embodiments, a polynucleotide-programmable nucleotide-binding domain can comprise a nickase domain. As used herein, the term "nickase" refers to a polynucleotide-programmable nucleotide-binding domain, including a nuclease domain, capable of cleaving only one of the two strands of a double-stranded nucleic acid molecule (e.g., DNA). In some embodiments, a nickase can be derived from a fully catalytically active (e.g., native) form of a polynucleotide-programmable nucleotide-binding domain by introducing one or more mutations into the active polynucleotide-programmable nucleotide-binding domain. For example, if a polynucleotide-programmable nucleotide-binding domain comprises a nickase domain derived from Cas9, the Cas9-derived nickase domain can comprise a D10A mutation and a histidine at position 840. In such an embodiment, residue H840 retains catalytic activity and is therefore capable of cleaving one strand of a nucleic acid duplex. In another example, a Cas9-derived nickase domain can comprise an H840A mutation, but the amino acid residue at position 10 remains D. In some embodiments, a nickase can be derived from a fully catalytically active (e.g., native) form of a polynucleotide-programmable nucleotide-binding domain by removing all or part of a nuclease domain that is not required for nickase activity. For example, if the polynucleotide-programmable nucleotide-binding domain comprises a nickase domain derived from Cas9, the Cas9-derived nickase domain can comprise a deletion of all or part of the RuvC domain or the HNH domain.

[0236] In some embodiments, the wild-type Cas9 corresponds to or comprises the following amino acid sequence: [ka] (Single underline: HNH domain; double underline: RuvC domain).

[0237] In some embodiments, the strand of a nucleic acid duplex target polynucleotide sequence that is cleaved by a base editor comprising a nickase domain (e.g., a Cas9-derived nickase domain, a Cas12-derived nickase domain) is the strand that is not edited by the base editor (i.e., the strand cleaved by the base editor is the opposite strand to the strand containing the base to be edited). In other embodiments, a base editor comprising a nickase domain (e.g., a Cas9-derived nickase domain, a Cas12-derived nickase domain) can cleave the strand of a DNA molecule that is targeted for editing. In such embodiments, the non-target strand is not cleaved.

[0238] In some embodiments, the Cas9 nuclease has an inactive (e.g., inactivated) DNA cleavage domain. That is, the Cas9 is a nickase referred to as a "nCas9" protein (for "nickase" Cas9). The Cas9 nickase can be a Cas9 protein capable of cleaving only one strand of a double-stranded nucleic acid molecule (e.g., a double-stranded DNA molecule). In some embodiments, the Cas9 nickase cleaves the target strand of the double-stranded nucleic acid molecule. This means that the Cas9 nickase cleaves the strand that is base-paired (complementary) to the gRNA (e.g., sgRNA) bound to the Cas9. In some embodiments, the Cas9 nickase comprises a D10A mutation and has a histidine at position 840. In some embodiments, the Cas9 nickase cleaves the non-target, non-base-edited strand of the double-stranded nucleic acid molecule. This means that the Cas9 nickase cleaves the strand that is not base-paired to the gRNA (e.g., sgRNA) bound to the Cas9. In some embodiments, the Cas9 nickase comprises an H840A mutation and has an aspartic acid residue at position 10, or a corresponding mutation. In some embodiments, the Cas9 nickase 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 the Cas9 nickases provided herein. Additional suitable Cas9 nickases will be apparent to those of skill in the art based on this disclosure and knowledge in the art, and are within the scope of this disclosure.

[0239] The amino acid sequence of an exemplary catalytic Cas9 nickase (nCas9) is as follows:

[0240] The Cas9 nuclease has two functional endonuclease domains: RuvC and HNH. Upon target binding, Cas9 undergoes a conformational change that positions the nuclease domains to cleave opposite strands of the target DNA. The end result of Cas9-mediated DNA cleavage is a double-strand break (DSB) within the target DNA (approximately 3–4 nucleotides upstream of the PAM sequence). The resulting DSB is then repaired by one of two general repair pathways: (1) the efficient but error-prone non-homologous end joining (NHEJ) pathway; or (2) the less efficient but highly fidelity homology-directed repair (HDR) pathway.

[0241] The "efficiency" of non-homologous end joining (NHEJ) and / or homology-directed repair (HDR) can be calculated by any convenient method. For example, in some embodiments, efficiency can be expressed as the percentage of successful HDR. For example, a Surveyor nuclease assay can be used to generate cleavage products, and the product-to-substrate ratio can be used to calculate the percentage. For example, a Surveyor nuclease enzyme can be used to directly cleave DNA containing the newly incorporated restriction sequence as a result of successful HDR. More cleaved substrates indicate a greater rate of HDR (higher HDR efficiency). As an illustrative example, the rate (percentage) of HDR can be calculated using the following formula: [(cleavage product) / (substrate + cleavage product)] (e.g., (b + c) / (a + b + c), where "a" is the band intensity of the DNA substrate and "b" and "c" are the cleavage products).

[0242] In some embodiments, efficiency can be expressed as the percentage of successful NHEJ. For example, a T7 endonuclease I assay can be used to generate cleavage products, and the product-to-substrate ratio can be used to calculate the percentage of NHEJ. T7 endonuclease I cleaves mismatched heteroduplex DNA resulting from hybridization of wild-type and mutant DNA strands (NHEJ generates small random insertions or deletions (indels) at the original cleavage site). More cleavages indicate a higher rate of NHEJ (higher NHEJ efficiency). As an illustrative example, the rate (percentage) of NHEJ can be calculated using the following formula: (1-(1-(b+c) / (a+b+c)) 1 / 2 ) × 100, where "a" is the band intensity of the DNA substrate, and "b" and "c" are the cleavage products (Ran et al., Cell. 2013 Sep. 12;154(6):1380-9; and Ran et al., Nat Protoc. 2013 Nov.;8(11):2281-2308).

[0243] The NHEJ repair pathway is the most active repair mechanism, frequently resulting in small nucleotide insertions or deletions (indels) at DSB sites. The random nature of NHEJ-mediated DSB repair is suggested to be of practical importance because a cell population expressing Cas9 and gRNA or guide polynucleotide may result in diverse mutations. In many embodiments, NHEJ generates small indels in the target DNA, resulting in amino acid deletions, insertions, or frameshift mutations, leading to premature termination codons in the open reading frame (ORF) of the target gene. The ideal end result is a loss-of-function mutation in the target gene.

[0244] While NHEJ-mediated DSB repair often disrupts the open reading frame of a gene, homology-directed repair (HDR) can be used to generate specific nucleotide changes, ranging from single nucleotide changes to larger insertions such as the addition of fluorophores or tags.

[0245] To utilize HDR for gene editing, a DNA repair template containing the desired sequence can be delivered to the cell type of interest along with gRNA(s) and Cas9 or Cas9 nickase. The repair template can contain the desired edit and additional homologous sequences immediately upstream and downstream of the target (termed left and right homology arms). The length of each homology arm can depend on the size of the modification to be introduced; larger insertions require longer homology arms. The repair template can be a single-stranded oligonucleotide, a double-stranded oligonucleotide, or a double-stranded DNA plasmid. HDR efficiency is generally low (less than 10% of modified alleles), even in cells expressing Cas9, gRNA, and an exogenous repair template. Because HDR occurs between the S and G2 phases of the cell cycle, synchronizing cells can increase HDR efficiency. Chemical or genetic inhibition of genes involved in NHEJ can also increase the frequency of HDR.

[0246] In some embodiments, the Cas9 is a modified Cas9. A given gRNA targeting sequence may have additional sites of partial homology throughout the genome. These sites are called off-targets and must be considered when designing the gRNA. In addition to optimizing the gRNA design, Cas9 can also be modified to enhance CRISPR specificity. Cas9 generates double-strand breaks (DSBs) by combining the activities of two nuclease domains, RuvC and HNH. Cas9 nickase, a D10A mutant of SpCas9, retains one nuclease domain and generates DNA nicks rather than DSBs. The nickase system can also be combined with HDR-mediated gene editing for specific gene editing.

[0247] Catalytically inactivated nuclease Also provided herein are base editors comprising a catalytically inactivated (i.e., incapable of cleaving a target polynucleotide sequence) polynucleotide-programmable nucleotide-binding domain. As used herein, the terms "catalytically inactivated" and "nuclease-inactivated" are used interchangeably to refer to a polynucleotide-programmable nucleotide-binding domain having one or more mutations and / or deletions that render it unable to cleave a strand of nucleic acid. In some embodiments, a catalytically inactivated polynucleotide-programmable nucleotide-binding domain base editor can lack nuclease activity as a result of specific point mutations in one or more nuclease domains. For example, in the case of a base editor comprising a Cas9 domain, Cas9 can contain both the D10A and H840A mutations. Such mutations inactivate both nuclease domains, thereby abolishing nuclease activity. In other embodiments, a catalytically inactivated polynucleotide-programmable nucleotide-binding domain can contain one or more deletions of all or part of a catalytic domain (e.g., the RuvC1 and / or HNH domain). In a further embodiment, the catalytically inactivated polynucleotide-programmable nucleotide-binding domain comprises a point mutation (e.g., D10A or H840A) and a deletion of all or part of the nuclease domain. dCas9 domains are known in the art and are described, for example, in Qi et al., "Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression." Cell. 2013;152(5):1173-83, the entire contents of which are incorporated herein by reference.

[0248] Additional suitable nuclease-inactive dCas9 domains will be apparent to those of skill in the art based on this disclosure and knowledge in the art and are within the scope of this disclosure. Exemplary suitable nuclease-inactive Cas9 domains include, but are not limited to, the D10A / H840A, D10A / D839A / H840A, and D10A / D839A / H840A / N863A mutant domains (see, e.g., Prashant et al., CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature Biotechnology. 2013;31(9):833-838, the entire contents of which are incorporated herein by reference).

[0249] In some embodiments, the dCas9 corresponds to or comprises a Cas9 amino acid sequence having one or more mutations that partially or entirely inactivate Cas9 nuclease activity. In some embodiments, the nuclease-inactive dCas9 domain comprises a D10X mutation and an H840X mutation in the amino acid sequence described herein, or a corresponding mutation in any of the amino acid sequences provided herein, where X is any amino acid change. In some embodiments, the nuclease-inactive dCas9 domain comprises a D10A mutation and an H840A mutation in the amino acid sequence described herein, or a corresponding mutation in any of the amino acid sequences provided herein. In some embodiments, the nuclease-inactive Cas9 domain comprises the amino acid sequence described in the cloning vector pPlatTET-gRNA2 (Accession No. BAV54124).

[0250] In some embodiments, the variant Cas9 protein is capable of cleaving the complementary strand of a guide target sequence but has a reduced ability to cleave the non-complementary strand of a double-stranded guide target sequence. For example, the variant Cas9 protein may have a mutation (amino acid substitution) that reduces the function of the RuvC domain. As a non-limiting example, in some embodiments, the variant Cas9 protein has D10A (aspartic acid to alanine at amino acid position 10), which allows it to cleave the complementary strand of a double-stranded guide target sequence but reduces its ability to cleave the non-complementary strand of a double-stranded guide target sequence (thus, when the variant Cas9 protein cleaves a double-stranded target nucleic acid, a single-strand break (SSB) is generated rather than a double-strand break (DSB)) (see, e.g., Jinek et al., Science. 2012 Aug. 17;337(6096):816-21).

[0251] In some embodiments, a variant Cas9 protein can cleave the non-complementary strand of a double-stranded guide target sequence but has a reduced ability to cleave the complementary strand of the guide target sequence. For example, the variant Cas9 protein can have a mutation (amino acid substitution) that reduces the function of the HNH domain (RuvC / HNH / RuvC domain motif). As a non-limiting example, in some embodiments, the variant Cas9 protein has an H840A (histidine to alanine at amino acid position 840) mutation, which allows it to cleave the non-complementary strand of a guide target sequence but reduces its ability to cleave the complementary strand of a guide target sequence (thus, when the variant Cas9 protein cleaves a double-stranded guide target sequence, an SSB occurs instead of a DSB). Such a Cas9 protein has a reduced ability to cleave a guide target sequence (e.g., a single-stranded guide target sequence) but retains the ability to bind to the guide target sequence (e.g., a single-stranded guide target sequence).

[0252] As another non-limiting example, in some embodiments, a variant Cas9 protein comprises a W476A and a W1126A mutation, resulting in a polypeptide with a reduced ability to cleave target DNA. Such a Cas9 protein has a reduced ability to cleave target DNA (e.g., single-stranded target DNA), but retains the ability to bind to target DNA (e.g., single-stranded target DNA).

[0253] As another non-limiting example, in some embodiments, a variant Cas9 protein has P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutations, which result in the polypeptide having a reduced ability to cleave target DNA. Such Cas9 proteins have a reduced ability to cleave target DNA (e.g., single-stranded target DNA), but retain the ability to bind to target DNA (e.g., single-stranded target DNA).

[0254] As another non-limiting example, in some embodiments, a variant Cas9 protein has H840A, W476A, and W1126A mutations, thereby reducing the polypeptide's ability to cleave target DNA. Such Cas9 proteins have reduced ability to cleave target DNA (e.g., single-stranded target DNA), but retain the ability to bind to target DNA (e.g., single-stranded target DNA). As another non-limiting example, in some embodiments, a variant Cas9 protein has H840A, D10A, W476A, and W1126A mutations, thereby reducing the polypeptide's ability to cleave target DNA. Such Cas9 proteins have reduced ability to cleave target DNA (e.g., single-stranded target DNA), but retain the ability to bind to target DNA (e.g., single-stranded target DNA). In some embodiments, a variant Cas9 has a restored catalytic His residue at position 840 of the Cas9 HNH domain (A840H).

[0255] As another non-limiting example, in some embodiments, a variant Cas9 protein has H840A, P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutations, thereby reducing the ability of the polypeptide to cleave target DNA. Such Cas9 proteins have a reduced ability to cleave target DNA (e.g., single-stranded target DNA), but retain the ability to bind to target DNA (e.g., single-stranded target DNA). As another non-limiting example, in some embodiments, a variant Cas9 protein has D10A, H840A, P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutations, thereby reducing the ability of the polypeptide to cleave target DNA. Such Cas9 proteins have a reduced ability to cleave target DNA (e.g., single-stranded target DNA) but retain the ability to bind to target DNA (e.g., single-stranded target DNA). In some embodiments, when a variant Cas9 protein has the W476A and W1126A mutations, or when a variant Cas9 protein has the P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutations, the variant Cas9 protein does not efficiently bind to a PAM sequence. Thus, in some such embodiments, when such a variant Cas9 protein is used in a method of binding, the method does not require a PAM sequence. In other words, in some embodiments, when such a variant Cas9 protein is used in a method of binding, the method may include a guide RNA but can be performed in the absence of a PAM sequence (thus, binding specificity is provided by the targeting segment of the guide RNA). Other residues may be mutated (i.e., inactivate one or other nuclease moiety) to achieve the above effect. By way of non-limiting example, residues D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987 may be modified (i.e., substituted). Mutations other than alanine substitutions are also suitable.

[0256] In some embodiments, variant Cas9 proteins with reduced catalytic activity (e.g., when the Cas9 protein has D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987 mutations, e.g., D10A, G12A, G17A, E762A, H840A, N854A, N863A, H982A, H983A, A984A, and / or D986A) can still site-specifically bind to target DNA (because they are still guided to the target DNA sequence by the guide RNA), so long as they retain the ability to interact with the guide RNA.

[0257] In some embodiments, the variant Cas protein can be spCas9, spCas9-VRQR, spCas9-VRER, xCas9(sp), saCas9, saCas9-KKH, spCas9-MQKSER, spCas9-LRKIQK, or spCas9-LRVSQL.

[0258] In some embodiments, the Cas9 domain is a Cas9 domain from Staphylococcus aureus (SaCas9). In some embodiments, the SaCas9 domain is a nuclease-active SaCas9, a nuclease-inactive SaCas9 (SaCas9d), or a SaCas9 nickase (SaCas9n). In some embodiments, the SaCas9 comprises an N579A mutation, or a corresponding mutation in any of the amino acid sequences provided in the Sequence Listing submitted herewith.

[0259] In some embodiments, the SaCas9 domain, SaCas9d domain, or SaCas9n domain can bind to a nucleic acid sequence having a non-canonical PAM. In some embodiments, the SaCas9 domain, SaCas9d domain, or SaCas9n domain can bind to a nucleic acid sequence having an NNGRRT or NNGRRV PAM sequence. In some embodiments, the SaCas9 domain comprises one or more of the following mutations: E781X, N967X, and R1014X, or a corresponding mutation in any of the amino acid sequences provided herein, where X is any amino acid. In some embodiments, the SaCas9 domain comprises one or more of the following mutations: E781K, N967K, and R1014H, or a corresponding mutation in any of the amino acid sequences provided herein. In some embodiments, the SaCas9 domain comprises the following mutations: E781K, N967K, or R1014H, or a corresponding mutation in any of the amino acid sequences provided herein.

[0260] In some embodiments, one of the Cas9 domains present in the fusion protein may be replaced with a guide nucleotide sequence-programmable DNA-binding protein domain that does not have a PAM sequence requirement. In some embodiments, the Cas9 is SaCas9. Residue A579 of SaCas9 can be mutated from N579 to obtain SaCas9 nickase. Residues K781, K967, and H1014 can be mutated from E781, N967, and R1014 to obtain SaKKH Cas9.

[0261] In some embodiments, a modified SpCas9 (SpCas9-MQKFRAER) was used that contains the amino acid substitutions D1135M, S1136Q, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337R and has specificity for the modified PAM5'-NGC-3'.

[0262] Alternatives to S. pyogenes Cas9 include RNA-guided endonucleases from the Cpf1 family that exhibit cleavage activity in mammalian cells. CRISPR from Prevotella and Francisella 1 (CRISPR / Cpf1) is a DNA editing technology similar to the CRISPR / Cas9 system. Cpf1 is an RNA-guided endonuclease of the class II CRISPR / Cas system. This adaptive immune mechanism is found in Prevotella and Francisella bacteria. The Cpf1 gene is associated with the CRISPR locus and encodes an endonuclease that uses guide RNA to locate and cleave viral DNA. Cpf1 is a smaller and simpler endonuclease than Cas9, overcoming some of the limitations of the CRISPR / Cas9 system. Unlike Cas9 nuclease, Cpf1-mediated DNA cleavage results in a double-strand break with a short 3' overhang. The zigzag cleavage pattern of Cpf1 may open up the possibility of directional gene transfer similar to traditional restriction enzyme cloning, thereby improving the efficiency of gene editing. Like the Cas9 variants and orthologs described above, Cpf1 can also expand the number of CRISPR targetable sites in AT-rich regions or AT-rich genomes lacking the NGG PAM site favored by SpCas9. The Cpf1 locus contains a mixed alpha / beta domain, RuvC-I followed by a helical region, RuvC-II, and a zinc finger-like domain. The Cpf1 protein possesses a RuvC-like endonuclease domain similar to the RuvC domain of Cas9.

[0263] Furthermore, unlike Cas9, Cpf1 lacks the HNH endonuclease domain, and the N-terminus of Cpf1 lacks the alpha-helical recognition lobe of Cas9. The structure of the Cpf1 CRISPR-Cas domain indicates that Cpf1 is functionally unique and classifies it as a Class 2, Type V CRISPR system. The Cpf1 locus encodes the Cas1, Cas2, and Cas4 proteins, and is more similar to Type I and Type III systems than Type II systems. Functional Cpf1 does not require a trans-activating CRISPR RNA (tracrRNA) and therefore only requires CRISPR (crRNA). Cpf1 is not only smaller than Cas9, but also has a smaller sgRNA molecule (almost half the nucleotides of Cas9), which is beneficial for genome editing. The Cpf1-crRNA complex cleaves target DNA or RNA by identifying the 5'-YTN-3' or 5'-TTN-3' protospacer-adjacent motif, in contrast to the G-rich PAM targeted by Cas9. After identifying the PAM, Cpf1 introduces sticky-end-like DNA double-strand breaks with 4- or 5-nucleotide overhangs.

[0264] In some embodiments, the Cas9 is a Cas9 variant with specificity for engineered PAM sequences. In some embodiments, additional Cas9 variants and PAM sequences are described in Miller, SM, et al. Continuous evolution of SpCas9 variants compatible with non-G PAMs, Nat. Biotechnol. (2020), the entire contents of which are incorporated herein by reference. In some embodiments, the Cas9 variant does not have a specific PAM requirement. In some embodiments, the Cas9 variant, e.g., an SpCas9 variant, has specificity for the NRNH PAM, where R is A or G and H is A, C, or T. In some embodiments, the SpCas9 variant has specificity for the PAM sequence AAA, TAA, CAA, GAA, TAT, GAT, or CAC. In some embodiments, the SpCas9 variant comprises an amino acid substitution at position 1114, 1134, 1135, 1137, 1139, 1151, 1180, 1188, 1211, 1218, 1219, 1221, 1249, 1256, 1264, 1290, 1318, 1317, 1320, 1321, 1323, 1332, 1333, 1335, 1337, or 1339, or a corresponding position. In some embodiments, the SpCas9 variant comprises an amino acid substitution at position 1114, 1135, 1218, 1219, 1221, 1249, 1320, 1321, 1323, 1332, 1333, 1335, or 1337, or a corresponding position. In some embodiments, the SpCas9 variant comprises an amino acid substitution at position 1114, 1134, 1135, 1137, 1139, 1151, 1180, 1188, 1211, 1219, 1221, 1256, 1264, 1290, 1318, 1317, 1320, 1323, 1333, or a corresponding position.In some embodiments, the SpCas9 variant comprises an amino acid substitution at positions 1114, 1131, 1135, 1150, 1156, 1180, 1191, 1218, 1219, 1221, 1227, 1249, 1253, 1286, 1293, 1320, 1321, 1332, 1335, 1339, or a corresponding position. In some embodiments, the SpCas9 variant comprises an amino acid substitution at positions 1114, 1127, 1135, 1180, 1207, 1219, 1234, 1286, 1301, 1332, 1335, 1337, 1338, 1349, or a corresponding position. Exemplary amino acid substitutions and PAM specificities of SpCas9 variants are shown in Tables 2A-2D. [Table 2A] Table 2B Table 2C

Table 2D

[0265] In some embodiments, a nucleic acid programmable DNA-binding protein (napDNAbp) is the sole effector of a microbial CRISPR-Cas system. Microbial CRISPR-Cas system sole effectors include, but are not limited to, Cas9, Cpf1, Cas12b / C2c1, and Cas12c / C2c3. Typically, microbial CRISPR-Cas systems are divided into class 1 and class 2 systems. Class 1 systems have multi-subunit effector complexes, while class 2 systems have single protein effectors. For example, Cas9 and Cpf1 are class 2 effectors. In addition to Cas9 and Cpf1, three distinct Class 2 CRISPR-Cas systems (Cas12b / C2c1 and Cas12c / C2c3) have been described by Shmakov et al., "Discovery and Functional Characterization of Diverse Class 2 CRISPR Cas Systems," Mol. Cell, 2015 Nov. 5;60(3):385-397, the entire contents of which are incorporated herein by reference. Two effectors in the Cas12b / C2c1 and Cas12c / C2c3 systems contain a RuvC-like endonuclease domain related to Cpf1. The third system contains an effector with two defined HEPN RNase domains. Unlike CRISPR RNA production by Cas12b / C2c1, mature CRISPR RNA production is independent of tracrRNA. Cas12b / C2c1 relies on both CRISPR RNA and tracrRNA for DNA cleavage.

[0266] In some embodiments, the napDNAbp is a circular permutation (eg, SEQ ID NO: 257).

[0267] The crystal structure of Alicyclobacillus acidoterrastris Cas12b / C2c1 (AacC2c1) has been reported in complex with a chimeric single-molecule guide RNA (sgRNA). See, e.g., Liu et al., "C2c1-sgRNA Complex Structure Reveals RNA-Guided DNA Cleavage Mechanism," Mol. Cell, 2017 Jan. 19;65(2):310-322, the entire contents of which are incorporated herein by reference. The crystal structure of Alicyclobacillus acidoterrestris C2c1as bound to target DNA has also been reported as a ternary complex. See, e.g., Yang et al., "PAM-dependent Target DNA Recognition and Cleavage by C2C1 CRISPR-Cas endonuclease," Cell, 2016 Dec. 15;167(7):1814-1828, the entire contents of which are incorporated herein by reference. Catalytically competent AacC2c1 conformations are captured independently within a single RuvC catalytic pocket for both target and non-target DNA strands, and Cas12b / C2c1-mediated cleavage generates a zigzag seven-nucleotide cut in the target DNA. Structural comparison between the Cas12b / C2c1 ternary complex and previously identified counterparts of Cas9 and Cpf1 demonstrates the diversity of mechanisms employed by the CRISPR-Cas9 system.

[0268] In some embodiments, the nucleic acid programmable DNA-binding protein (napDNAbp) of any of the fusion proteins provided herein can be a Cas12b / C2c1 or Cas12c / C2c3 protein. In some embodiments, the napDNAbp is a Cas12b / C2c1 protein. In some embodiments, the napDNAbp is a Cas12c / C2c3 protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is 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 at least 99.5% identical to a native Cas12b / C2c1 or Cas12c / C2c3 protein. In some embodiments, the napDNAbp is a native Cas12b / C2c1 or Cas12c / C2c3 protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is 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 at least 99.5% identical to any one of the napDNAbp sequences provided herein. It should be understood that Cas12b / C2c1 or Cas12c / C2c3 from other bacterial species can also be used in accordance with the present disclosure.

[0269] In some embodiments, napDNAbp refers to Cas12c. In some embodiments, the Cas12c protein is Cas12c1 (SEQ ID NO: 266) or a variant of Cas12c1. In some embodiments, the Cas12 protein is Cas12c2 (SEQ ID NO: 267) or a variant of Cas12c2. In some embodiments, the Cas12 protein is the Cas12c protein from Oleiphilus sp. HI0009 (i.e., OspCas12c; SEQ ID NO: 268) or a variant of OspCas12c. These Cas12c molecules are described in Yan et al., "Functionally Diverse Type V CRISPR-Cas Systems," Science, 2019 Jan. 4;363:88-91, the entire contents of which are incorporated herein by reference. In some embodiments, the napDNAbp comprises an amino acid sequence that is 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 at least 99.5% identical to a native Cas12c1, Cas12c2, or OspCas12c protein. In some embodiments, the napDNAbp is a native Cas12c1, Cas12c2, or OspCas12c protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is 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 at least 99.5% identical to any Cas12c1, Cas12c2, or OspCas12c protein described herein. It should be understood that Cas12c1, Cas12c2, or OspCas12c from other bacterial species can also be used in accordance with the present disclosure.

[0270] In some embodiments, napDNAbp refers to Cas12g, Cas12h, or Cas12i, as described, for example, in Yan et al., "Functionally Diverse Type V CRISPR-Cas Systems," Science, 2019 Jan. 4;363:88-91, the entire contents of each of which are incorporated herein by reference. Exemplary Cas12g, Cas12h, and Cas12i polypeptide sequences are provided in the Sequence Listing as SEQ ID NOs: 269-272. By aggregating over 10 terabytes of sequence data, a new classification of type V Cas proteins, including Cas12g, Cas12h, and Cas12i, has been identified that shows weak similarity to previously characterized class V proteins. In some embodiments, the Cas12 protein is Cas12g or a variant of Cas12g. In some embodiments, the Cas12 protein is Cas12h or a variant of Cas12h. In some embodiments, the Cas12 protein is Cas12i or a variant of Cas12i. It should be understood that other RNA-guided DNA-binding proteins can also be used as napDNAbp and are within the scope of the present disclosure. In some embodiments, the napDNAbp comprises an amino acid sequence that is 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 at least 99.5% identical to a native Cas12g, Cas12h, or Cas12i protein. In some embodiments, the napDNAbp is a native Cas12g, Cas12h, or Cas12i protein.In some embodiments, the napDNAbp comprises an amino acid sequence that is 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 at least 99.5% identical to any Cas12g, Cas12h, or Cas12i protein described herein. It should be understood that Cas12g, Cas12h, or Cas12i from other bacterial species can also be used in accordance with the present disclosure. In some embodiments, the Cas12i is Cas12i1 or Cas12i2.

[0271] In some embodiments, the nucleic acid-programmable DNA-binding protein (napDNAbp) of the fusion protein provided herein can be a Cas12j / CasΦ protein. Cas12j / CasΦ is described in Pausch et al., "CRISPR-CasΦ from Huge Phages is a Hypercompact Genome Editor," Science, July 17, 2020, Vol. 369, Issue 6501, pp. 333-337, which is incorporated herein by reference in its entirety. In some embodiments, the napDNAbp comprises an amino acid sequence that is 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 at least 99.5% identical to a native Cas12j / CasΦ protein. In some embodiments, the napDNAbp is a native Cas12j / CasΦ protein. In some embodiments, the napDNAbp is a nuclease-inactive ("deactivated") Cas12j / CasΦ protein. It should be understood that Cas12j / CasΦ from other species can also be used in accordance with the present disclosure.

[0272] Fusion proteins containing internal inserts Provided herein are fusion proteins comprising a heterologous polypeptide fused to a nucleic acid-programmable nucleic acid binding protein, e.g., a napDNAbp. The heterologous polypeptide can be a polypeptide not found in the native or wild-type napDNAbp polypeptide sequence. The heterologous polypeptide can be fused to the napDNAbp at the C-terminus of the napDNAbp, at the N-terminus of the napDNAbp, or inserted at an internal position of the napDNAbp. In some embodiments, the heterologous polypeptide is a deaminase (e.g., cytidine adenosine deaminase) or a functional fragment thereof. For example, the fusion protein can comprise a deaminase flanked by N-terminal and C-terminal fragments of a Cas9 or Cas12 (e.g., Cas12b / C2c1) polypeptide. In some embodiments, the cytidine deaminase is an APOBEC deaminase (e.g., APOBEC1). In some embodiments, the adenosine deaminase is TadA (e.g., TadA*7.10 or TadA*8). In some embodiments, TadA is TadA*8 or TadA*9. The TadA sequences described herein (e.g., TadA7.10 or TadA*8) are suitable deaminases for the fusion proteins described above.

[0273] In some embodiments, the fusion protein comprises the following structure: NH2-[N-terminal fragment of napDNAbp]-[deaminase]-[C-terminal fragment of napDNAbp]-COOH; NH2-[N-terminal fragment of Cas9]-[adenosine deaminase]-[C-terminal fragment of Cas9]-COOH; NH2-[N-terminal fragment of Cas12]-[adenosine deaminase]-[C-terminal fragment of Cas12]-COOH; NH2-[N-terminal fragment of Cas9]-[cytidine deaminase]-[C-terminal fragment of Cas9]-COOH; NH2-[N-terminal fragment of Cas12]-[cytidine deaminase]-[C-terminal fragment of Cas12]-COOH; where each occurrence of "]-[" is an optional linker.

[0274] The deaminase can be a circularly permuted deaminase. For example, the deaminase can be a circularly permuted adenosine deaminase. In some embodiments, the deaminase is a circularly permuted TadA that is circularly permuted at amino acid residues 116, 136, or 65 numbered in the TadA reference sequence.

[0275] The fusion protein can comprise two or more deaminases. The fusion protein can comprise, for example, one, two, three, four, five, or more deaminases. In some embodiments, the fusion protein comprises one or two deaminases. The two or more deaminases in the fusion protein can be adenosine deaminase, cytidine deaminase, or a combination thereof. The two or more deaminases can be homodimers or heterodimers. The two or more deaminases can be inserted in tandem into the napDNAbp. In some embodiments, the two or more deaminases do not have to be tandemly inserted into the napDNAbp.

[0276] In some embodiments, the napDNAbp in the fusion protein is a Cas9 polypeptide or a fragment thereof. The Cas9 polypeptide can be a variant Cas9 polypeptide. In some embodiments, the Cas9 polypeptide is a Cas9 nickase (nCas9) polypeptide or a fragment thereof. In some embodiments, the Cas9 polypeptide is a nuclease-deactivated Cas9 (dCas9) polypeptide or a fragment thereof. The Cas9 polypeptide in the fusion protein can be a full-length Cas9 polypeptide. In some cases, the Cas9 polypeptide in the fusion protein need not be a full-length Cas9 polypeptide. The Cas9 polypeptide can be truncated, for example, at the N-terminus or C-terminus relative to the native Cas9 protein. The Cas9 polypeptide can be a circularly permuted Cas9 protein. The Cas9 polypeptide can be a fragment, portion, or domain of a Cas9 polypeptide and still be capable of binding to a target polynucleotide and a guide nucleic acid sequence.

[0277] In some embodiments, the Cas9 polypeptide is Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1 Cas9 (St1Cas9), or a fragment or variant of any of the Cas9 polypeptides described herein.

[0278] In some embodiments, the fusion protein comprises an adenosine deaminase domain and a cytidine deaminase domain inserted into Cas9. In some embodiments, the adenosine deaminase is fused into Cas9 and the cytidine deaminase is fused to the C-terminus. In some embodiments, the adenosine deaminase is fused into Cas9 and the cytidine deaminase is fused to the N-terminus. In some embodiments, the cytidine deaminase is fused into Cas9 and the adenosine deaminase is fused to the C-terminus. In some embodiments, the cytidine deaminase is fused into Cas9 and the adenosine deaminase is fused to the N-terminus.

[0279] Exemplary structures of fusion proteins of adenosine deaminase and cytidine deaminase with Cas9 are provided below: NH2-[Cas9(adenosine deaminase)]-[cytidine deaminase]-COOH; NH2-[cytidine deaminase]-[Cas9(adenosine deaminase)]-COOH; NH2-[Cas9(cytidine deaminase)]-[adenosine deaminase]-COOH; or NH2-[adenosine deaminase]-[Cas9 (cytidine deaminase)]-COOH.

[0280] In some embodiments, the "-" used in the above general structures indicates the presence of an optional linker.

[0281] In various embodiments, the catalytic domain has a DNA-modifying activity (e.g., deaminase activity), such as adenosine deaminase activity. In some embodiments, the adenosine deaminase is TadA (e.g., TadA*7.10). In some embodiments, TadA is TadA*8. In some embodiments, TadA*8 is fused into Cas9 and a cytidine deaminase is fused to the C-terminus. In some embodiments, TadA*8 is fused into Cas9 and a cytidine deaminase is fused to the N-terminus. In some embodiments, a cytidine deaminase is fused into Cas9 and TadA*8 is fused to the C-terminus. In some embodiments, a cytidine deaminase is fused into Cas9 and TadA*8 is fused to the N-terminus. Exemplary structures of fusion proteins of TadA*8 and cytidine deaminase with Cas9 are provided below: NH2-[Cas9(TadA*8)]-[cytidine deaminase]-COOH; NH2-[cytidine deaminase]-[Cas9(TadA*8)]-COOH; NH2-[Cas9(cytidine deaminase)]-[TadA*8]-COOH; or NH2-[TadA*8]-[Cas9(cytidine deaminase)]-COOH.

[0282] In some embodiments, the "-" used in the above general structures indicates the presence of an optional linker.

[0283] A heterologous polypeptide (e.g., a deaminase) can be inserted into the napDNAbp (e.g., Cas9 or Cas12 (e.g., Cas12b / C2c1)) at a suitable position such that the napDNAbp retains its ability to bind to a target polynucleotide and a guide nucleic acid. The deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) can be inserted into the napDNAbp without impairing the function of the deaminase (e.g., base editing activity) or the napDNAbp (e.g., the ability to bind to a target nucleic acid and a guide nucleic acid). The deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) can be inserted into the napDNAbp, for example, in a disordered region shown by crystallographic studies or in a region containing a high temperature factor or B factor. Regions of proteins that are poorly ordered, disordered, or unstructured, such as solvent-exposed regions and loops, can be used for insertion without compromising structure or function. Deaminases (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) can be inserted into napDNAbp in flexible loop regions or solvent-exposed regions. In some embodiments, deaminases (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) are inserted into flexible loops of Cas9 or Cas12b / C2c1 polypeptides.

[0284] In some embodiments, the insertion location of the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is determined by B-factor analysis of the crystal structure of the Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted into a region of the Cas9 polypeptide that contains a higher-than-average B-factor (e.g., a higher B-factor relative to the whole protein or a protein domain containing disordered regions). The B-factor or temperature factor can indicate fluctuations from the average positions of the atoms (e.g., as a result of temperature-dependent atomic vibrations or static disorder in the crystal lattice). A high B-factor (e.g., a higher-than-average B-factor) of a backbone atom can indicate a region of relatively high local mobility. Such a region can be used to insert the deaminase without compromising structure or function. A deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) can be inserted at a position containing a residue having a Cα atom with a B factor that is 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or greater than the average B factor of the entire protein. A deaminase (e.g., an adenosine deaminase, a cytidine deaminase, or an adenosine deaminase and a cytidine deaminase) can be inserted at a position containing a residue having a Cα atom with a B factor that is 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200% or greater than the average B factor of the Cas9 protein domain containing those residues. Cas9 polypeptide positions containing higher than average B factors can include, for example, residues 768, 792, 1052, 1015, 1022, 1026, 1029, 1067, 1040, 1054, 1068, 1246, 1247, and 1248, as numbered in the above Cas9 reference sequence.Regions of the Cas9 polypeptide containing higher than average B factors can include, for example, residues 792-872, 792-906, and 2-791, as numbered in the above Cas9 reference sequence.

[0285] A heterologous polypeptide (e.g., a deaminase) can be inserted into the napDNAbp at an amino acid residue selected from the group consisting of 768, 791, 792, 1015, 1016, 1022, 1023, 1026, 1029, 1040, 1052, 1054, 1067, 1068, 1069, 1246, 1247, and 1248, as numbered in the above Cas9 reference sequence, or the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the heterologous polypeptide is inserted between amino acid positions 768-769, 791-792, 792-793, 1015-1016, 1022-1023, 1026-1027, 1029-1030, 1040-1041, 1052-1053, 1054-1055, 1067-1068, 1068-1069, 1247-1248, or 1248-1249, as numbered in the above Cas9 reference sequence, or their corresponding amino acid positions. In some embodiments, the heterologous polypeptide is inserted between amino acid positions 769-770, 792-793, 793-794, 1016-1017, 1023-1024, 1027-1028, 1030-1031, 1041-1042, 1053-1054, 1055-1056, 1068-1069, 1069-1070, 1248-1249, or 1249-1250, as numbered in the above Cas9 reference sequence, or their corresponding amino acid positions. In some embodiments, the heterologous polypeptide replaces an amino acid residue selected from the group consisting of 768, 791, 792, 1015, 1016, 1022, 1023, 1026, 1029, 1040, 1052, 1054, 1067, 1068, 1069, 1246, 1247, and 1248, as numbered in the above Cas9 reference sequence, or the corresponding amino acid residue in another Cas9 polypeptide. It should be understood that reference to the above Cas9 reference sequence for insertion positions is for exemplary purposes.The insertions discussed herein are not limited to the Cas9 polypeptide sequences of the above Cas9 reference sequences, but also include insertions at the corresponding positions of variant Cas9 polypeptides, such as Cas9 nickase (nCas9), nuclease-deactivated Cas9 (dCas9), Cas9 variants lacking the nuclease domain, truncated Cas9, or Cas9 domains lacking a partial or complete HNH domain.

[0286] A heterologous polypeptide (e.g., a deaminase) can be inserted into the napDNAbp at an amino acid residue selected from the group consisting of 768, 792, 1022, 1026, 1040, 1068, and 1247, as numbered in the above Cas9 reference sequence, or the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the heterologous polypeptide is inserted between amino acid positions 768-769, 792-793, 1022-1023, 1026-1027, 1029-1030, 1040-1041, 1068-1069, or 1247-1248, as numbered in the above Cas9 reference sequence, or their corresponding amino acid positions. In some embodiments, the heterologous polypeptide is inserted between amino acid positions 769-770, 793-794, 1023-1024, 1027-1028, 1030-1031, 1041-1042, 1069-1070, or 1248-1249, as numbered in the above Cas9 reference sequence, or their corresponding amino acid positions. In some embodiments, the heterologous polypeptide replaces an amino acid residue selected from the group consisting of 768, 792, 1022, 1026, 1040, 1068, and 1247, as numbered in the above Cas9 reference sequence, or the corresponding amino acid residue in another Cas9 polypeptide.

[0287] A heterologous polypeptide (e.g., a deaminase) can be inserted into the napDNAbp at an amino acid residue described herein, or the corresponding amino acid residue of another Cas9 polypeptide. In one embodiment, the heterologous polypeptide (e.g., a deaminase) can be inserted into the napDNAbp at an amino acid residue selected from the group consisting of 1002, 1003, 1025, 1052-1056, 1242-1247, 1061-1077, 943-947, 686-691, 569-578, 530-539, and 1060-1077, as numbered in the Cas9 reference sequence above, or the corresponding amino acid residue of another Cas9 polypeptide. The deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) can be inserted N-terminally or C-terminally to, or replace, a residue. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted C-terminal to the residue.

[0288] In some embodiments, the adenosine deaminase (e.g., TadA) is inserted at an amino acid residue selected from the group consisting of 1015, 1022, 1029, 1040, 1068, 1247, 1054, 1026, 768, 1067, 1248, 1052, and 1246 numbered in the above Cas9 reference sequence, or the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the adenosine deaminase (e.g., TadA) is inserted at residues 792-872, 792-906, or 2-791 numbered in the above Cas9 reference sequence, or in place of the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the adenosine deaminase is inserted N-terminally to an amino acid selected from the group consisting of 1015, 1022, 1029, 1040, 1068, 1247, 1054, 1026, 768, 1067, 1248, 1052, and 1246 numbered in the above Cas9 reference sequence, or the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the adenosine deaminase is inserted C-terminally to an amino acid selected from the group consisting of 1015, 1022, 1029, 1040, 1068, 1247, 1054, 1026, 768, 1067, 1248, 1052, and 1246 numbered in the above Cas9 reference sequence, or the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the adenosine deaminase is inserted to replace an amino acid selected from the group consisting of 1015, 1022, 1029, 1040, 1068, 1247, 1054, 1026, 768, 1067, 1248, 1052, and 1246, as numbered in the above Cas9 reference sequence, or the corresponding amino acid residue in another Cas9 polypeptide.

[0289] In some embodiments, the cytidine deaminase (e.g., APOBEC1) is inserted at an amino acid residue selected from the group consisting of 1016, 1023, 1029, 1040, 1069, and 1247 numbered in the Cas9 reference sequence above, or the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the cytidine deaminase is inserted N-terminally to an amino acid residue selected from the group consisting of 1016, 1023, 1029, 1040, 1069, and 1247 numbered in the Cas9 reference sequence above, or the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the cytidine deaminase is inserted C-terminally to an amino acid residue selected from the group consisting of 1016, 1023, 1029, 1040, 1069, and 1247 numbered in the Cas9 reference sequence above, or the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the cytidine deaminase is inserted to replace an amino acid selected from the group consisting of 1016, 1023, 1029, 1040, 1069, and 1247, numbered in the above Cas9 reference sequence, or the corresponding amino acid residue in another Cas9 polypeptide.

[0290] In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted at amino acid residue 768 numbered in the above Cas9 reference sequence, or the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted N-terminally to amino acid residue 768 numbered in the above Cas9 reference sequence, or the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted C-terminally to amino acid residue 768 numbered in the above Cas9 reference sequence, or the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted to replace amino acid residue 768, as numbered in the above Cas9 reference sequence, or the corresponding amino acid residue in another Cas9 polypeptide.

[0291] In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted at amino acid residue 791 or 792, as numbered in the above Cas9 reference sequence, or at the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted N-terminally to amino acid residue 791 or 792, as numbered in the above Cas9 reference sequence, or at the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted C-terminally to amino acid 791, or N-terminally to amino acid 792, as numbered in the above Cas9 reference sequence, or at the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted to replace amino acid 791, or to replace amino acid 792, as numbered in the above Cas9 reference sequence, or to replace the corresponding amino acid residue in another Cas9 polypeptide.

[0292] In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted at amino acid residue 1016 numbered in the above Cas9 reference sequence, or the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted N-terminal to amino acid residue 1016 numbered in the above Cas9 reference sequence, or the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted C-terminal to amino acid residue 1016 numbered in the above Cas9 reference sequence, or the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted to replace amino acid residue 1016, as numbered in the above Cas9 reference sequence, or the corresponding amino acid residue in another Cas9 polypeptide.

[0293] In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted at amino acid residue 1022 or 1023 numbered in the above Cas9 reference sequence, or at the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted N-terminally to amino acid residue 1022 or 1023 numbered in the above Cas9 reference sequence, or at the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted C-terminal to amino acid residue 1022, or to amino acid residue 1023, as numbered in the above Cas9 reference sequence, or to the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted to replace amino acid residue 1022, or to replace amino acid residue 1023, as numbered in the above Cas9 reference sequence, or to replace the corresponding amino acid residue in another Cas9 polypeptide.

[0294] In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted at amino acid residue 1026, or at amino acid residue 1029, as numbered in the above Cas9 reference sequence, or at the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted N-terminally to amino acid residue 1026, or at amino acid residue 1029, as numbered in the above Cas9 reference sequence, or at the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted C-terminal to amino acid residue 1026, or to amino acid residue 1029, as numbered in the above Cas9 reference sequence, or to the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted to replace amino acid residue 1026, or to replace amino acid residue 1029, as numbered in the above Cas9 reference sequence, or to replace the corresponding amino acid residue in another Cas9 polypeptide.

[0295] In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted at amino acid residue 1040 numbered in the above Cas9 reference sequence, or the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted N-terminal to amino acid residue 1040 numbered in the above Cas9 reference sequence, or the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted C-terminal to amino acid residue 1040 numbered in the above Cas9 reference sequence, or the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted to replace amino acid residue 1040, as numbered in the above Cas9 reference sequence, or the corresponding amino acid residue in another Cas9 polypeptide.

[0296] In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted at amino acid residue 1052 or 1054, as numbered in the above Cas9 reference sequence, or at the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted N-terminally to amino acid residue 1052 or 1054, as numbered in the above Cas9 reference sequence, or at the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted C-terminal to amino acid residue 1052 or 1054, as numbered in the Cas9 reference sequence above, or the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted to replace amino acid residue 1052 or 1054, as numbered in the Cas9 reference sequence above, or the corresponding amino acid residue in another Cas9 polypeptide.

[0297] In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted at amino acid residue 1067, or at amino acid residue 1068, or at amino acid residue 1069, as numbered in the above Cas9 reference sequence, or at the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted N-terminally to amino acid residue 1067, or at amino acid residue 1068, or at amino acid residue 1069, as numbered in the above Cas9 reference sequence, or at the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted C-terminally to amino acid residue 1067, or to amino acid residue 1068, or to amino acid residue 1069, as numbered in the above Cas9 reference sequence, or at the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted to replace amino acid residue 1067, or to replace amino acid residue 1068, or to replace amino acid residue 1069, as numbered in the above Cas9 reference sequence, or at the corresponding amino acid residue in another Cas9 polypeptide.

[0298] In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted at amino acid residue 1246, or at amino acid residue 1247, or at amino acid residue 1248, as numbered in the above Cas9 reference sequence, or at the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted N-terminally to amino acid residue 1246, or at amino acid residue 1247, or at amino acid residue 1248, as numbered in the above Cas9 reference sequence, or at the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted C-terminal to amino acid residue 1246, or to amino acid residue 1247, or to amino acid residue 1248, as numbered in the Cas9 reference sequence above, or at the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted to replace amino acid residue 1246, or to replace amino acid residue 1247, or to replace amino acid residue 1248, as numbered in the Cas9 reference sequence above, or at the corresponding amino acid residue in another Cas9 polypeptide.

[0299] In some embodiments, the heterologous polypeptide (e.g., a deaminase) is inserted into a flexible loop of a Cas9 polypeptide. The flexible loop portion can be selected from the group consisting of amino acid residues 530-537, 569-570, 686-691, 943-947, 1002-1025, 1052-1077, 1232-1247, or 1298-1300, as numbered in the Cas9 reference sequence above, or the corresponding amino acid residues in another Cas9 polypeptide. The flexible loop portion can be selected from the group consisting of amino acid residues 1-529, 538-568, 580-685, 692-942, 948-1001, 1026-1051, 1078-1231, or 1248-1297, as numbered in the Cas9 reference sequence above, or the corresponding amino acid residues in another Cas9 polypeptide.

[0300] A heterologous polypeptide (e.g., adenine deaminase) can be inserted into the region of a Cas9 polypeptide corresponding to the following numbered amino acid residues in the Cas9 reference sequence: 1017-1069, 1242-1247, 1052-1056, 1060-1077, 1002-1003, 943-947, 530-537, 568-579, 686-691, 1242-1247, 1298-1300, 1066-1077, 1052-1056, or 1060-1077, or at the corresponding amino acid residues in another Cas9 polypeptide.

[0301] A heterologous polypeptide (e.g., adenine deaminase) can be inserted in place of the deleted region of a Cas9 polypeptide. The deleted region can correspond to the N-terminal or C-terminal portion of the Cas9 polypeptide. In some embodiments, the deleted region corresponds to residues 792-872 as numbered in the above Cas9 reference sequence, or the corresponding amino acid residues in another Cas9 polypeptide. In some embodiments, the deleted region corresponds to residues 792-906 as numbered in the above Cas9 reference sequence, or the corresponding amino acid residues in another Cas9 polypeptide. In some embodiments, the deleted region corresponds to residues 2-791 as numbered in the above Cas9 reference sequence, or the corresponding amino acid residues in another Cas9 polypeptide. In some embodiments, the deleted region corresponds to residues 1017-1069 as numbered in the above Cas9 reference sequence, or the corresponding amino acid residues.

[0302] Exemplary internal fusion base editors are provided in Table 3 below: [Table 3]

[0303] A heterologous polypeptide (e.g., a deaminase) can be inserted within a structural or functional domain of a Cas9 polypeptide. A heterologous polypeptide (e.g., a deaminase) can be inserted between two structural or functional domains of a Cas9 polypeptide. A heterologous polypeptide (e.g., a deaminase) can be inserted in place of a structural or functional domain of a Cas9 polypeptide, for example, after deleting the domain from the Cas9 polypeptide. A structural or functional domain of a Cas9 polypeptide can include, for example, RuvC I, RuvC II, RuvC III, Rec1, Rec2, PI, or HNH.

[0304] In some embodiments, the Cas9 polypeptide lacks one or more domains selected from the group consisting of RuvC I, RuvC II, RuvC III, Rec1, Rec2, PI, or HNH domain. In some embodiments, the Cas9 polypeptide lacks a nuclease domain. In some embodiments, the Cas9 polypeptide lacks an HNH domain. In some embodiments, the Cas9 polypeptide lacks a portion of the HNH domain such that the HNH activity of the Cas9 polypeptide is reduced or eliminated. In some embodiments, the Cas9 polypeptide comprises a deletion of the nuclease domain, and a deaminase is inserted to replace the nuclease domain. In some embodiments, the HNH domain is deleted and a deaminase is inserted instead. In some embodiments, one or more of the RuvC domains are deleted and a deaminase is inserted instead.

[0305] A fusion protein comprising a heterologous polypeptide can be flanked by N- and C-terminal fragments of napDNAbp. In some embodiments, the fusion protein comprises a deaminase flanked by N- and C-terminal fragments of a Cas9 polypeptide. The N- or C-terminal fragment can bind to a target polynucleotide sequence. The C-terminus of the N- or C-terminal fragment can comprise a portion of the flexible loop of the Cas9 polypeptide. The C-terminus of the N- or C-terminal fragment can comprise a portion of the alpha-helical structure of the Cas9 polypeptide. The N- or C-terminal fragment can comprise a DNA-binding domain. The N- or C-terminal fragment can comprise a RuvC domain. The N- or C-terminal fragment can comprise an HNH domain. In some embodiments, neither the N- or C-terminal fragment comprises an HNH domain.

[0306] In some embodiments, the C-terminus of the N-terminal Cas9 fragment includes amino acids adjacent to the target nucleobase when the fusion protein deaminates the target nucleobase. In some embodiments, the N-terminus of the C-terminal Cas9 fragment includes amino acids adjacent to the target nucleobase when the fusion protein deaminates the target nucleobase. The insertion position of a different deaminase can be different to ensure proximity between the target nucleobase and the C-terminus of the N-terminal Cas9 fragment or an amino acid within the N-terminus of the C-terminal Cas9 fragment. For example, the insertion position of a deaminase can be an amino acid residue selected from the group consisting of 1015, 1022, 1029, 1040, 1068, 1247, 1054, 1026, 768, 1067, 1248, 1052, and 1246, as numbered in the above Cas9 reference sequence, or the corresponding amino acid residue in another Cas9 polypeptide.

[0307] The N-terminal Cas9 fragment of the fusion protein (i.e., the N-terminal Cas9 fragment flanking the deaminase in the fusion protein) can comprise the N-terminus of the Cas9 polypeptide. The N-terminal Cas9 fragment of the fusion protein can comprise at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, or 1300 amino acids in length. The N-terminal Cas9 fragment of the fusion protein can comprise a sequence corresponding to amino acid residues 1-56, 1-95, 1-200, 1-300, 1-400, 1-500, 1-600, 1-700, 1-718, 1-765, 1-780, 1-906, 1-918, or 1-1100 numbered in the above Cas9 reference sequence, or the corresponding amino acid residues of another Cas9 polypeptide. An N-terminal Cas9 fragment can comprise amino acid residues 1-56, 1-95, 1-200, 1-300, 1-400, 1-500, 1-600, 1-700, 1-718, 1-765, 1-780, 1-906, 1-918, or 1-1100, as numbered in the above Cas9 reference sequences, or a sequence comprising 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 at least 99.5% sequence identity to the corresponding amino acid residues of another Cas9 polypeptide.

[0308] The C-terminal Cas9 fragment of the fusion protein (i.e., the C-terminal Cas9 fragment that flanks the deaminase in the fusion protein) can comprise the C-terminus of the Cas9 polypeptide. The C-terminal Cas9 fragment of the fusion protein can comprise at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, or 1300 amino acids in length. The C-terminal Cas9 fragment of the fusion protein can comprise a sequence corresponding to amino acid residues 1099-1368, 918-1368, 906-1368, 780-1368, 765-1368, 718-1368, 94-1368, or 56-1368 numbered in the above Cas9 reference sequence, or the corresponding amino acid residues of another Cas9 polypeptide. An N-terminal Cas9 fragment can comprise amino acid residues 1099-1368, 918-1368, 906-1368, 780-1368, 765-1368, 718-1368, 94-1368, or 56-1368, as numbered in the above Cas9 reference sequences, or a sequence comprising 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 at least 99.5% sequence identity to the corresponding amino acid residues of another Cas9 polypeptide.

[0309] The N-terminal Cas9 fragment and the C-terminal Cas9 fragment of the fusion protein taken together may not correspond to the full-length native Cas9 polypeptide sequence, e.g., as set forth in the Cas9 reference sequence above.

[0310] The fusion proteins described herein can achieve targeted deamination with reduced deamination at non-target sites (e.g., off-target sites), such as reduced spurious deamination throughout the genome. The fusion proteins described herein can achieve targeted deamination with reduced bystander deamination at non-target sites. Unwanted or off-target deamination can be reduced by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%, for example, compared to a terminal fusion protein comprising a deaminase fused to the N-terminus or C-terminus of a Cas9 polypeptide. Unwanted or off-target deamination can be reduced by at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold, for example, compared to a terminal fusion protein comprising a deaminase fused to the N-terminus or C-terminus of a Cas9 polypeptide.

[0311] In some embodiments, the deaminase of the fusion protein (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) deaminates no more than two nucleobases within the region of the R-loop. In some embodiments, the deaminase of the fusion protein deaminates no more than three nucleobases within the region of the R-loop. In some embodiments, the deaminase of the fusion protein deaminates no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleobases within the region of the R-loop. An R-loop is a triple-stranded nucleic acid structure comprising a DNA:RNA hybrid, a DNA:DNA, or an RNA:RNA complementary structure, and an associated single-stranded DNA. As used herein, an R-loop can be formed when a target polynucleotide contacts a CRISPR complex or a base editing complex, and a portion of a guide polynucleotide, e.g., a guide RNA, hybridizes with and displaces a portion of a target polynucleotide, e.g., a target DNA. In some embodiments, the R-loop comprises a spacer sequence and a hybridization region of the target DNA complement sequence. The R-loop region can be about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleobase pairs in length. In some embodiments, the R-loop region is about 20 nucleobase pairs in length. It should be understood that, as used herein, the R-loop region is not limited to the target DNA strand that hybridizes with the guide polynucleotide. For example, editing of target nucleobases within an R-loop region may be on the DNA strand containing the complementary strand of the guide RNA, or on the DNA strand that is opposite the strand complementary to the guide RNA. In some embodiments, editing within the R-loop region involves editing nucleobases on the non-complementary strand (protospacer strand) to the guide RNA in the target DNA sequence.

[0312] The fusion proteins described herein can achieve targeted deamination in an editing window that differs from standard base editing. In some embodiments, the target nucleobase is located about 1 to about 20 bases upstream of the PAM sequence in the target polynucleotide sequence. In some embodiments, the target nucleobase is located about 2 to about 12 bases upstream of the PAM sequence in the target polynucleotide sequence. In some embodiments, the target nucleobase is located about 1 to 9 base pairs, about 2 to 10 base pairs, about 3 to 11 base pairs, about 4 to 12 base pairs, about 5 to 13 base pairs, about 6 to 14 base pairs, about 7 to 15 base pairs, about 8 to 16 base pairs, about 9 to 17 base pairs, about 10 to 18 base pairs, about 11 to 19 base pairs, about 12 to 20 base pairs, about 1 to 7 base pairs, or about 2 to 8 base pairs, about 3 to 9 base pairs, about 4 to 10 base pairs, about 5 to 11 base pairs, about 6 to 12 base pairs, about 7 to 13 base pairs, about 8 to 14 base pairs, about 9 to 15 base pairs, about 10 to 16 base pairs, about 11 to 17 base pairs, about 12 to 18 base pairs, about 13 to 19 base pairs, about 14 to 20 base pairs, about 1 to 5 base pairs, about 2 to 6 base pairs, about 3 to 7 base pairs, about 4 to 8 base pairs base pairs, about 5 to 9 base pairs, about 6 to 10 base pairs, about 7 to 11 base pairs, about 8 to 12 base pairs, about 9 to 13 base pairs, about 10 to 14 base pairs, about 11 to 15 base pairs, about 12 to 16 base pairs, about 13 to 17 base pairs, about 14 to 18 base pairs, about 15 to 19 base pairs, about 16 to 20 base pairs, about 1 to 3 base pairs, about 2 to 4 base pairs, about 3 to 5 base pairs, about 4 to 6 base pairs , about 5-7 base pairs, about 6-8 base pairs, about 7-9 base pairs, about 8-10 base pairs, about 9-11 base pairs, about 10-12 base pairs, about 11-13 base pairs, about 12-14 base pairs, about 13-15 base pairs, about 14-16 base pairs, about 15-17 base pairs, about 16-18 base pairs, about 17-19 base pairs, or about 18-20 base pairs away from or upstream of the PAM sequence. In some embodiments, the target nucleobase is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more base pairs away from or upstream of the PAM sequence. In some embodiments, the target nucleobase is about 1, 2, 3, 4, 5, 6, 7, 8, or 9 base pairs upstream of the PAM sequence. In some embodiments, the target nucleobase is about 2, 3, 4, or 6 base pairs upstream of the PAM sequence.

[0313] A fusion protein can contain two or more heterologous polypeptides. For example, the fusion protein can additionally contain one or more UGI domains and / or one or more nuclear localization signals. Two or more heterologous domains can be inserted in tandem. Two or more heterologous domains can be inserted in positions in the NapDNAbp that do not tandemly position the heterologous domains.

[0314] The fusion protein can include a linker between the deaminase and the napDNAbp polypeptide. The linker can be a peptide or a non-peptide linker. For example, the linker can be XTEN, (GGGS)n (SEQ ID NO: 1308), (GGGGS)n (SEQ ID NO: 109), (G)n, (EAAAK)n (SEQ ID NO: 1309), (GGS)n, or SGSETPGTSESATPES (SEQ ID NO: 56). In some embodiments, the fusion protein includes a linker between the N-terminal Cas9 fragment and the deaminase. In some embodiments, the fusion protein includes a linker between the C-terminal Cas9 fragment and the deaminase. In some embodiments, the N-terminal and C-terminal fragments of the napDNAbp are linked to the deaminase with a linker. In some embodiments, the N-terminal and C-terminal fragments are connected to the deaminase domain without a linker. In some embodiments, the fusion protein includes a linker between the N-terminal Cas9 fragment and the deaminase, but does not include a linker between the C-terminal Cas9 fragment and the deaminase. In some embodiments, the fusion protein comprises a linker between the C-terminal Cas9 fragment and the deaminase, but does not comprise a linker between the N-terminal Cas9 fragment and the deaminase.

[0315] In some embodiments, the napDNAbp in the fusion protein is a Cas12 polypeptide, such as Cas12b / C2c1, or a fragment thereof. The Cas12 polypeptide can be a variant Cas12 polypeptide. In other embodiments, the N-terminal or C-terminal fragment of the Cas12 polypeptide comprises a nucleic acid programmable DNA-binding domain or a RuvC domain. In other embodiments, the fusion protein comprises a linker between the Cas12 polypeptide and the catalytic domain. In other embodiments, the amino acid sequence of the linker is GGSGGS (SEQ ID NO:273) or GSSGSETPGTSESATPESSG (SEQ ID NO:1310). In other embodiments, the linker is a rigid linker. In other embodiments of the above aspects, the linker is encoded by GGAGGCTCTGGAGGAAGC (SEQ ID NO:1311) or GGCTCTTCTGGATCTGAAACACCTGGCACAAGCGAGAGCGCCACCCCTGAGAGCTCTGGC (SEQ ID NO:1312).

[0316] Fusion proteins containing a heterologous catalytic domain flanked by N-terminal and C-terminal fragments of a Cas12 polypeptide are also useful for base editing in the methods described herein. Fusion proteins containing Cas12 and one or more deaminase domains, such as adenosine deaminase, or fusion proteins containing an adenosine deaminase domain flanked by Cas12 sequences, are also useful for highly specific and efficient base editing of target sequences. In one embodiment, a chimeric Cas12 fusion protein contains a heterologous catalytic domain (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) inserted within a Cas12 polypeptide. In some embodiments, the fusion protein comprises an adenosine deaminase domain and a cytidine deaminase domain inserted within Cas12. In some embodiments, the adenosine deaminase is fused within Cas12 and the cytidine deaminase is fused to the C-terminus. In some embodiments, adenosine deaminase is fused into Cas12 and cytidine deaminase is fused to the N-terminus. In some embodiments, cytidine deaminase is fused into Cas12 and adenosine deaminase is fused to the C-terminus. In some embodiments, cytidine deaminase is fused into Cas12 and adenosine deaminase is fused to the N-terminus. Exemplary structures of fusion proteins of adenosine deaminase and cytidine deaminase with Cas12 are provided below: NH2-[Cas12(adenosine deaminase)]-[cytidine deaminase]-COOH; NH2-[cytidine deaminase]-[Cas12(adenosine deaminase)]-COOH; NH2-[Cas12(cytidine deaminase)]-[adenosine deaminase]-COOH; or NH2-[adenosine deaminase]-[Cas12(cytidine deaminase)]-COOH;

[0317] In some embodiments, the "-" used in the above general structures indicates the presence of an optional linker.

[0318] In various embodiments, the catalytic domain has a DNA-modifying activity (e.g., deaminase activity), such as adenosine deaminase activity. In some embodiments, the adenosine deaminase is TadA (e.g., TadA*7.10). In some embodiments, TadA is TadA*8. In some embodiments, TadA*8 is fused into Cas12 and a cytidine deaminase is fused to the C-terminus. In some embodiments, TadA*8 is fused into Cas12 and a cytidine deaminase is fused to the N-terminus. In some embodiments, a cytidine deaminase is fused into Cas12 and TadA*8 is fused to the C-terminus. In some embodiments, a cytidine deaminase is fused into Cas12 and TadA*8 is fused to the N-terminus. Exemplary structures of fusion proteins of TadA*8 and cytidine deaminase with Cas12 are provided below: N-[Cas12(TadA*8)]-[cytidine deaminase]-C; N-[cytidine deaminase]-[Cas12(TadA*8)]-C; N-[Cas12(cytidine deaminase)]-[TadA*8]-C; or N-[TadA*8]-[Cas12(cytidine deaminase)]-C.

[0319] In some embodiments, the "-" used in the above general structures indicates the presence of an optional linker.

[0320] In other embodiments, the fusion protein contains one or more catalytic domains. In other embodiments, at least one of the one or more catalytic domains is inserted within a Cas12 polypeptide or fused to the N-terminus or C-terminus of Cas12. In other embodiments, at least one of the one or more catalytic domains is inserted within a loop, alpha-helical region, unstructured portion, or solvent-exposed portion of a Cas12 polypeptide. In other embodiments, the Cas12 polypeptide is Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas12g, Cas12h, Cas12i, or Cas12j / CasΦ. In other embodiments, the Cas12 polypeptide has at least about 85% amino acid sequence identity to Bacillus hisashii Cas12b, Bacillus thermoamylovorans Cas12b, Bacillus sp. V3-13 Cas12b, or Alicyclobacillus acidiphilus Cas12b (SEQ ID NO: 259). In other embodiments, the Cas12 polypeptide has at least about 90% amino acid sequence identity to Bacillus hisashii Cas12b (SEQ ID NO:260), Bacillus thermoamylovorans Cas12b, Bacillus sp. V3-13 Cas12b, or Alicyclobacillus acidiphilus Cas12b. In other embodiments, the Cas12 polypeptide has at least about 95% amino acid sequence identity to Bacillus hisashii Cas12b, Bacillus thermoamylovorans Cas12b (SEQ ID NO:265), Bacillus sp. V3-13 Cas12b (SEQ ID NO:264), or Alicyclobacillus acidiphilus Cas12b.In other embodiments, the Cas12 polypeptide comprises or consists essentially of a fragment of Bacillus hisashii Cas12b, Bacillus thermoamylovorans Cas12b, Bacillus sp. V3-13 Cas12b, or Alicyclobacillus acidiphilus Cas12b. In embodiments, the Cas12 polypeptide comprises BvCas12b (V4), which in some embodiments is expressed as a poly-A tail of 5' mRNA Cap---5' UTR---bhCas12b---termination sequence---3' UTR---120 (SEQ ID NOs: 261-263).

[0321] In other embodiments, the catalytic domain is inserted between amino acid positions 153-154, 255-256, 306-307, 980-981, 1019-1020, 534-535, 604-605, or 344-345 of BhCas12b, or the corresponding amino acid residues of Cas12a, Cas12c, Cas12d, Cas12e, Cas12g, Cas12h, Cas12i, or Cas12j / CasΦ. In other embodiments, the catalytic domain is inserted between amino acids P153 and S154 of BhCas12b. In other embodiments, the catalytic domain is inserted between amino acids K255 and E256 of BhCas12b. In other embodiments, the catalytic domain is inserted between amino acids D980 and G981 of BhCas12b. In other embodiments, the catalytic domain is inserted between amino acids K1019 and L1020 of BhCas12b. In other embodiments, the catalytic domain is inserted between amino acids F534 and P535 of BhCas12b. In other embodiments, the catalytic domain is inserted between amino acids K604 and G605 of BhCas12b. In other embodiments, the catalytic domain is inserted between amino acids H344 and F345 of BhCas12b. In other embodiments, the catalytic domain is inserted between amino acid positions 147 and 148, 248 and 249, 299 and 300, 991 and 992, or 1031 and 1032 of BvCas12b, or at the corresponding amino acid residues of Cas12a, Cas12c, Cas12d, Cas12e, Cas12g, Cas12h, Cas12i, or Cas12j / CasΦ. In another embodiment, the catalytic domain is inserted between amino acids P147 and D148 of BvCas12b. In another embodiment, the catalytic domain is inserted between amino acids G248 and G249 of BvCas12b. In another embodiment, the catalytic domain is inserted between amino acids P299 and E300 of BvCas12b. In another embodiment, the catalytic domain is inserted between amino acids G991 and E992 of BvCas12b. In another embodiment, the catalytic domain is inserted between amino acids K1031 and M1032 of BvCas12b.In other embodiments, the catalytic domain is inserted between amino acid positions 157 and 158, 258 and 259, 310 and 311, 1008 and 1009, or 1044 and 1045 of AaCas12b, or at the corresponding amino acid residues of Cas12a, Cas12c, Cas12d, Cas12e, Cas12g, Cas12h, Cas12i, or Cas12j / CasΦ. In other embodiments, the catalytic domain is inserted between amino acids P157 and G158 of AaCas12b. In other embodiments, the catalytic domain is inserted between amino acids V258 and G259 of AaCas12b. In other embodiments, the catalytic domain is inserted between amino acids D310 and P311 of AaCas12b. In other embodiments, the catalytic domain is inserted between amino acids G1008 and E1009 of AaCas12b. In other embodiments, the catalytic domain is inserted between amino acids G1044 and K1045 of AaCas12b.

[0322] In other embodiments, the fusion protein contains a nuclear localization signal (e.g., a bipartite nuclear localization signal). In other embodiments, the amino acid sequence of the nuclear localization signal is MAPKKKRKVGIHGVPAA (SEQ ID NO: 1313). In other embodiments of the above aspect, the nuclear localization signal is encoded by the following sequence: ATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCC (SEQ ID NO: 1314). In other embodiments, the Cas12b polypeptide contains a mutation that silences the catalytic activity of the RuvC domain. In other embodiments, the Cas12b polypeptide contains a D574A, D829A, and / or D952A mutation. In other embodiments, the fusion protein further contains a tag (e.g., an influenza hemagglutinin tag).

[0323] In some embodiments, the fusion protein comprises a napDNAbp domain (e.g., a Cas12-derived domain) comprising an internally fused nucleobase editing domain (e.g., a deaminase domain, e.g., all or part of an adenosine deaminase domain). In some embodiments, the napDNAbp is Cas12b. In some embodiments, the base editor comprises a BhCas12b domain comprising an internally fused TadA*8 domain inserted into a locus provided in Table 4 below. [Table 4]

[0324] As a non-limiting example, an adenosine deaminase (e.g., TadA*8.13) can be inserted into BhCas12b to generate a fusion protein (e.g., TadA*8.13-BhCas12b) that effectively edits nucleic acid sequences.

[0325] In some embodiments, the base editing system described herein is an ABE with TadA inserted into Cas9. The polypeptide sequences of related ABEs with TadA inserted into Cas9 are provided in the accompanying sequence listing as SEQ ID NOs: 1315-1360.

[0326] In some embodiments, adenosine deaminase base editors were generated by inserting TadA or a variant thereof into a Cas9 polypeptide at a specific position.

[0327] Exemplary fusion proteins are described, without limitation, in International PCT Application No. PCT / US2020 / 016285 and U.S. Provisional Patent Applications Nos. 62 / 852,228 and 62 / 852,224, the contents of which are incorporated herein by reference in their entireties.

[0328] Editing A to G In some embodiments, the base editors described herein comprise an adenosine deaminase domain. The adenosine deaminase domain of such base editors can facilitate editing of adenine (A) nucleobases to guanine (G) nucleobases by deaminating A to form inosine (I), which exhibits the base-pairing properties of G. Adenosine deaminase is capable of deaminating (i.e., removing the amine group from) the adenine of deoxyadenosine residues in deoxyribonucleic acid (DNA). In some embodiments, the A-to-G base editor further comprises an inhibitor of inosine base excision repair, such as a uracil glycosylase inhibitor (UGI) domain or a catalytically inactive inosine-specific nuclease. Without wishing to be bound by any particular theory, the UGI domain or catalytically inactive inosine-specific nuclease can inhibit or prevent base excision repair of deaminated adenosine residues (e.g., inosine), which may improve the activity or efficiency of the base editor.

[0329] Base editors comprising adenosine deaminase can act on any polynucleotide, including DNA, RNA, and DNA-RNA hybrids. In certain embodiments, base editors comprising adenosine deaminase can deaminate target A in polynucleotides comprising RNA. For example, a base editor can comprise an adenosine deaminase domain capable of deaminating target A in RNA polynucleotides and / or DNA-RNA hybrid polynucleotides. In one embodiment, the adenosine deaminase incorporated in the base editor comprises all or a portion of an adenosine deaminase acting on RNA (ADAR, e.g., ADAR1 or ADAR2) or an adenosine deaminase acting on tRNA (ADAT). Base editors comprising an adenosine deaminase domain can also deaminate A nucleobases in DNA polynucleotides. In one embodiment, the adenosine deaminase domain of the base editor comprises all or a portion of ADAT containing one or more mutations that enable ADAT to deaminate target A in DNA. For example, a base editor can include all or a portion of ADAT from Escherichia coli (EcTadA) containing one or more of the following mutations: D108N, A106V, D147Y, E155V, L84F, H123Y, I156F, or the corresponding mutations of another adenosine deaminase. Exemplary ADAT homolog polypeptide sequences are provided in the Sequence Listing as SEQ ID NOs: 1363-1370.

[0330] The adenosine deaminase can be derived from any suitable organism (e.g., E. coli). In some embodiments, the adenosine deaminase is derived from a prokaryote. In some embodiments, the adenosine deaminase is derived from a bacterium. In some embodiments, the adenosine deaminase is derived from Escherichia coli, Staphylococcus aureus, Salmonella typhi, Shewanella putrefaciens, Haemophilus influenzae, Caulobacter crescentus, or Bacillus subtilis. In some embodiments, the adenosine deaminase is E. coli. In some embodiments, the adenosine deaminase is a naturally occurring adenosine deaminase that includes one or more mutations (e.g., mutations in ecTadA) corresponding to any of the mutations provided herein. Corresponding residues in any homologous proteins can be identified, for example, by sequence alignment and sequencing of the homologous residues. Mutations in any naturally occurring adenosine deaminase (e.g., with homology to ecTadA) that correspond to any of the mutations described herein (e.g., any of the mutations identified in ecTadA) can be made as appropriate.

[0331] In some embodiments, the adenosine deaminase 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 the amino acid sequences described in any of the adenosine deaminases provided herein. It should be understood that the adenosine deaminases provided herein can include one or more mutations (e.g., any of the mutations provided herein). The present disclosure provides any deaminase domain that has any of the mutations described herein or a combination thereof, in addition to a certain percent identity. In some embodiments, the adenosine deaminase comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more mutations compared to a reference sequence or any of the adenosine deaminases provided herein. In some embodiments, the adenosine deaminase comprises an amino acid sequence having at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, or at least 170 identical contiguous amino acid residues compared to any one of the amino acid sequences known in the art or described herein.

[0332] It should be understood that any of the mutations provided herein (e.g., based on the TadA reference sequence) can be introduced into other adenosine deaminases, such as E. coli TadA (ecTadA), S. aureus TadA (saTadA), or other adenosine deaminases (e.g., bacterial adenosine deaminases). One skilled in the art will appreciate that additional deaminases can be similarly aligned to identify homologous amino acid residues at which mutations as provided herein can be made. Thus, any of the mutations identified in the TadA reference sequence can be made in other adenosine deaminases (e.g., ecTadA) that have homologous amino acid residues. It should also be understood that any of the mutations provided herein, individually or in any combination, can be made in the TadA reference sequence or another adenosine deaminase.

[0333] In some embodiments, the adenosine deaminase comprises a D108X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a D108G, D108N, D108V, D108A, or D108Y mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase. However, it should be understood that additional deaminases can be similarly aligned to identify homologous amino acid residues that can be mutated as provided herein.

[0334] In some embodiments, the adenosine deaminase comprises an A106X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an A106V mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA).

[0335] In some embodiments, the adenosine deaminase comprises an E155X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where the presence of X denotes any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an E155D, E155G, or E155V mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA).

[0336] In some embodiments, the adenosine deaminase comprises a D147X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where the presence of X denotes any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a D147Y mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA).

[0337] In some embodiments, the adenosine deaminase comprises an A106X, E155X, or D147X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA), where X represents any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an E155D, E155G, or E155V mutation. In some embodiments, the adenosine deaminase comprises D147Y.

[0338] It should also be understood that any of the mutations provided herein, individually or in any combination, can be made to ecTadA or another adenosine deaminase. For example, an adenosine deaminase can contain the D108N, A106V, E155V, and / or D147Y mutations of the TadA reference sequence, or the corresponding mutations of another adenosine deaminase (e.g., ecTadA). In some embodiments, the adenosine deaminase includes the following mutations of the TadA reference sequence (mutation groups are separated by ";"), or the corresponding mutations of another adenosine deaminase: D108N and A106V; D108N and E155V; D108N and D147Y; A106V and E155V; A106V and D147Y; E155V and D147Y; D108N, A106V, and E155V; D108N, A106V, and D147Y; D108N, E155V, and D147Y; A106V, E155V, and D147Y; and D108N, A106V, E155V, and D147Y. However, it should be understood that any combination of the corresponding mutations provided herein can be made in an adenosine deaminase (eg, ecTadA).

[0339] In some embodiments, the adenosine deaminase comprises one or more H8X, T17X, ​​L18X, W23X, L34X, W45X, R51X, A56X, E59X, E85X, M94X, I95X, V102X, F104X, A106X, R107X, D108X, K110X, M118X, N127X, A138X, F149X, M151X, R153X, Q154X, I156X, and / or K157X mutations of the TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase, where the occurrence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of the H8Y, T17S, L18E, W23L, L34S, W45L, R51H, A56E or A56S, E59G, E85K or E85G, M94L, I95L, V102A, F104L, A106V, R107C or R107H or R107P, D108G or D108N or D108V or D108A or D108Y, K110I, M118K, N127S, A138V, F149Y, M151V, R153C, Q154L, I156D, and / or K157R mutations of the TadA reference sequence, or one or more corresponding mutations of another adenosine deaminase.

[0340] In some embodiments, the adenosine deaminase comprises one or more H8X, D108X, and / or N127X mutations of the TadA reference sequence, or one or more corresponding mutations of another adenosine deaminase, where X indicates the presence of any amino acid. In some embodiments, the adenosine deaminase comprises one or more H8Y, D108N, and / or N127S mutations of the TadA reference sequence, or one or more corresponding mutations of another adenosine deaminase.

[0341] In some embodiments, the adenosine deaminase comprises one or more of the H8X, R26X, M61X, L68X, M70X, A106X, D108X, A109X, N127X, D147X, R152X, Q154X, E155X, K161X, Q163X, and / or T166X mutations of the TadA reference sequence, or one or more corresponding mutations of another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of the H8Y, R26W, M61I, L68Q, M70V, A106T, D108N, A109T, N127S, D147Y, R152C, Q154H or Q154R, E155G or E155V or E155D, K161Q, Q163H, and / or T166P mutations of the TadA reference sequence, or one or more corresponding mutations of another adenosine deaminase.

[0342] In some embodiments, the adenosine deaminase comprises one, two, three, four, five, or six mutations selected from the group consisting of H8X, D108X, N127X, D147X, R152X, and Q154X of the TadA reference sequence, or the corresponding one or more mutations of another adenosine deaminase (e.g., ecTadA), where X indicates the presence of any amino acid other than the corresponding amino acid of the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, five, six, seven, or eight mutations selected from the group consisting of H8X, M61X, M70X, D108X, N127X, Q154X, E155X, and Q163X of the TadA reference sequence, or the corresponding one or more mutations of another adenosine deaminase (e.g., ecTadA), where X indicates the presence of any amino acid other than the corresponding amino acid of the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, or five mutations selected from the group consisting of H8X, D108X, N127X, E155X, and T166X of the TadA reference sequence, or the corresponding one or more mutations of another adenosine deaminase (e.g., ecTadA), where X indicates the presence of any amino acid other than the corresponding amino acid of the wild-type adenosine deaminase.

[0343] In some embodiments, the adenosine deaminase comprises one, two, three, four, five, or six mutations selected from the group consisting of H8X, A106X, and D108X, or the corresponding one or more mutations in another adenosine deaminase, where X represents the presence of any amino acid other than the corresponding amino acid in wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, five, six, seven, or eight mutations selected from the group consisting of H8X, R26X, L68X, D108X, N127X, D147X, and E155X, or the corresponding one or more mutations in another adenosine deaminase, where X represents the presence of any amino acid other than the corresponding amino acid in wild-type adenosine deaminase.

[0344] In some embodiments, the adenosine deaminase comprises one, two, three, four, five, six, or seven mutations selected from the group consisting of H8X, R126X, L68X, D108X, N127X, D147X, and E155X of the TadA reference sequence, or the corresponding one or more mutations of another adenosine deaminase, where X represents the presence of any amino acid other than the corresponding amino acid of the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, or five mutations selected from the group consisting of H8X, D108X, A109X, N127X, and E155X of the TadA reference sequence, or the corresponding one or more mutations of another adenosine deaminase, where X represents the presence of any amino acid other than the corresponding amino acid of the wild-type adenosine deaminase.

[0345] In some embodiments, the adenosine deaminase comprises one, two, three, four, five, or six mutations selected from the group consisting of H8Y, D108N, N127S, D147Y, R152C, and Q154H of the TadA reference sequence, or the corresponding one or more mutations of another adenosine deaminase (e.g., ecTadA). In some embodiments, the adenosine deaminase comprises one, two, three, four, five, six, seven, or eight mutations selected from the group consisting of H8Y, M61I, M70V, D108N, N127S, Q154R, E155G, and Q163H of the TadA reference sequence, or the corresponding one or more mutations of another adenosine deaminase (e.g., ecTadA). In some embodiments, the adenosine deaminase comprises one, two, three, four, or five mutations selected from the group consisting of H8Y, D108N, N127S, E155V, and T166P of the TadA reference sequence, or the corresponding one or more mutations of another adenosine deaminase (e.g., ecTadA). In some embodiments, the adenosine deaminase comprises one, two, three, four, five, or six mutations selected from the group consisting of H8Y, A106T, D108N, N127S, E155D, and K161Q of the TadA reference sequence, or the corresponding one or more mutations of another adenosine deaminase (e.g., ecTadA). In some embodiments, the adenosine deaminase comprises one, two, three, four, five, six, seven, or eight mutations selected from the group consisting of H8Y, R26W, L68Q, D108N, N127S, D147Y, and E155V of the TadA reference sequence, or the corresponding one or more mutations of another adenosine deaminase (e.g., ecTadA). In some embodiments, the adenosine deaminase comprises one, two, three, four, or five mutations selected from the group consisting of H8Y, D108N, A109T, N127S, and E155G of the TadA reference sequence, or the corresponding one or more mutations of another adenosine deaminase (e.g., ecTadA).

[0346] In some embodiments, the adenosine deaminase comprises one or more or one or more corresponding mutations of another adenosine deaminase. In some embodiments, the adenosine deaminase comprises a D108N, D108G, or D108V mutation of the TadA reference sequence, or a corresponding mutation of another adenosine deaminase. In some embodiments, the adenosine deaminase comprises an A106V and D108N mutation of the TadA reference sequence, or a corresponding mutation of another adenosine deaminase. In some embodiments, the adenosine deaminase comprises an R107C and D108N mutation of the TadA reference sequence, or a corresponding mutation of another adenosine deaminase. In some embodiments, the adenosine deaminase comprises an H8Y, D108N, N127S, D147Y, and Q154H mutation of the TadA reference sequence, or a corresponding mutation of another adenosine deaminase. In some embodiments, the adenosine deaminase comprises the H8Y, D108N, N127S, D147Y, and E155V mutations of the TadA reference sequence, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises the D108N, D147Y, and E155V mutations of the TadA reference sequence, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises the H8Y, D108N, and N127S mutations of the TadA reference sequence, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises the A106V, D108N, D147Y, and E155V mutations of the TadA reference sequence, or corresponding mutations in another adenosine deaminase (e.g., ecTadA).

[0347] In some embodiments, the adenosine deaminase comprises one or more of the S2X, H8X, I49X, L84X, H123X, N127X, I156X, and / or K160X mutations of the TadA reference sequence, or one or more corresponding mutations of another adenosine deaminase, where the occurrence of X denotes any amino acid other than the corresponding amino acid of the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of the S2A, H8Y, I49F, L84F, H123Y, N127S, I156F, and / or K160S mutations of the TadA reference sequence, or one or more corresponding mutations of another adenosine deaminase (e.g., ecTadA).

[0348] In some embodiments, the adenosine deaminase comprises an L84X mutant adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase, hi some embodiments, the adenosine deaminase comprises an L84F mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA).

[0349] In some embodiments, the adenosine deaminase comprises an H123X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an H123Y mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0350] In some embodiments, the adenosine deaminase comprises an I156X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an I156F mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0351] In some embodiments, the adenosine deaminase comprises one, two, three, four, five, six, or seven mutations selected from the group consisting of L84X, A106X, D108X, H123X, D147X, E155X, and I156X of the TadA reference sequence, or the corresponding one or more mutations of another adenosine deaminase, where X represents the presence of any amino acid other than the corresponding amino acid of the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, five, or six mutations selected from the group consisting of S2X, I49X, A106X, D108X, D147X, and E155X of the TadA reference sequence, or the corresponding one or more mutations of another adenosine deaminase, where X represents the presence of any amino acid other than the corresponding amino acid of the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, or five mutations selected from the group consisting of H8X, A106X, D108X, N127X, and K160X of the TadA reference sequence, or the corresponding one or more mutations of another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid of the wild-type adenosine deaminase.

[0352] In some embodiments, the adenosine deaminase comprises one, two, three, four, five, six, or seven mutations selected from the group consisting of L84F, A106V, D108N, H123Y, D147Y, E155V, and I156F of the TadA reference sequence, or the corresponding one or more mutations of another adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, five, or six mutations selected from the group consisting of S2A, I49F, A106V, D108N, D147Y, and E155V of the TadA reference sequence.

[0353] In some embodiments, the adenosine deaminase comprises one, two, three, four, or five mutations selected from the group consisting of H8Y, A106T, D108N, N127S, and K160S of the TadA reference sequence, or the corresponding one or more mutations of another adenosine deaminase.

[0354] In some embodiments, the adenosine deaminase comprises one or more of the E25X, R26X, R107X, A142X, and / or A143X mutations of the TadA reference sequence, or one or more corresponding mutations of another adenosine deaminase, where the occurrence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of the E25M, E25D, E25A, E25R, E25V, E25S, E25Y, R26G, R26N, R26Q, R26C, R26L, R26K, R107P, R107K, R107A, R107N, R107W, R107H, R107S, A142N, A142D, A142G, A143D, A143G, A143E, A143L, A143W, A143M, A143S, A143Q, and / or A143R mutations of the TadA reference sequence, or one or more corresponding mutations of another adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of the mutations described herein corresponding to the TadA reference sequence, or one or more corresponding mutations of another adenosine deaminase.

[0355] In some embodiments, the adenosine deaminase comprises an E25X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an E25M, E25D, E25A, E25R, E25V, E25S, or E25Y mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA).

[0356] In some embodiments, the adenosine deaminase comprises an R26X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an R26G, R26N, R26Q, R26C, R26L, or R26K mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA).

[0357] In some embodiments, the adenosine deaminase comprises an R107X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an R107P, R107K, R107A, R107N, R107W, R107H, or R107S mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA).

[0358] In some embodiments, the adenosine deaminase comprises an A142X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an A142N, A142D, or A142G mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA).

[0359] In some embodiments, the adenosine deaminase comprises an A143X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an A143D, A143G, A143E, A143L, A143W, A143M, A143S, A143Q, and / or A143R mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA).

[0360] In some embodiments, the adenosine deaminase comprises one or more of the H36X, N37X, P48X, I49X, R51X, M70X, N72X, D77X, E134X, S146X, Q154X, K157X, and / or K161X mutations of the TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase, where the occurrence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of the H36L, N37T, N37S, P48T, P48L, I49V, R51H, R51L, M70L, N72S, D77G, E134G, S146R, S146C, Q154H, K157N, and / or K161T mutations of the TadA reference sequence, or one or more corresponding mutations of another adenosine deaminase (e.g., ecTadA).

[0361] In some embodiments, the adenosine deaminase comprises an H36X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an H36L mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0362] In some embodiments, the adenosine deaminase comprises an N37X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an N37T or N37S mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0363] In some embodiments, the adenosine deaminase comprises a P48X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a P48T or P48L mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0364] In some embodiments, the adenosine deaminase comprises an R51X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an R51H or R51L mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0365] In some embodiments, the adenosine deaminase comprises a S146X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a S146R or S146C mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0366] In some embodiments, the adenosine deaminase comprises a K157X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a K157N mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0367] In some embodiments, the adenosine deaminase comprises a P48X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a P48S, P48T, or P48A mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0368] In some embodiments, the adenosine deaminase comprises an A142X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an A142N mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0369] In some embodiments, the adenosine deaminase comprises a W23X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a W23R or W23L mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0370] In some embodiments, the adenosine deaminase comprises an R152X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X represents any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an R152P or R52H mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase.

[0371] In one embodiment, the adenosine deaminase can include the mutations H36L, R51L, L84F, A106V, D108N, H123Y, S146C, D147Y, E155V, I156F, and K157N. In some embodiments, the adenosine deaminase includes the following combinations of mutations relative to the TadA reference sequence, where each mutation in the combination is separated by an "_" and each combination of mutations is within parentheses: (A106V_D108N), (R107C_D108N), (H8Y_D108N_N127S_D147Y_Q154H), (H8Y_D108N_N127S_D147Y_E155V), (D108N_D147Y_E155V), (H8Y_D108N_N127S), (H8Y_D108N_N127S_D147Y_Q154H), (A106V_D108N_D147Y_E155V), (D108Q_D147Y_E155V), (D108M_D147Y_E155V), (D108L_D147Y_E155V), (D108K_D147Y_E155V), (D108I_D147Y_E155V), (D108F_D147Y_E155V), (A106V_D108N_D147Y), (A106V_D108M_D147Y_E155V), (E59A_A106V_D108N_D147Y_E155V), (E59A catalyst deactivation_A106V_D108N_D147Y_E155V), (L84F_A106V_D108N_H123Y_D147Y_E155V_I156Y), (L84F_A106V_D108N_H123Y_D147Y_E155V_I156F), (D103A_D104N), (G22P_D103A_D104N), (D103A_D104N_S138A), (R26G_L84F_A106V_R107H_D108N_H123Y_A142N_A143D_D147Y_E155V_I156F), (E25G_R26G_L84F_A106V_R107H_D108N_H123Y_A142N_A143D_D147Y_E155V_I156F), (E25D_R26G_L84F_A106V_R107K_D108N_H123Y_A142N_A143G_D147Y_E155 V_I156F), (R26Q_L84F_A106V_D108N_H123Y_A142N_D147Y_E155V_I156F), (E25M_R26G_L84F_A106V_R107P_D108N_H123Y_A142N_A143D_D147Y_E155V_I156F), (R26C_L84F_A106V_R107H _D108N_H123Y_A142N_D147Y_E155V_I156F), (L84F_A106V_D108N_H123Y_A142N_A143L_D147Y_E155V_I156F), (R26G_L84F_A106V_D108N_H123Y_A142N_D147Y_E155V_I156F), (E25A_R26G_L84F_A106V_R107N_D108N_H123Y_A142N_A143E_D147Y_E155V_I156 F), (R26G_L84F_A106V_R107H_D108N_H123Y_A142N_A143D_D147Y_E155V_I156F), (A106V_D108N_A142N_D147Y_E155V)、 (R26G_A106V_D108N_A142N_D147Y_E155V)、 (E25D_R26G_A106V_R107K_D108N_A142N_A143G_D147Y_E155V)、(R26G_A106V_D108N_R107H_A142N_A143D_D147Y_E155V)、 (E25D_R26G_A106V_D108N_A142N_D147Y_E155V)、 (A106V_R107K_D108N_A142N_D147Y_E155V)、 (A106V_D108N_A142N_A143G_D147Y_E155V)、 (A106V_D108N_A142N_A143L_D147Y_E155V)、 (H36L_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N)、 (N37T_P48T_M70L_L84F_A106V_D108N_H123Y_D147Y_I49V_E155V_I156F)、 (N37S_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F_K161T)、 (H36L_L84F_A106V_D108N_H123Y_D147Y_Q154H_E155V_I156F)、 (N72S_L84F_A106V_D108N_H123Y_S146R_D147Y_E155V_I156F)、 (H36L_P48L_L84F_A106V_D108N_H123Y_E134G_D147Y_E155V_I156F)、 (H36L_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F_K157N) (H36L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F)、 (L84F_A106V_D108N_H123Y_S146R_D147Y_E155V_I156F_K161T)、 (N37S_R51H_D77G_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F)、 (R51L_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F_K157N)、 (D24G_Q71R_L84F_H96L_A106V_D108N_H123Y_D147Y_E155V_I156F_K160E)、 (H36L_G67V_L84F_A106V_D108N_H123Y_S146T_D147Y_E155V_I156F)、 (Q71L_L84F_A106V_D108N_H123Y_L137M_A143E_D147Y_E155V_I156F)、 (E25G_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F_Q159L)、 (L84F_A91T_F104I_A106V_D108N_H123Y_D147Y_E155V_I156F)、 (N72D_L84F_A106V_D108N_H123Y_G125A_D147Y_E155V_I156F)、 (P48S_L84F_S97C_A106V_D108N_H123Y_D147Y_E155V_I156F)、 (W23G_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F)、 (D24G_P48L_Q71R_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F_Q159L)、 (L84F_A106V_D108N_H123Y_A142N_D147Y_E155V_I156F)、 (H36L_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_E155V_I156F_K157N)、(N37S_L84F_A106V_D108N_H123Y_A142N_D147Y_E155V_I156F_K161T)、 (L84F_A106V_D108N_D147Y_E155V_I156F)、 (R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N_K161T)、 (L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K161T)、 (L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N_K160E_K161T)、 (L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N_K160E)、(R74Q_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F)、 (R74A_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F)、 (L84F_A106V_D108N_H123Y_D147Y_E155V_I156F)、 (R74Q_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F)、 (L84F_R98Q_A106V_D108N_H123Y_D147Y_E155V_I156F)、 (L84F_A106V_D108N_H123Y_R129Q_D147Y_E155V_I156F)、 (P48S_L84F_A106V_D108N_H123Y_A142N_D147Y_E155V_I156F)、 (P48S_A142N)、 (P48T_I49V_L84F_A106V_D108N_H123Y_A142N_D147Y_E155V_I156F_L157N)、 (P48T_I49V_A142N)、 (H36L_P48S_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N)、(H36L_P48S_R51L_L84F_A106V_D108N_H123Y_S146C_A142N_D147Y_E155V_I156F (H36L_P48T_I49V_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N)、 (H36L_P48T_I49V_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_E155V_I156F_K157N)、 (H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F _K157N)、 (H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_E155V_I156F_K157N)、(H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_A142N_D147Y_E155V_I156F_K157N)、 (W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N)、(W23R_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N)、(W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146R_D147Y_E155V_I156F_K161T)、 (H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152H_E155V_I156F_K157N)、 (H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152P_E155V_I156F_K157N), (W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152P_E155V_I156F_K157N), (W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142A_S146C_D147Y_E155V_I156F_K157N), (W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142A_S146C_D147Y_R152P_E155V_I156F_K157N), (W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146R_D147Y_E155V_I156F_K161T), (W23R_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152P_E155V_I156F_K157N), (H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_R152P_E155V_I156F_K157N).

[0372] In some embodiments, the TadA deaminase is a TadA variant. In some embodiments, the TadA variant is TadA*7.10. In certain embodiments, the fusion protein comprises a single TadA*7.10 domain (e.g., provided as a monomer). In other embodiments, the fusion protein comprises TadA*7.10 and TadA(wt), and is capable of forming a heterodimer. In one embodiment, a fusion protein of the invention comprises wild-type TadA linked to TadA*7.10 linked to a Cas9 nickase.

[0373] In some embodiments, TadA*7.10 comprises at least one modification. In some embodiments, the adenosine deaminase comprises a modification in the following sequence: TadA*7.10 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 8)

[0374] In some embodiments, TadA*7.10 comprises modifications at amino acids 82 and / or 166. In certain embodiments, TadA*7.10 comprises one or more of the following modifications: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R. In other embodiments, variants of TadA*7.10 include: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123H+Y147R; V82S+Y123H+Y147R; V82S+Y123H 3H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R.

[0375] In some embodiments, the adenosine deaminase variant (e.g., TadA*8) comprises a deletion. In some embodiments, the adenosine deaminase variant comprises a C-terminal deletion. In particular embodiments, the adenosine deaminase variant comprises a C-terminal deletion starting at residues 149, 150, 151, 152, 153, 154, 155, 156, and 157 relative to the TadA reference sequence, TadA*7.10, or a corresponding mutation in another TadA.

[0376] In other embodiments, the adenosine deaminase variant (e.g., TadA*8) is a monomer that includes one or more of the following modifications relative to the TadA reference sequence TadA*7.10: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R, or the corresponding mutations in another TadA. In another embodiment, the adenosine deaminase variant (TadA*8) has the following sequence relative to TadA*7.10 of the TadA reference sequence: Y147T+Q154R;Y147T+Q154S;Y147R+Q154S;V82S+Q154S;V82S+Y147R;V82S+Q154R;V82S+Y123H;I76Y+V82S;V82S+Y123H+Y147T;V82S+Y123H+Y147R;V82S +Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R, or a corresponding mutation in another TadA.

[0377] In other embodiments, the adenosine deaminase variant is a homodimer comprising two adenosine deaminase domains (e.g., TadA*8), each of which has one or more of the following modifications relative to TadA*7.10 of the TadA reference sequence: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R, or corresponding mutations in another TadA. In other embodiments, the adenosine deaminase variant comprises two adenosine deaminase domains (e.g., TadA*8), each of which has the following sequence changes relative to TadA*7.10 of the TadA reference sequence: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R, or a corresponding mutation in another TadA.

[0378] In other embodiments, the adenosine deaminase variant is a heterodimer of a wild-type adenosine deaminase domain and an adenosine deaminase variant domain containing one or more of the following modifications Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R relative to the TadA reference sequence, TadA*7.10, or the corresponding mutations in another TadA (e.g., TadA*8). In other embodiments, the adenosine deaminase variant has a wild-type adenosine deaminase domain and the following amino acid sequences relative to the TadA reference sequence, TadA*7.10: Y147T+Q154R;Y147T+Q154S;Y147R+Q154S;V82S+Q154S;V82S+Y147R;V82S+Q154R;V82S+Y123H;I76Y+V82S;V82S+Y123H+Y147T;V82S+Y123H+Y147R;V82S+Y123H+Q154R Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R, or a heterodimer with an adenosine deaminase variant domain containing the corresponding mutations of another TadA (e.g., TadA*8).

[0379] In other embodiments, the adenosine deaminase variant is a heterodimer of a TadA*7.10 domain and an adenosine deaminase variant domain (e.g., TadA*8) that contains one or more of the following modifications relative to TadA*7.10 in the TadA reference sequence: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R, or the corresponding mutations in another TadA. In another embodiment, the adenosine deaminase variant has a TadA*7.10 domain and the following amino acid sequences relative to TadA*7.10 of the TadA reference sequence: Y147T+Q154R;Y147T+Q154S;Y147R+Q154S;V82S+Q154S;V82S+Y147R;V82S+Q154R;V82S+Y123H;I76Y+V82S;V82S+Y123H+Y147T;V82S+Y123H+Y147R;V82S+Y123H+Q154R;Y1 Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R, or a heterodimer with an adenosine deaminase variant domain containing the corresponding mutations of another TadA (e.g., TadA*8).

[0380] In certain embodiments, the adenosine deaminase heterodimer comprises a TadA*8 domain and an adenosine deaminase domain selected from Staphylococcus aureus (S. aureus) TadA, Bacillus subtilis (B. subtilis) TadA, Salmonella typhimurium (S. typhimurium) TadA, Shewanella putrefaciens (S. putrefaciens) TadA, Haemophilus influenzae F3031 (H. influenzae) TadA, Caulobacter crescentus (C. crescentus) TadA, Geobacter sulfurreducens (G. sulfurreducens) TadA, or TadA*7.10.

[0381] In some embodiments, the adenosine deaminase is TadA*8. In one embodiment, the adenosine deaminase is TadA*8 that comprises or consists essentially of the following sequence or a fragment thereof having adenosine deaminase activity: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCTFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 12)

[0382] In some embodiments, TadA*8 is truncated. In some embodiments, the truncated TadA*8 lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 N-terminal amino acid residues compared to full-length TadA*8. In some embodiments, the truncated TadA*8 lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 C-terminal amino acid residues compared to full-length TadA*8. In some embodiments, the adenosine deaminase variant is full-length TadA*8.

[0383] In some embodiments, TadA*8 is TadA*8.1, TadA*8.2, TadA*8.3, TadA*8.4, TadA*8.5, TadA*8.6, TadA*8.7, TadA*8.8, TadA*8.9, TadA*8.10, TadA*8.11, TadA*8.12, TadA*8.13, TadA*8.14, TadA*8.15, TadA*8.16, TadA*8.17, TadA*8.18, TadA*8.19, TadA*8.20, TadA*8.21, TadA*8.22, TadA*8.23, or TadA*8.24.

[0384] In other embodiments, a base editor of the disclosure comprises an adenosine deaminase variant (e.g., TadA*8) monomer that includes one or more of the following modifications relative to TadA*7.10 of the TadA reference sequence: R26C, V88A, A109S, T111R, D119N, H122N, Y147D, F149Y, T166I, and / or D167N, or the corresponding mutations in another TadA. In another embodiment, the adenosine deaminase variant (TadA*8) monomer has the following sequence relative to the TadA reference sequence TadA*7.10: R26C+A109S+T111R+D119N+H122N+Y147D+F149Y+T166I+D167N; V88A+A109S+T111R+D119N+H122N+F149Y+T166I+D167 N; R26C+A109S+T111R+D119N+H122N+F149Y+T166I+D167N; V88A+T111R+D119N+F149Y; and A109S+T111R+D119N+H122N+Y147D+F149Y+T166I+D167N, or the corresponding mutations in another TadA.

[0385] In other embodiments, the base editor comprises a heterodimer of a wild-type adenosine deaminase domain and an adenosine deaminase variant domain that includes one or more of the following modifications R26C, V88A, A109S, T111R, D119N, H122N, Y147D, F149Y, T166I and / or D167N relative to the TadA reference sequence, TadA*7.10, or the corresponding mutations in another TadA (e.g., TadA*8). In other embodiments, the base editors include the following bases relative to the wild-type adenosine deaminase domain and the TadA reference sequence TadA*7.10: R26C+A109S+T111R+D119N+H122N+Y147D+F149Y+T166I+D167N; V88A+A109S+T111R+D119N+H122N+F149Y+T166I+D167N; R26C+A109S+T111R+D119N+H122N ... 9N+H122N+F149Y+T166I+D167N; V88A+T111R+D119N+F149Y; and A109S+T111R+D119N+H122N+Y147D+F149Y+T166I+D167N, or a heterodimer with an adenosine deaminase variant domain containing the corresponding mutations of another TadA (e.g., TadA*8).

[0386] In other embodiments, the base editor comprises a heterodimer of a TadA*7.10 domain and an adenosine deaminase variant domain that includes one or more of the following modifications R26C, V88A, A109S, T111R, D119N, H122N, Y147D, F149Y, T166I, and / or D167N relative to TadA*7.10 of the TadA reference sequence, or the corresponding mutations in another TadA (e.g., TadA*8). In other embodiments, the base editor comprises the TadA*7.10 domain and, relative to TadA*7.10 of the TadA reference sequence, R26C+A109S+T111R+D119N+H122N+Y147D+F149Y+T166I+D167N;V88A+A109S+T111R+D119N+H122N+F149Y+T166I+D167N;R26C+A109S+T111R+D119N +H122N+F149Y+T166I+D167N; V88A+T111R+D119N+F149Y; and A109S+T111R+D119N+H122N+Y147D+F149Y+T166I+D167N, or a heterodimer with an adenosine deaminase variant domain containing the corresponding mutations of another TadA (e.g., TadA*8).

[0387] In some embodiments, TadA*8 is a variant shown in Table 5. Table 5 lists the position number of a specific amino acid in the TadA amino acid sequence and the amino acid in TadA-7.10 adenosine deaminase present at that position. Table 5 also lists the amino acid changes of TadA variants relative to TadA-7.10 according to phage-mediated non-linear evolution (PANCE) and phage-mediated continuous evolution (PACE) methods, as described in M. Richter et al., 2020, Nature Biotechnology, doi.org / 10.1038 / s41587-020-0453-z, the entire contents of which are incorporated herein by reference. In some embodiments, TadA*8 is TadA*8a, TadA*8b, TadA*8c, TadA*8d, or TadA*8e. In some embodiments, TadA*8 is TadA*8e. [Table 5]

[0388] In one embodiment, a fusion protein of the invention comprises wild-type TadA linked to an adenosine deaminase variant described herein (e.g., TadA*8) linked to a Cas9 nickase. In certain embodiments, the fusion protein comprises a single TadA*8 domain (e.g., provided as a monomer). In other embodiments, the fusion protein comprises TadA*8 and TadA(wt), capable of forming a heterodimer.

[0389] In some embodiments, the adenosine deaminase 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 the amino acid sequences described in any of the adenosine deaminases provided herein. It should be understood that the adenosine deaminases provided herein may comprise one or more mutations (e.g., any of the mutations provided herein). The present disclosure provides any deaminase domain that has any of the mutations described herein or a combination thereof, in addition to a certain percent identity. In some embodiments, the adenosine deaminase comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more mutations compared to a reference sequence or any of the adenosine deaminases provided herein. In some embodiments, the adenosine deaminase comprises an amino acid sequence having at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, or at least 170 identical contiguous amino acid residues compared to any one of the amino acid sequences known in the art or described herein.

[0390] In certain embodiments, TadA*8 contains one or more mutations at any of the following positions shown in bold: In other embodiments, TadA*8 contains one or more mutations at any of the underlined positions: [ka]

[0391] For example, TadA*8 contains modifications at amino acid positions 82 and / or 166 (e.g., V82S, T166R) relative to TadA*7.10 of the TadA reference sequence, alone or in combination with any one or more of the following: Y147T, Y147R, Q154S, Y123H, and / or Q154R, or the corresponding mutations in another TadA. In certain embodiments, the combination of modifications is, relative to the TadA reference sequence TadA*7.10, Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123H+Y147R; V82 Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R, or the corresponding mutation in another TadA.

[0392] In some embodiments, TadA*8 is truncated. In some embodiments, the truncated TadA*8 lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 N-terminal amino acid residues compared to full-length TadA*8. In some embodiments, the truncated TadA*8 lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 C-terminal amino acid residues compared to full-length TadA*8. In some embodiments, the adenosine deaminase variant is full-length TadA*8.

[0393] In one embodiment, a fusion protein of the invention comprises wild-type TadA linked to an adenosine deaminase variant described herein (e.g., TadA*8) linked to a Cas9 nickase. In particular embodiments, the fusion protein comprises a single TadA*8 domain (e.g., provided as a monomer). In other embodiments, the base editor comprises TadA*8 and TadA(wt), and is capable of forming a heterodimer.

[0394] In certain embodiments, the fusion protein comprises a single (e.g., provided as a monomer) TadA*8. In some embodiments, TadA*8 is linked to a Cas9 nickase. In some embodiments, the fusion protein of the invention comprises a heterodimer of wild-type TadA (TadA(wt)) linked to TadA*8. In other embodiments, the fusion protein of the invention comprises a heterodimer of TadA*7.10 linked to TadA*8. In some embodiments, the base editor is ABE8 comprising a TadA*8 variant monomer. In some embodiments, the base editor is ABE8 comprising a heterodimer of TadA*8 and TadA(wt). In some embodiments, the base editor is ABE8 comprising a heterodimer of TadA*8 and TadA*7.10. In some embodiments, the base editor is ABE8 comprising a heterodimer of TadA*8. In some embodiments, TadA*8 is selected from Table 11, 13, or 14. In some embodiments, ABE8 is selected from Table 13, 14, or 16.

[0395] In some embodiments, the adenosine deaminase is a TadA*9 variant. In some embodiments, the adenosine deaminase is a TadA*9 variant selected from the variants described below with reference to the following sequence (designated TadA*7.10): [ka]

[0396] In some embodiments, the adenosine deaminase comprises one or more of the following modifications: R21N, R23H, E25F, N38G, L51W, P54C, M70V, Q71M, N72K, Y73S, V82T, M94V, P124W, T133K, D139L, D139M, C146R, and A158K. The one or more modifications are underlined and bolded in the sequence above.

[0397] In some embodiments, the adenosine deaminase has the following combination of modifications: V82S+Q154R+Y147R; V82S+Q154R+Y123H; V82S+Q154R+Y147R+Y123H; Q154R+Y147R+Y123H+I76Y+V82S; V82S+I76Y; V82S+Y147R; V82S+Y147R+Y123H; V82S+Q154R+Y123H; Q154R+Y147R+Y123H+I76Y ;V82S+Y147R;V82S+Y147R+Y123H;V82S+Q154R+Y123H;V82S+Q154R+Y147R;V82S+Q154R+Y147R;Q154R+Y147R+Y123H+I76Y;Q154R+Y147R+Y123H+I76Y+V82S;I76Y_V82S_Y123H_Y147R_Q154R;Y147R+Q154R+H123H;and V82S+Q154R.

[0398] In some embodiments, the adenosine deaminase has the following combinations of modifications: E25F+V82S+Y123H, T133K+Y147R+Q154R; E25F+V82S+Y123H+Y147R+Q154R; L51W+V82S+Y123H+C146R+Y147R+Q154R; Y73S+V82S+Y123H+Y147R+Q154R; P54C+V82S+Y123H+Y1 47R+Q154R;N38G+V82T+Y123H+Y147R+Q154R;N72K+V82S+Y123H+D139L+Y147R+Q154R;E25F+V82S+Y123H+D1 39M+Y147R+Q154R;Q71M+V82S+Y123H+Y147R+Q154R;E25F+V82S+Y123H+T133K+Y147R+Q154R;E25F+V82S+Y12 3H+Y147R+Q154R;V82S+Y123H+P124W+Y147R+Q154R;L51W+V82S+Y123H+C146R+Y147R+Q154R;P54C+V82S+Y1 23H+Y147R+Q154R;Y73S+V82S+Y123H+Y147R+Q154R;N38G+V82T+Y123H+Y147R+Q154R;R23H+V82S+Y123H+Y1 47R+Q154R; R21N+V82S+Y123H+Y147R+Q154R; V82S+Y123H+Y147R+Q154R+A158K; N72K+V82S+Y123H+D139L+Y147R+Q154R; E25F+V82S+Y123H+D139M+Y147R+Q154R; and M70V+V82S+M94V+Y123H+Y147R+Q154R.

[0399] In some embodiments, the adenosine deaminase has the following combinations of modifications: Q71M+V82S+Y123H+Y147R+Q154R; E25F+I76Y+V82S+Y123H+Y147R+Q154R; I76Y+V82T+Y123H+Y147R+Q154R; N38G+I76Y+V82S+Y123H+Y147R+Q154R; R23H+I76Y+V82S+Y123H+Y147R+Q154R; P54C+I76Y+V82S+Y123H+Y147R+Q154R; R21N+I76Y+V82S +Y123H+Y147R+Q154R;I76Y+V82S+Y123H+D139M+Y147R+Q154R;Y73S+I7 6Y+V82S+Y123H+Y147R+Q154R;E25F+I76Y+V82S+Y123H+Y147R+Q154R;I 76Y+V82T+Y123H+Y147R+Q154R;N38G+I76Y+V82S+Y123H+Y147R+Q154R; R23H+I76Y+V82S+Y123H+Y147R+Q154R;P54C+I76Y+V82S+Y123H+Y147R+ Q154R;R21N+I76Y+V82S+Y123H+Y147R+Q154R;I76Y+V82S+Y123H+D139M+Y147R+Q154R;Y73S+I76Y+V82S+Y123H+Y147R+Q154R;andV82S+Q154R;N72K_V82S+Y123H+Y147R+Q154R;Q71M_V82S+Y123H+Y147R+Q154R;V82S+Y123H+T133K+Y147R+Q154R;V82S+Y123H+T133K+Y147R+Q154R+A158K;M 70V+Q71M+N72K+V82S+Y123H+Y147R+Q154R; N72K_V82S+Y123H+Y147R+Q154R; Q71M_V82S+Y123H+Y147R+Q154R; M70V+V82S+M94V+Y123H+Y147R+Q154R; V82S+Y123H+T133K+Y147R+Q154R; V82S+Y123H+T133K+Y147R+Q154R+A158K; and M70V+Q71M+N72K+V82S+Y123H+Y147R+Q154R. In some embodiments, the adenosine deaminase is expressed as a monomer.In other embodiments, the adenosine deaminase is expressed as a heterodimer. In some embodiments, the deaminase or other polypeptide sequence lacks methionine, for example, when included as a component of a fusion protein. This may result in a change in position number. However, those skilled in the art will understand that such corresponding mutations refer to the same mutation, for example, Y73S and Y72S, and D139M and D138M.

[0400] In some embodiments, the TadA*9 variant comprises a modification described in Table 17 described herein. In some embodiments, the TadA*9 variant is a monomer. In some embodiments, the TadA*9 variant is a heterodimer with a wild-type TadA adenosine deaminase. In some embodiments, the TadA*9 variant is a heterodimer with another TadA variant (e.g., TadA*8, TadA*9). Further details of the TadA*9 adenosine deaminase are described in International PCT Application No. PCT / 2020 / 049975, which is incorporated herein by reference in its entirety.

[0401] Any of the mutations provided herein, and any additional mutations (e.g., based on the ecTadA amino acid sequence), can be introduced into any other adenosine deaminase. Any of the mutations provided herein, individually or in any combination, can be made into the TadA reference sequence or another adenosine deaminase (e.g., ecTadA).

[0402] Details of the A to G nucleobase editing protein are described in International PCT Application No. PCT / 2017 / 045381 (WO2018 / 027078) and Gaudelli, NM, et al., "Programmable base editing of A ·T to G ·C in genomic DNA without DNA cleavage" Nature, 551, 464-471 (2017), the entire contents of which are incorporated herein by reference.

[0403] C to T Edit In some embodiments, a base editor disclosed herein comprises a fusion protein comprising a cytidine deaminase capable of deaminating a targeted cytidine (C) base in a polynucleotide to generate a uridine (U) that has the base-pairing properties of thymine. In some embodiments, for example, when the polynucleotide is double-stranded (e.g., DNA), the uridine base can be replaced (e.g., by cellular repair mechanisms) with a thymidine base, resulting in a C:G to T:A transition. In other embodiments, deamination of C to U in a nucleic acid by a base editor cannot be accompanied by a U to T substitution.

[0404] Deamination of a targeted C in a polynucleotide to produce a U is a non-limiting example of the type of base editing that can be performed by the base editors described herein. In another example, a base editor comprising a cytidine deaminase domain can mediate the conversion of a cytosine (C) base to a guanine (G) base. For example, a U in a polynucleotide generated by deamination of a cytidine by the cytidine deaminase domain of a base editor can be excised from the polynucleotide by a base excision repair mechanism (e.g., by a uracil DNA glycosylase (UDG) domain) to generate an abasic site. The nucleobase opposite the abasic site can then be replaced (e.g., by a base repair mechanism) with another base, such as C, by, for example, a translesion polymerase. Typically, the nucleobase opposite the abasic site is replaced with C, although other substitutions (e.g., A, G, or T) can also occur.

[0405] Thus, in some embodiments, base editors described herein comprise a deamination domain (e.g., a cytidine deaminase domain) capable of deaminating a targeted C in a polynucleotide to a U. Furthermore, as described below, base editors can comprise additional domains that, in some embodiments, facilitate the conversion of the U resulting from deamination to a T or a G. For example, a base editor comprising a cytidine deaminase domain can further comprise a uracil glycosylase inhibitor (UGI) domain, which mediates the substitution of a U for a T, completing the C-to-T base editing event. In another example, a base editor can incorporate a translesion polymerase to improve the efficiency of C-to-G base editing, as the translesion polymerase can facilitate the incorporation of a C opposite the abasic site (i.e., a G will be incorporated at the abasic site, completing the C-to-G base editing event).

[0406] Base editors that include a cytidine deaminase domain can deaminate target Cs in any polynucleotide, including DNA, RNA, and DNA-RNA hybrids. Typically, cytidine deaminases catalyze C nucleobases located in single-stranded portions of a polynucleotide. In some embodiments, the entire polynucleotide containing the target C can be single-stranded. For example, a cytidine deaminase incorporated into a base editor can deaminate a target C in a single-stranded RNA polynucleotide. In other embodiments, base editors that include a cytidine deaminase domain can act on double-stranded polynucleotides, but the target C can be located in a portion of the polynucleotide that is single-stranded during the deamination reaction. For example, in embodiments in which the NAGPB domain includes a Cas9 domain, some nucleotides may be left unpaired during formation of the Cas9-gRNA-target DNA complex, resulting in the formation of a Cas9 "R-loop complex." These unpaired nucleotides can form single-stranded DNA bubbles that can serve as substrates for single-strand-specific nucleotide deaminase enzymes (e.g., cytidine deaminase).

[0407] In some embodiments, the base editor cytidine deaminase can comprise all or part of an apolipoprotein B mRNA editing complex (APOBEC) family deaminase. APOBEC is an evolutionarily conserved family of cytidine deaminases. Members of this family are C-to-U editing enzymes. The N-terminal domain of APOBEC-like proteins is the catalytic domain, and the C-terminal domain is the pseudocatalytic domain. More specifically, the catalytic domain is a zinc-dependent cytidine deaminase domain, which is important for cytidine deamination. APOBEC family members include APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D (now referred to as "APOBEC3E"), APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, and activation-induced (cytidine) deaminase. In some embodiments, the deaminase incorporated into the base editor comprises all or a portion of an APOBEC1 deaminase. In some embodiments, the deaminase incorporated into the base editor comprises all or a portion of an APOBEC2 deaminase. In some embodiments, the deaminase incorporated into the base editor comprises all or a portion of an APOBEC3 deaminase. In some embodiments, the deaminase incorporated into the base editor comprises all or a portion of an APOBEC3A deaminase. In some embodiments, the deaminase incorporated into the base editor comprises all or a portion of an APOBEC3B deaminase. In some embodiments, the deaminase incorporated into the base editor comprises all or a portion of an APOBEC3C deaminase. In some embodiments, the deaminase incorporated into the base editor comprises all or a portion of an APOBEC3D deaminase. In some embodiments, the deaminase incorporated into the base editor comprises all or a portion of an APOBEC3E deaminase. In some embodiments, the deaminase incorporated into the base editor comprises all or a portion of an APOBEC3F deaminase.In some embodiments, the deaminase incorporated into the base editor comprises all or a portion of an APOBEC3G deaminase. In some embodiments, the deaminase incorporated into the base editor comprises all or a portion of an APOBEC3H deaminase. In some embodiments, the deaminase incorporated into the base editor comprises all or a portion of an APOBEC4 deaminase. In some embodiments, the deaminase incorporated into the base editor comprises all or a portion of an activation-induced deaminase (AID). In some embodiments, the deaminase incorporated into the base editor comprises all or a portion of cytidine deaminase 1 (CDA1). It should be understood that a base editor can comprise a deaminase derived from any suitable organism (e.g., human or rat). In some embodiments, the deaminase domain of a base editor is derived from a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase domain of a base editor is derived from a rat (e.g., rat APOBEC1). In some embodiments, the deaminase domain of the base editor is human APOBEC1. In some embodiments, the deaminase domain of the base editor is pmCDA1.

[0408] Other exemplary deaminases that can be fused to Cas9 according to aspects of the present disclosure are provided below. In embodiments, the deaminase is activation-induced deaminase (AID). It should be understood that in some embodiments, the active domain of each sequence can be used, for example, a domain that does not contain a localization signal (nuclear export signal, nuclear localization sequence without a cytoplasmic localization signal).

[0409] Some aspects of the present disclosure are based on the recognition that modulating the catalytic activity of the deaminase domain of any of the fusion proteins described herein, for example, by introducing a point mutation into the deaminase domain, affects the processivity of the fusion protein (e.g., a base editor). For example, mutations that reduce, but do not eliminate, the catalytic activity of the deaminase domain within a base-editing fusion protein make it less likely that the deaminase domain will catalyze the deamination of residues adjacent to the target residue, thereby narrowing the deamination window. Narrowing the deamination window can prevent unnecessary deamination of residues adjacent to a particular target residue, thereby reducing or preventing off-target effects.

[0410] For example, in some embodiments, an APOBEC deaminase incorporated into a base editor can include one or more mutations selected from the group consisting of H121X, H122X, R126X, R126X, R118X, W90X, W90X, and R132X of rAPOBEC1, or one or more corresponding mutations of another APOBEC deaminase, where X is any amino acid. In some embodiments, an APOBEC deaminase incorporated into a base editor can include one or more mutations selected from the group consisting of H121R, H122R, R126A, R126E, R118A, W90A, W90Y, and R132E of rAPOBEC1, or one or more corresponding mutations of another APOBEC deaminase.

[0411] In some embodiments, the APOBEC deaminase incorporated into the base editor can comprise one or more mutations selected from the group consisting of D316X, D317X, R320X, R320X, R313X, W285X, W285X, R326X of hAPOBEC3G, or one or more corresponding mutations of another APOBEC deaminase, where X is any amino acid. In some embodiments, any of the fusion proteins provided herein comprises an APOBEC deaminase that comprises one or more mutations selected from the group consisting of D316R, D317R, R320A, R320E, R313A, W285A, W285Y, R326E of hAPOBEC3G, or one or more corresponding mutations of another APOBEC deaminase.

[0412] In some embodiments, the APOBEC deaminase incorporated into the base editor can comprise the H121R and H122R mutations of rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor can comprise an APOBEC deaminase comprising the R126A mutation of rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor can comprise an APOBEC deaminase comprising the R126E mutation of rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor can comprise an APOBEC deaminase comprising the R118A mutation of rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor can comprise an APOBEC deaminase comprising a W90A mutation in rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor can comprise an APOBEC deaminase comprising a W90Y mutation in rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor can comprise an APOBEC deaminase comprising a R132E mutation in rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor can comprise an APOBEC deaminase comprising a W90Y and R126E mutations in rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase.In some embodiments, the APOBEC deaminase incorporated into the base editor can comprise an APOBEC deaminase that comprises the R126E and R132E mutations of rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor can comprise an APOBEC deaminase that comprises the W90Y and R132E mutations of rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor can comprise an APOBEC deaminase that comprises the W90Y, R126E, and R132E mutations of rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase.

[0413] In some embodiments, the APOBEC deaminase incorporated into the base editor can comprise an APOBEC deaminase comprising the D316R and D317R mutations of hAPOBEC3G, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprises an APOBEC deaminase comprising the R320A mutation of hAPOBEC3G, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor can comprise an APOBEC deaminase comprising the R320E mutation of hAPOBEC3G, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, the APOBEC deaminase incorporated into the base editor can comprise an APOBEC deaminase comprising the R313A mutation of hAP...

Claims

1. A recombinant rabies virus genome comprising a nucleic acid encoding a therapeutic transgene, the genome lacks a G gene encoding a rabies virus glycoprotein or a functional variant thereof; the genome lacks an L gene encoding rabies virus polymerase or a functional variant thereof; and The genome comprises: N gene encoding rabies virus nucleoprotein or a functional variant thereof; The P gene encoding the rabies virus phosphoprotein or a functional variant thereof, and / or Lacking the M gene encoding the rabies virus matrix protein or a functional variant thereof; The recombinant rabies virus genome.

2. A recombinant rabies virus particle comprising a rabies virus glycoprotein and the recombinant rabies virus genome of claim 1.

3. Rabies virus glycoprotein, and a recombinant rabies virus genome comprising a nucleic acid encoding a therapeutic transgene, the genome lacks a G gene encoding a rabies virus glycoprotein or a functional variant thereof; the genome lacks an L gene encoding rabies virus polymerase or a functional variant thereof; and The genome comprises: N gene encoding rabies virus nucleoprotein or a functional variant thereof; The P gene encoding the rabies virus phosphoprotein or a functional variant thereof, and / or M gene encoding rabies virus matrix protein or a functional variant thereof The recombinant rabies virus particle,

4. each of said genes is operably linked to a transcriptional regulatory element, optionally said transcriptional regulatory element comprising a transcription initiation signal, optionally said transcription initiation signal being exogenous to said rabies virus or endogenous to said rabies virus; and / or A recombinant genome as described in claim 1 or a recombinant viral particle as described in claims 2 or 3, wherein each of the genes is operably linked to a transcription termination polyadenylation signal.

5. the therapeutic transgene comprises a sequence encoding a nucleic acid editing system or a component thereof; Optionally, the nucleic acid editing system or components thereof comprise a Clustered Regulatory Interspaced Short Palindromic Repeat (CRISPR) system, a zinc finger nuclease (ZFN), a meganuclease, and a Transcription Activator-Like Effector Nuclease (TALEN); Optionally, the CRISPR system comprises a nucleobase editor comprising a polynucleotide programmable nucleotide binding domain and a nucleobase editing domain; Optionally, the nucleobase editing domain comprises an adenosine deaminase, a cytidine deaminase, or a functional variant thereof; Optionally, the adenosine deaminase comprises a TadA deaminase derived from any of the adenosine base editors listed in Table 10, Table 11, Table 12, Table 13, Table 14, or Table 15; and / or Publish | |.|、||||||||、||||||||、||||#.                E E !l [###、|||||||、||###!0、!!EBB!BBBBBBBBBB|. |.||、||||||、|||||. 4 likely�Ence \‐‼‼^E then observing plance of EnceEffiyE V.^ ¼xi meter BC E^ ¼^ personality.ning ¼^ personality.ning. ¼^ personality. E44 always |.||||||||#、|||||||. E\ asked!list ü∼‼∼∼‼∼itive |||||、||||||||、|||||、|!!Bfo4、｢B Eｼ.!EE�������listmentl __、|||||||、||||||.|、|BBB!BBBBBBBBBBBB [...................................... [|||||||、|||||||||、||||｢、!|BB8@ [#_、|#####_、|######｢｢a ____________________________________________ our). The -ﻍe--- always The 、||##|||___、||##_、、!!BB!!B、!｢ BE8.22-m、ABE8.23-m、ABE8.24-m、AB . -*----l öB�^_kofixl *V�- always always EEE nceE E plance of the (1)|、|||||、|||#、 the -*8- always dnd fin ______________________________________________________ our / our___ our___ our_____ our_| Theｨ-*nceE^B�^agonE always dE, [||、||||||||、||||!.4、!BB!4、!、ABE9.6, ABE9.7, ABE9.8, ABE9.9, ABE9.10, ABE9.11, ABE9.12, ABE9.1 3, ABE9.14, ABE9.15, ABE9.16, ABE9.17, ABE9.18, ABE9.19, ABE9.2, A BE9.21, ABE9.22, ABE9.23, ABE9.24, ABE9.25, ABE9.26, ABE9.27, ABE 9.28, ABE9.29, ABE9.30, ABE9.31, ABE9.32, ABE9.33, ABE9.34, ABE9.3 and / or an adenosine deaminase derived from any one of the adenosine base editors ABE9.5, ABE9.36, ABE9.37, ABE9.38, ABE9.39, ABE9.40, ABE9.41, ABE9.42, ABE9.43, ABE9.44, ABE9.45, ABE9.46, ABE9.47, ABE9.48, ABE9.49, ABE9.50, ABE9.51, ABE9.52, ABE9.53, ABE9.54, ABE9.55, ABE9.56, ABE9.57, and ABE9.58; and / or 5. The recombinant genome of claim 1 or 4, wherein the cytidine deaminase is selected from the group consisting of Petromyzon marinus cytosine deaminase 1 (PmCDA1), activation-induced cytidine deaminase (AID), and APOBEC. A recombinant viral particle according to any one of claims 2, 3 and 4.

6. the polynucleotide programmable nucleotide binding domain comprises a Cas9 polypeptide, a Cas12 polypeptide, or a functional variant thereof; Optionally, the CRISPR system further comprises a guide RNA (gRNA) or a nucleic acid sequence encoding a gRNA.

7. The nucleic acid encoding the therapeutic transgene is about 300 bp, about 400 bp, about 500 bp, about 600 bp, about 700 bp, about 800 bp, about 900 bp, about 1,000 bp, about 1,100 bp, about 1,200 bp, about 1,300 bp, about 1,400 bp, about 1,500 bp, about 1,600 bp, about 1,700 bp, about 1,800 bp, about 1,900 bp, about 2,000 bp, about 2,100 bp 8. The recombinant genome of any one of claims 1, 4 to 6 or the recombinant viral particle of any one of claims 2 to 6, wherein the nucleic acid sequence of the recombinant genome of any one of claims 1, 4 to 6 is greater than about 2,200 bp, about 2,300 bp, about 2,400 bp, about 2,500 bp, about 2,600 bp, about 2,700 bp, about 2,800 bp, about 2,900 bp, about 3,000 bp, about 4,500 bp, about 8,500 bp, or about 10,000 bp.

8. A pharmaceutical composition comprising the recombinant virus particle according to any one of claims 2 to 7.

9. A method for expressing a therapeutic transgene in a target cell, comprising transducing the target cell with a recombinant viral particle according to any one of claims 2 to 7.

10. 1. A method for expressing a nucleobase editor in a target cell, comprising transducing a recombinant rabies viral particle into the target cell, the recombinant viral particle comprising: a rabies virus glycoprotein or a variant thereof; a recombinant rabies virus genome comprising a nucleic acid encoding a nucleobase editor comprising a polynucleotide-programmable nucleotide-binding domain and a nucleobase editing domain; the genome lacks the G gene encoding the rabies virus glycoprotein or a functional variant thereof, and / or The genome lacks an L gene encoding rabies virus polymerase or a functional variant thereof; The method.

11. The genome comprises: N gene encoding rabies virus nucleoprotein or a functional variant thereof; A P gene encoding the rabies virus phosphoprotein or a functional variant thereof; and M gene encoding rabies virus matrix protein or functional variant thereof and / or The genome comprises: N gene encoding rabies virus nucleoprotein or a functional variant thereof; The P gene encoding the rabies virus phosphoprotein or a functional variant thereof, and / or M gene encoding rabies virus matrix protein or a functional variant thereof The method of claim 10, wherein the

12. 1. A packaging system for the recombinant preparation of rabies virus particles, said packaging system comprising: N gene encoding rabies virus nucleoprotein or a functional variant thereof; P gene encoding rabies virus phosphoprotein or a functional variant thereof; An L gene encoding rabies virus polymerase or a functional variant thereof, and containing a recombinant rabies virus genome, the genome lacks a G gene encoding a rabies virus glycoprotein or a functional variant thereof; the genome lacks an L gene encoding rabies virus polymerase or a functional variant thereof; The packaging system.

13. the N, P, and L genes are each contained in a separate vector; or The N, P, and L genes are contained within a single vector. Optionally, the single vector comprises a first expression cassette comprising the N and P genes, and a second expression cassette comprising the L gene; Optionally, The first expression cassette comprises, from 5' to 3': Transcription factors, The P gene, and The N gene Contains; or The first expression cassette comprises, from 5' to 3': Transcription factors, The P gene, Ribosome skipping elements, and The N gene Including, Optionally, the ribosome skipping element is an IRES element or Or it is a 2A element, The packaging system of claim 12.

14. 14. The packaging system of claim 12 or 13, further comprising an M gene encoding a rabies virus matrix protein or a functional variant thereof.

15. 15. A method for producing a recombinant rabies virus particle, comprising introducing into a cell a packaging system according to any one of claims 12 to 14 under conditions operable to package the recombinant rabies virus genome to form the recombinant rabies virus particle.

16. N gene encoding rabies virus nucleoprotein or a functional variant thereof; A P gene encoding the rabies virus phosphoprotein or a functional variant thereof; and L gene encoding rabies virus polymerase or a functional variant thereof Including, A recombinant rabies virus particle packaging cell in which the N gene, the P gene, and the L gene are stably integrated.

17. Use of a recombinant rabies virus according to any one of claims 2 to 7 or a pharmaceutical composition according to claim 8 in the manufacture of a medicament for treating a disease or disorder in a subject.

18. The pharmaceutical composition of claim 8 for treating a disease or disorder in a subject.

19. The pharmaceutical composition of claim 18, wherein the disease or disorder is a disease or disorder of the nervous system, or a disease or disorder of the eye.

20. 1. A method for delivering a nucleobase editor to a subject, comprising administering to the subject a recombinant rabies viral particle, the recombinant viral particle comprising: a rabies virus glycoprotein or a variant thereof; a recombinant rabies virus genome comprising a nucleic acid encoding a nucleobase editor comprising a polynucleotide-programmable nucleotide-binding domain and a nucleobase editing domain; the genome lacks the G gene encoding the rabies virus glycoprotein or a functional variant thereof, and / or The genome lacks an L gene encoding rabies virus polymerase or a functional variant thereof; The method.