Base editor systems and methods of use thereof

Abstract

This application relates to a system for strand-specific editing of DNA (including human mitochondrial DNA). The system provided herein includes a single-strand nickase and a deaminase, which can be bound individually or together to a double-stranded DNA-binding polypeptide to achieve DNA editing within the editing region. Also provided herein are components of the DNA editing system and methods for using the system.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims priority to and the benefit of International Application No. PCT / CN2022 / 144031, filed December 30, 2022, and International Application No. PCT / CN2023 / 088117, filed April 13, 2023, the entire contents of which are incorporated herein by reference. [Technical Field]

[0002] The present application relates to a system for strand-specific editing of DNA (including human mitochondrial DNA). The system provided herein includes a single-stranded nickase and a deaminase, which are individually or together bound to a double-stranded DNA-binding polypeptide to allow DNA editing to occur within the edited region. Also provided herein are components of the system for editing DNA described herein and methods for using the system. [Background technology]

[0003] Mitochondrial DNA (mtDNA) exists in multiple copies, and heterogeneity is evident in most human cells with mitochondrial diseases. mtDNA mutations are associated with many human diseases, approximately 95% of which are point mutations. In some mitochondrial diseases, wild-type and mutant mtDNA coexist, and the ratio of wild-type to mutant mtDNA usually correlates with the severity of the clinical phenotype. Theoretically, mtDNA editing systems could be used to treat such mtDNA-related diseases. The CRISPR system has been widely used for nuclear genome editing. However, applying this system to mitochondrial genome editing remains unfeasible due to the lack of an efficient method for delivering guide RNA to this organelle. Therefore, powerful mtDNA base editing technologies are urgently needed to elucidate potential pathogenic mechanisms and correct disease-causing mutations to establish treatments. Summary of the Invention [Means for solving the problem]

[0004] In some aspects, the present disclosure provides base editor systems comprising a first unit comprising a first double-stranded (ds) DNA-binding polypeptide bound to a single-stranded (ss-) nickase and a second unit comprising a second dsDNA-binding polypeptide bound to a deaminase, wherein the base editor system is configured such that when the first dsDNA-binding polypeptide of the first unit and the second dsDNA-binding polypeptide of the second unit are simultaneously bound to dsDNA, the ss-nickase and the deaminase can be precisely localized to their respective sites of action within the same editing region of the dsDNA.

[0005] In some embodiments, the ss-nickase of the first unit recognizes a recognition sequence present within the dsDNA editing region. In some embodiments, the recognition sequence of the ss-nickase is a palindromic recognition sequence. In some embodiments, the recognition sequence of the ss-nickase is 5'-GATC-3'. In some embodiments, the recognition sequence of the ss-nickase is a non-palindromic recognition sequence. In some embodiments, the recognition sequence of the ss-nickase is 5'-GATD-3', where D is a base selected from G, A, and T. In some embodiments, the recognition sequence of the ss-nickase is 5'-GAGTC-3'. In some embodiments, the recognition sequence of the ss-nickase is hemimethylated.

[0006] In some embodiments, the first dsDNA-binding polypeptide of the first unit comprises a transcription activator-like effector (TALE) domain. In some embodiments, the first dsDNA-binding polypeptide of the first unit comprises a zinc finger (ZF) domain. In some embodiments, the first dsDNA-binding polypeptide of the first unit specifically binds to a portion of dsDNA upstream of an edited region in a dsDNA sequence. In some embodiments, the first dsDNA-binding polypeptide of the first unit specifically binds to a portion of dsDNA downstream of an edited region in a dsDNA sequence.

[0007] In some embodiments, the site of action of the ss-nickase is located on the same strand of dsDNA as that bound to the first dsDNA-binding polypeptide of the first unit. In some embodiments, the first dsDNA-binding polypeptide of the first unit is bound to a dsDNA position 5 to 9 bases away from the recognition sequence of the ss-nickase. In some embodiments, the site of action of the ss-nickase is located on the complementary strand of dsDNA as that bound to the first dsDNA-binding polypeptide of the first unit. In some embodiments, the first dsDNA-binding polypeptide of the first unit is bound to a dsDNA position 0 to 4 bases away from the recognition sequence of the ss-nickase.

[0008] In some embodiments, the ss-nickases are heterologous. In some embodiments, the ss-nickases of the first unit are type I, type II, type III, or type IV nickases. In some embodiments, the ss-nickases are MutH or Nt.BspD6I, or nickases derived therefrom.

[0009] In some embodiments, the second dsDNA-binding polypeptide of the second unit comprises a transcription activator-like effector (TALE) domain. In some embodiments, the second dsDNA-binding polypeptide of the second unit comprises a zinc finger (ZF) domain. In some embodiments, the second dsDNA-binding polypeptide binds to a dsDNA strand to which the first dsDNA-binding polypeptide of the first unit is not bound. In some embodiments, the second dsDNA-binding polypeptide of the second unit specifically binds to a dsDNA downstream of the edited region in the dsDNA sequence. In some embodiments, the second dsDNA-binding polypeptide of the second unit specifically binds to a dsDNA upstream of the edited region in the dsDNA sequence.

[0010] In some embodiments, the site of action of the deaminase is part of the unnicked strand of dsDNA. In some embodiments, the deaminase is heterologous. In some embodiments, the deaminase is a single-stranded deaminase (ss-deaminase). In some embodiments, the deaminase of the second unit is a cytosine to uracil deaminase, a 5-methylcytosine to thymine deaminase, a guanine to xanthine deaminase, an adenine to hypoxanthine deaminase, or an adenine to inosine deaminase. In some embodiments, the deaminase is TadA8e, APOBEC, or AID.

[0011] In some embodiments, the edited region in the dsDNA is 1-24 base pairs in length. In some embodiments, the site of action of the ss-nickase is separated from the site of action of the deaminase by 10 base pairs or less.

[0012] In some embodiments, the first unit is a fusion polypeptide, and the first dsDNA-binding polypeptide of the first unit is fused to the ss-nickase of the first unit. In some embodiments, the first dsDNA-binding polypeptide of the first unit is fused to the C-terminus of the ss-nickase of the first unit. In some embodiments, the first dsDNA-binding polypeptide of the first unit is fused to the N-terminus of the ss-nickase of the first unit. In some embodiments, the first unit further comprises a linker connecting the first dsDNA-binding polypeptide and the ss-nickase. In some embodiments, the second unit is a fusion polypeptide, and the second dsDNA-binding polypeptide of the second unit is fused to the deaminase of the second unit. In some embodiments, the second dsDNA-binding polypeptide of the second unit is fused to the C-terminus of the deaminase of the second unit. In some embodiments, the second dsDNA-binding polypeptide of the second unit is fused to the N-terminus of the deaminase of the second unit. In some embodiments, the second unit further comprises a linker connecting the second dsDNA-binding polypeptide and the deaminase. In some embodiments, the linker of the first unit and / or the linker of the second unit comprises a polypeptide linker. In some embodiments, the length of the polypeptide linker is between 2 and 100 amino acid residues.

[0013] In some embodiments, the first dsDNA-binding polypeptide is non-covalently bound to the ss-nickase of the first unit, and in some embodiments, the second dsDNA-binding polypeptide is non-covalently bound to the deaminase of the second unit.

[0014] In some embodiments, the first unit further comprises a mitochondrial localization signal (MLS). In some embodiments, the MLS is located at the N-terminus of the first unit. In some embodiments, the second unit further comprises a mitochondrial localization signal (MLS). In some embodiments, the MLS is located at the N-terminus of the second unit.

[0015] In some embodiments, the dsDNA is circularized dsDNA. In some embodiments, the dsDNA is mitochondrial DNA (mtDNA). In some embodiments, the dsDNA is in a B-DNA conformation.

[0016] In another aspect, the description provides a base editor system comprising a single-stranded (ss-) nickase, a deaminase, and a double-stranded (ds) DNA-binding polypeptide, wherein the ss-nickase, deaminase, and dsDNA-binding polypeptide form a complex, and the complex is configured such that, when the dsDNA-binding polypeptide is bound to dsDNA, the ss-nickase and deaminase can be precisely localized to their respective sites of action within the same editing region of the dsDNA.

[0017] In other aspects, the description provides non-naturally occurring polynucleotides encoding a first unit comprising a first double-stranded (ds) DNA-binding polypeptide bound to a single-stranded (ss-) nickase, and / or a second unit comprising a second dsDNA-binding polypeptide bound to a deaminase.

[0018] In another aspect, the description provides a non-naturally occurring polynucleotide that encodes a first unit comprising a first double-stranded (ds) DNA-binding polypeptide bound to a single-stranded (ss-) nickase and a second unit comprising a second dsDNA-binding polypeptide bound to a deaminase, wherein the first and second units are configured, when expressed, such that when the first dsDNA-binding polypeptide of the first unit and the second dsDNA-binding polypeptide of the second unit are simultaneously bound to dsDNA, the ss-nickase and deaminase can be precisely localized to their respective sites of action within the same editing region of dsDNA.

[0019] In another aspect, the description provides a non-naturally occurring polynucleotide that encodes a single-stranded (ss-) nickase, a deaminase, and a double-stranded (ds) DNA-binding polypeptide, wherein the ss-nickase, deaminase, and dsDNA-binding polypeptide, when expressed, form a complex that is configured such that, when the dsDNA-binding polypeptide is bound to dsDNA, the ss-nickase and deaminase can be precisely localized to their respective sites of action within the same editing region of dsDNA.

[0020] In another aspect, the description provides methods of editing a target nucleotide within an edited region in a cell, the method comprising delivering any of the base editor systems described herein, or a polynucleotide encoding same, to a cell. In some embodiments, the edited region is located in mitochondrial DNA.

[0021] In another aspect, the description provides a method of treating an individual suffering from a disease associated with a DNA mutation, the method comprising administering to the individual one or more polynucleotides encoding a first unit comprising a first double-stranded (ds)DNA-binding polypeptide bound to a single-stranded (ss-)nickase and a second unit comprising a second dsDNA-binding polypeptide bound to a deaminase, wherein the first and second units are configured, when expressed in the individual, such that when bound to dsDNA, the first dsDNA-binding polypeptide of the first unit and the second dsDNA-binding polypeptide of the second unit are capable of precisely localizing the ss-nickase and the deaminase to their respective sites of action within the same edited region of the dsDNA. In some embodiments, the mutated DNA is mitochondrial DNA.

[0022] In another aspect, the description provides a kit for a base editor system, the kit comprising: a first unit comprising a first double-stranded (ds) DNA-binding polypeptide bound to a single-stranded (ss-) nickase; and a second unit comprising a second dsDNA-binding polypeptide bound to a deaminase, wherein the base editor system is configured such that when the first dsDNA-binding polypeptide of the first unit and the second dsDNA-binding polypeptide of the second unit are simultaneously bound to dsDNA, the ss-nickase and the deaminase can be precisely localized to their respective sites of action within the same editing region of dsDNA.

[0023] In another aspect, the description provides kits for base editor systems, the kits comprising one or more polynucleotides encoding a first unit comprising a first double-stranded (ds) DNA-binding polypeptide bound to a single-stranded (ss-) nickase and a second unit comprising a second dsDNA-binding polypeptide bound to a deaminase, wherein the base editor system, when expressed, is configured such that when the first dsDNA-binding polypeptide of the first unit and the second dsDNA-binding polypeptide of the second unit are simultaneously bound to dsDNA, the ss-nickase and the deaminase can be precisely localized to their respective sites of action within the same edited region of dsDNA.

[0024] In another aspect, the description provides a non-naturally occurring polypeptide having nickase activity, wherein the non-naturally occurring polypeptide comprises a variant of the amino acid sequence of SEQ ID NO:1 with E91A and F94A mutations (SEQ ID NO:2). In some embodiments, the non-naturally occurring polypeptide is isolated.

[0025] In another aspect, the present specification provides a polynucleotide encoding the non-naturally occurring polypeptide of SEQ ID NO:2.

[0026] All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.

[0027] It should also be understood that those skilled in the art could make changes in form and detail of the embodiments described herein without departing from the scope of the present disclosure. Moreover, although various advantages, aspects, and objectives have been described with reference to various embodiments, the scope of the present disclosure should not be limited by these advantages, aspects, and objectives. [Brief explanation of the drawings]

[0028] [Figure 1A] Figure 1A shows the mitochondrial A-to-G editing efficiency of HEK293T cells treated with paired TALE-TadA8e(V106W) at the MT-RNR2 site. [Figure 1B] Figure 1B shows the mitochondrial A-to-G editing efficiency of HEK293T cells treated with paired TALE-TadA8e(V106W) at the MT-ND1 site. [Figure 1C] Figure 1C shows the mitochondrial A-to-G editing efficiency of HEK293T cells treated with paired TALE-TadA8e(V106W) at the MT-ND4 site. [Figure 1D] Figure 1D shows the mitochondrial A-to-G editing efficiency of HEK293T cells treated with the left TALE-MutH and the right TALE-TadA8e(V106W) at the MT-RNR2 site. [Figure 1E] Figure 1E shows the mitochondrial A-to-G editing efficiency of HEK293T cells treated with the left TALE-MutH and the right TALE-TadA8e(V106W) at the MT-ND1 site. [Figure 1F] Figure 1F shows the mitochondrial A-to-G editing efficiency of HEK293T cells treated with the left TALE-MutH and the right TALE-TadA8e(V106W) at the MT-ND4 site. [Figure 1G]Figure 1G shows the mitochondrial A-to-G editing efficiency in HEK293T cells treated with the left TALE-MutH(D70A) and the right TALE-TadA8e(V106W) in MT-RNR2. [Figure 1H] Figure 1H shows the mitochondrial A-to-G editing efficiency of HEK293T cells treated with the left TALE-MutH(D70A) and the right TALE-TadA8e(V106W) in MT-ND1. [Figure 1I] Figure 1I shows the mitochondrial A-to-G editing efficiency of HEK293T cells treated with the left TALE-MutH(D70A) and the right TALE-TadA8e(V106W) in MT-ND4. [Figure 1J] Figure 1J shows the product distribution of mitochondrial A-to-G editing in HEK293T cells treated with the left TALE-MutH and the right TALE-TadA8e(V106W) at the MT-RNR2 site. [Figure 1K] Figure 1K shows the product distribution of mitochondrial A-to-G editing in HEK293T cells treated with the left TALE-MutH and the right TALE-TadA8e(V106W) at the MT-ND1 site. [Figure 1L] Figure 1L shows the product distribution of mitochondrial A-to-G editing in HEK293T cells treated with the left TALE-MutH and the right TALE-TadA8e(V106W) at the MT-ND4 site. [Figure 1M] Figure 1M shows a time course analysis of mitochondrial A-to-G editing efficiency in HEK293T cells treated with the left TALE-MutH and the right TALE-TadA8e(V106W) at the MT-RNR2 site. [Figure 1N] Figure 1N shows a time course analysis of the editing efficiency of mitochondrial A-to-G editing in HEK293T cells treated with the left TALE-MutH and the right TALE-TadA8e(V106W) at the MT-ND1 site. [Figure 1O]Figure 1O shows a time course analysis of mitochondrial A-to-G editing efficiency in HEK293T cells treated with the left TALE-MutH and the right TALE-TadA8e(V106W) at the MT-ND4 site. [Figure 1P] Figure 1P shows a predicted model for improving mitochondrial DNA editing efficiency by combining nickase and TadA8e(V106W). TALE-nickase binds to target DNA and nicks dsDNA. Nicked dsDNA is prone to form single-stranded DNA structures. TALE-TadA8e(V106W) binds to target DNA and efficiently deaminates adenine on ssDNA. The generated inosine is permanently converted to guanine after DNA repair or DNA replication. [Figure 2A] Figure 2A shows the different orientations of mitoABEMutH when MutH moves 3 bp away from 5'-GATC-3' to edit adenines on different strands at MT-RNR2 site 1, MT-ND1 site 1, and MT-ND4 site 1. Top panel: TALE-fused MutH on the left and TALE-fused TadA8e(V106W) on the right. Bottom panel: TALE-fused TadA8e(V106W) on the left and TALE-fused MutH on the right. Values ​​and errors reflect the mean ± standard deviation of n = 3 independent biological replicates. [Figure 2B] Figure 2B shows the different orientations of mitoABEMutH when editing adenines on different strands, 5 bp away from 5'-GATC-3', at MT-RNR2 site 2 and MT-ND4 site 1. Top panel: Left TALE-fused TadA8e(V106W) and right TALE-fused MutH. Bottom panel: Left TALE-fused MutH and right TALE-fused TadA8e(V106W). Values ​​and errors reflect the mean ± standard deviation of n=3 independent biological replicates. [Figure 2C] Figure 2C shows the editing efficiency of mitoABEMutH with MutH and TadA8e(V106W) at various distances from 5'-GATC-3' in MT-ND4. Values ​​and errors reflect the mean ± standard deviation of n = 3 independent biological replicates. [Figure 2D] Figure 2D shows the editing efficiency of mitoABEMutH with MutH and TadA8e(V106W) at various distances from 5′-GATC-3′ at MT-RNR2 site 1. [Figure 2E] Figure 2E shows the editing efficiency of mitoABEMutH with MutH and TadA8e(V106W) at various distances from 5′-GATC-3′ in MT-ND4. [Figure 2F] Figure 2F shows strand-specific nicking of MutH at various distances from 5'-GATC-3'. When the distance between TALE-MutH and 5'-GATC-3' is 0-4 bp, the complementary strand is nicked, resulting in editing of the TALE-MutH recognition strand. When the distance between TALE-MutH and 5'-GATC-3' is 5-9 bp, the TALE-MutH recognition strand is nicked, resulting in editing of the complementary strand. [Figure 2G] Figure 2G shows the editing efficiency of the TALE-MutH (left) and TALE-TadA8e(V106W) systems, which contain a linker sequence between the TALE and MutH, at the MT-ND1 site. [Figure 2H] Figure 2H shows the editing efficiency of the TALE-MutH (left) and TALE-TadA8e(V106W) systems, which contain a linker sequence between the TALE and MutH, at the MT-ND4 site. [Figure 2I] Figure 2I shows the editing efficiency of the TALE-TadA8e(V106W) (left) and TALE-MutH (right) systems, which contain a linker sequence between the TALE and MutH, at the MT-ND1 site. [Figure 2J] Figure 2J shows the editing efficiency of the TALE-TadA8e(V106W) (left) and TALE-MutH (right) systems, which contain a linker sequence between the TALE and MutH, at the MT-ND4 site. [Figure 3A] Figure 3A shows the crystal structure of key amino acids of MutH interacting with hemimethylated 5′-GATC-3′. [Figure 3B] Figure 3B shows the editing efficiency of MutH mutants (including K48A, E91A, F94A, R184A, Y212S, and the double mutations E91A and F94A) combined with TadA8e(V106W) at the 5′-GATC-3′ position. [Figure 3C] Figure 3C shows the editing efficiency of the target spacer region at 5′-GATA-3′ with different orientations and distances of mitoABEMutH*. [Figure 3D] Figure 3D shows the editing efficiency of the target spacer region at different orientations and distances of mitoABEMutH* at 5'-GATG-3'. [Figure 3E] Figure 3E shows the editing efficiency of the target spacer region at 5'-GATT-3' with different orientations and distances of mitoABEMutH*. [Figure 3F] Figure 3F shows the editing efficiency of targeting the 5'-GATA-3' spacer region at different orientations and distances from 5'-GATD-3' in mitoABEMutH. All data points are shown from n=3 biologically independent experiments. [Figure 3G] Figure 3G shows the editing efficiency of the target spacer region at 5'-GATT-3', varying in orientation and distance from 5'-GATD-3' in mitoABEMutH. All data points are shown from n=3 biologically independent experiments. [Figure 3H] Figure 3H shows the editing efficiency of targeting the 5'-GATT-3' spacer region at different orientations and distances from 5'-GATD-3' in mitoABEMutH. All data points are shown from n=3 biologically independent experiments. [Figure 3I] Figure 3I shows the designable target range of TALE-MutH in human mitochondria. [Figure 3J] Figure 3J shows the designable target range of TALE-MutH* in human mitochondria. Numbers 0, 2, 4, and 6 indicate the frequency of MutH and MutH* recognition sequences within a 40-bp region. [Figure 4A]Figure 4A shows the predicted structures of the screened nickases, BsaI, BsmBI, BsmAI, Nb.BsrDI, Nt.CviPII, and BspQI. The arrows indicate the cleavage sites, and the C-termini of the proteins are the cleavage domains of the selected corresponding proteins. Full-length Nt.CviPII was used. [Figure 4B] Figure 4B shows the results of a mitochondrial base editing screen using nickases without sequence constraints. All data points from n=3 biologically independent experiments are shown. [Figure 4C] Figure 4C shows the editing efficiency of different mitochondrial sites when TALE-Nt.BspD6I(C) (left) is combined with TALE-TadA8e(V106W) (right). All data points are shown from n=3 biologically independent experiments. [Figure 4D] Figure 4D shows the editing efficiency of different mitochondrial sites when TALE-Nt.BspD6I(C) (right) is combined with TALE-TadA8e(V106W) (left). All data points are shown from n=3 biologically independent experiments. [Figure 4E] Figure 4E shows the editing efficiency of the left TALE-Nt.BspD6I(C) and right TALE-TadA8e(V100W) systems, which contain a linker sequence between the TALE and Nt.BspD6I(C), at MT-ND5 site 2. [Figure 4F] Figure 4F shows the editing efficiency of the TALE-Nt.BspD6I(C) (left) and TALE-TadA8e(V100W) (right) systems, which contain a linker sequence between the TALE and Nt.BspD6I(C), at the MT-ND4 site. [Figure 4G] Figure 4G shows the editing efficiency of the TALE-TadA8e(V100W) (left) and TALE-Nt.BspD6I(C) (right) systems, which contain a linker sequence between the TALE and Nt.BspD6I(C), at the MT-ND1 site. [Figure 4H]Figure 4H shows the editing efficiency of the TALE-TadA8e(V100W) (left) and TALE-Nt.BspD6I(C) (right) systems, which contain a linker sequence between the TALE and Nt.BspD6I(C), at the MT-ND4 site. [Figure 5A] Figure 5A shows the editing efficiency of mitoCBE in MT-ND4. [Figure 5B] Figure 5B shows the editing efficiency of mitoCBE at MT-RNR2 site 3. [Figure 5C] Figure 5C shows the editing efficiency of mitoCBE at MT-RNR2 site 1. [Figure 5D] Figures 5D–5F compare the overview of mitoCBE and DdCBE editing at MT-ND4 (Figure 5D), MT-RNR2 site 3 (Figure 5E), and MT-RNR2 site 1 (Figure 5F). For each position, the editing percentages of DdCBE (left G1397-N), DdCBE (left G1397-C), and mitoCBE (right MutH) are displayed sequentially as three consecutive bars. [Figure 5E] Figures 5D–5F compare the overview of mitoCBE and DdCBE editing at MT-ND4 (Figure 5D), MT-RNR2 site 3 (Figure 5E), and MT-RNR2 site 1 (Figure 5F). For each position, the editing percentages of DdCBE (left G1397-N), DdCBE (left G1397-C), and mitoCBE (right MutH) are displayed sequentially as three consecutive bars. [Figure 5F] Figures 5D–5F compare the overview of mitoCBE and DdCBE editing at MT-ND4 (Figure 5D), MT-RNR2 site 3 (Figure 5E), and MT-RNR2 site 1 (Figure 5F). For each position, the editing percentages of DdCBE (left G1397-N), DdCBE (left G1397-C), and mitoCBE (right MutH) are displayed sequentially as three consecutive bars. [Figure 6A]Figure 6A shows the editing efficiency of monomeric mitoABEs in MT-ND1, including TALE-MutH-TadA8e(V106W), TALE-TadA8e(V106W)-MutH, and TALE-Nt.BspD6I-TadA8e(V106W), and TALE-TadA8e(V106W)-Nt.BspD6. [Figure 6B] Figure 6B shows the editing efficiency of monomeric mitoABEs, including TALE-MutH-TadA8e(V106W), TALE-TadA8e(V106W)-MutH, TALE-Nt.BspD6I-TadA8e(V106W), and TALE-TadA8e(V106W)-Nt.BspD6I, on MT-ND4. The boxes in Figures 6A and 6B represent the editing windows of dimeric mitoABEs. [Figure 6C] Figure 6C shows the editing efficiency of monomeric mitoCBEs, including TALE-MutH-rAPOBEC1-UGI, TALE-rAPOBEC1-UGI-MutH, TALE-Nt.BspD6I(C)-rAPOBEC1-UGI, and TALE-rAPOBEC1-UGI-Nt.BspD6I(C), on MT-ND1. [Figure 6D] Figure 6D shows the editing efficiency of monomeric mitoCBEs, including TALE-MutH-rAPOBEC1-UGI, TALE-rAPOBEC1-UGI-MutH, TALE-Nt.BspD6I(C)-rAPOBEC1-UGI, and TALE-rAPOBEC1-UGI-Nt.BspD6I(C), on MT-ND4. All data points are from three biologically independent experiments. [Figure 7A]Figures 7A–7K show the results of untreated HEK293T cells (Figure 7A) and those treated with non-targeting mitoABEMutH (Figure 7B), non-targeting mitoABENt.BspD6I(C) (Figure 7C), and those treated with MT-ND4-targeting mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7D), and those treated with MT-ND4-targeting mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7E). mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7E), mitoABEMutH targeting MT-RNR2 (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7F), mitoABEMutH targeting MT-RNR2 (left TALE-TadA8e(V106W) and right TALE-MutH) (Figure 7G), mitoABEMutH targeting MT-ND1 (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7H). Figure 7H shows the average frequency and mitochondrial genome location of each unique single nucleotide mutation (SNV) in HEK293T cells treated with mitoABENt.BspD6I(C) (left TALE-Nt.BspD6I(C) and right TALE-TadA8e(V106W)) targeting MT-ND1, mitoABENt.BspD6I(C) (left TALE-TadA8e(V106W) and right TALE-Nt.BspD6I(C)) targeting MT-ND4, mitoCBEMutH (left TALE-rAPOBEC1-2×UGI and right TALE-MutH) targeting MT-RNR2. [Figure 7B]Figures 7A–7K show the results of untreated HEK293T cells (Figure 7A) and those treated with non-targeting mitoABEMutH (Figure 7B), non-targeting mitoABENt.BspD6I(C) (Figure 7C), and those treated with MT-ND4-targeting mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7D), and those treated with MT-ND4-targeting mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7E). mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7E), mitoABEMutH targeting MT-RNR2 (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7F), mitoABEMutH targeting MT-RNR2 (left TALE-TadA8e(V106W) and right TALE-MutH) (Figure 7G), mitoABEMutH targeting MT-ND1 (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7H). Figure 7H shows the average frequency and mitochondrial genome location of each unique single nucleotide mutation (SNV) in HEK293T cells treated with mitoABENt.BspD6I(C) (left TALE-Nt.BspD6I(C) and right TALE-TadA8e(V106W)) targeting MT-ND1, mitoABENt.BspD6I(C) (left TALE-TadA8e(V106W) and right TALE-Nt.BspD6I(C)) targeting MT-ND4, mitoCBEMutH (left TALE-rAPOBEC1-2×UGI and right TALE-MutH) targeting MT-RNR2. [Figure 7C]Figures 7A–7K show the results of untreated HEK293T cells (Figure 7A) and those treated with non-targeting mitoABEMutH (Figure 7B), non-targeting mitoABENt.BspD6I(C) (Figure 7C), and those treated with MT-ND4-targeting mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7D), and those treated with MT-ND4-targeting mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7E). mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7E), mitoABEMutH targeting MT-RNR2 (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7F), mitoABEMutH targeting MT-RNR2 (left TALE-TadA8e(V106W) and right TALE-MutH) (Figure 7G), mitoABEMutH targeting MT-ND1 (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7H). Figure 7H shows the average frequency and mitochondrial genome location of each unique single nucleotide mutation (SNV) in HEK293T cells treated with mitoABENt.BspD6I(C) (left TALE-Nt.BspD6I(C) and right TALE-TadA8e(V106W)) targeting MT-ND1, mitoABENt.BspD6I(C) (left TALE-TadA8e(V106W) and right TALE-Nt.BspD6I(C)) targeting MT-ND4, mitoCBEMutH (left TALE-rAPOBEC1-2×UGI and right TALE-MutH) targeting MT-RNR2. [Figure 7D]Figures 7A–7K show the results of untreated HEK293T cells (Figure 7A) and those treated with non-targeting mitoABEMutH (Figure 7B), non-targeting mitoABENt.BspD6I(C) (Figure 7C), and those treated with MT-ND4-targeting mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7D), and those treated with MT-ND4-targeting mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7E). mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7E), mitoABEMutH targeting MT-RNR2 (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7F), mitoABEMutH targeting MT-RNR2 (left TALE-TadA8e(V106W) and right TALE-MutH) (Figure 7G), mitoABEMutH targeting MT-ND1 (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7H). Figure 7H shows the average frequency and mitochondrial genome location of each unique single nucleotide mutation (SNV) in HEK293T cells treated with mitoABENt.BspD6I(C) (left TALE-Nt.BspD6I(C) and right TALE-TadA8e(V106W)) targeting MT-ND1, mitoABENt.BspD6I(C) (left TALE-TadA8e(V106W) and right TALE-Nt.BspD6I(C)) targeting MT-ND4, mitoCBEMutH (left TALE-rAPOBEC1-2×UGI and right TALE-MutH) targeting MT-RNR2. [Figure 7E]Figures 7A–7K show the results of untreated HEK293T cells (Figure 7A) and those treated with non-targeting mitoABEMutH (Figure 7B), non-targeting mitoABENt.BspD6I(C) (Figure 7C), and those treated with MT-ND4-targeting mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7D), and those treated with MT-ND4-targeting mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7E). mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7E), mitoABEMutH targeting MT-RNR2 (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7F), mitoABEMutH targeting MT-RNR2 (left TALE-TadA8e(V106W) and right TALE-MutH) (Figure 7G), mitoABEMutH targeting MT-ND1 (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7H). Figure 7H shows the average frequency and mitochondrial genome location of each unique single nucleotide mutation (SNV) in HEK293T cells treated with mitoABENt.BspD6I(C) (left TALE-Nt.BspD6I(C) and right TALE-TadA8e(V106W)) targeting MT-ND1, mitoABENt.BspD6I(C) (left TALE-TadA8e(V106W) and right TALE-Nt.BspD6I(C)) targeting MT-ND4, mitoCBEMutH (left TALE-rAPOBEC1-2×UGI and right TALE-MutH) targeting MT-RNR2. [Figure 7F]Figures 7A–7K show the results of untreated HEK293T cells (Figure 7A) and those treated with non-targeting mitoABEMutH (Figure 7B), non-targeting mitoABENt.BspD6I(C) (Figure 7C), and those treated with MT-ND4-targeting mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7D), and those treated with MT-ND4-targeting mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7E). mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7E), mitoABEMutH targeting MT-RNR2 (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7F), mitoABEMutH targeting MT-RNR2 (left TALE-TadA8e(V106W) and right TALE-MutH) (Figure 7G), mitoABEMutH targeting MT-ND1 (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7H). Figure 7H shows the average frequency and mitochondrial genome location of each unique single nucleotide mutation (SNV) in HEK293T cells treated with mitoABENt.BspD6I(C) (left TALE-Nt.BspD6I(C) and right TALE-TadA8e(V106W)) targeting MT-ND1, mitoABENt.BspD6I(C) (left TALE-TadA8e(V106W) and right TALE-Nt.BspD6I(C)) targeting MT-ND4, mitoCBEMutH (left TALE-rAPOBEC1-2×UGI and right TALE-MutH) targeting MT-RNR2. [Figure 7G]Figures 7A–7K show the results of untreated HEK293T cells (Figure 7A) and those treated with non-targeting mitoABEMutH (Figure 7B), non-targeting mitoABENt.BspD6I(C) (Figure 7C), and those treated with MT-ND4-targeting mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7D), and those treated with MT-ND4-targeting mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7E). mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7E), mitoABEMutH targeting MT-RNR2 (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7F), mitoABEMutH targeting MT-RNR2 (left TALE-TadA8e(V106W) and right TALE-MutH) (Figure 7G), mitoABEMutH targeting MT-ND1 (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7H). Figure 7H shows the average frequency and mitochondrial genome location of each unique single nucleotide mutation (SNV) in HEK293T cells treated with mitoABENt.BspD6I(C) (left TALE-Nt.BspD6I(C) and right TALE-TadA8e(V106W)) targeting MT-ND1, mitoABENt.BspD6I(C) (left TALE-TadA8e(V106W) and right TALE-Nt.BspD6I(C)) targeting MT-ND4, mitoCBEMutH (left TALE-rAPOBEC1-2×UGI and right TALE-MutH) targeting MT-RNR2. [Figure 7H]Figures 7A–7K show the results of untreated HEK293T cells (Figure 7A) and those treated with non-targeting mitoABEMutH (Figure 7B), non-targeting mitoABENt.BspD6I(C) (Figure 7C), and those treated with MT-ND4-targeting mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7D), and those treated with MT-ND4-targeting mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7E). mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7E), mitoABEMutH targeting MT-RNR2 (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7F), mitoABEMutH targeting MT-RNR2 (left TALE-TadA8e(V106W) and right TALE-MutH) (Figure 7G), mitoABEMutH targeting MT-ND1 (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7H). Figure 7H shows the average frequency and mitochondrial genome location of each unique single nucleotide mutation (SNV) in HEK293T cells treated with mitoABENt.BspD6I(C) (left TALE-Nt.BspD6I(C) and right TALE-TadA8e(V106W)) targeting MT-ND1, mitoABENt.BspD6I(C) (left TALE-TadA8e(V106W) and right TALE-Nt.BspD6I(C)) targeting MT-ND4, mitoCBEMutH (left TALE-rAPOBEC1-2×UGI and right TALE-MutH) targeting MT-RNR2. [Figure 7I]Figures 7A–7K show the results of untreated HEK293T cells (Figure 7A) and those treated with non-targeting mitoABEMutH (Figure 7B), non-targeting mitoABENt.BspD6I(C) (Figure 7C), and those treated with MT-ND4-targeting mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7D), and those treated with MT-ND4-targeting mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7E). mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7E), mitoABEMutH targeting MT-RNR2 (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7F), mitoABEMutH targeting MT-RNR2 (left TALE-TadA8e(V106W) and right TALE-MutH) (Figure 7G), mitoABEMutH targeting MT-ND1 (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7H). Figure 7H shows the average frequency and mitochondrial genome location of each unique single nucleotide mutation (SNV) in HEK293T cells treated with mitoABENt.BspD6I(C) (left TALE-Nt.BspD6I(C) and right TALE-TadA8e(V106W)) targeting MT-ND1, mitoABENt.BspD6I(C) (left TALE-TadA8e(V106W) and right TALE-Nt.BspD6I(C)) targeting MT-ND4, mitoCBEMutH (left TALE-rAPOBEC1-2×UGI and right TALE-MutH) targeting MT-RNR2. [Figure 7J]Figures 7A–7K show the results of untreated HEK293T cells (Figure 7A) and those treated with non-targeting mitoABEMutH (Figure 7B), non-targeting mitoABENt.BspD6I(C) (Figure 7C), and those treated with MT-ND4-targeting mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7D), and those treated with MT-ND4-targeting mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7E). mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7E), mitoABEMutH targeting MT-RNR2 (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7F), mitoABEMutH targeting MT-RNR2 (left TALE-TadA8e(V106W) and right TALE-MutH) (Figure 7G), mitoABEMutH targeting MT-ND1 (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7H). Figure 7H shows the average frequency and mitochondrial genome location of each unique single nucleotide mutation (SNV) in HEK293T cells treated with mitoABENt.BspD6I(C) (left TALE-Nt.BspD6I(C) and right TALE-TadA8e(V106W)) targeting MT-ND1, mitoABENt.BspD6I(C) (left TALE-TadA8e(V106W) and right TALE-Nt.BspD6I(C)) targeting MT-ND4, mitoCBEMutH (left TALE-rAPOBEC1-2×UGI and right TALE-MutH) targeting MT-RNR2. [Figure 7K]Figures 7A–7K show the results of untreated HEK293T cells (Figure 7A) and those treated with non-targeting mitoABEMutH (Figure 7B), non-targeting mitoABENt.BspD6I(C) (Figure 7C), and those treated with MT-ND4-targeting mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7D), and those treated with MT-ND4-targeting mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7E). mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7E), mitoABEMutH targeting MT-RNR2 (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7F), mitoABEMutH targeting MT-RNR2 (left TALE-TadA8e(V106W) and right TALE-MutH) (Figure 7G), mitoABEMutH targeting MT-ND1 (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7H). Figure 7H shows the average frequency and mitochondrial genome location of each unique single nucleotide mutation (SNV) in HEK293T cells treated with mitoABENt.BspD6I(C) (left TALE-Nt.BspD6I(C) and right TALE-TadA8e(V106W)) targeting MT-ND1, mitoABENt.BspD6I(C) (left TALE-TadA8e(V106W) and right TALE-Nt.BspD6I(C)) targeting MT-ND4, mitoCBEMutH (left TALE-rAPOBEC1-2×UGI and right TALE-MutH) targeting MT-RNR2. [Figure 7L] Figure 7L shows the average coverage of high-throughput sequencing of the mitochondrial genome. [Figure 7M] Figure 7M shows the average coverage of high-throughput sequencing of the nuclear genome. [Figure 7N]Figure 7N shows the average nuclear genome frequency of each unique single nucleotide variation (SNV) in the EGFP group (control), non-targeted group, and targeted group. All data in Figures 7A-7K and 7N are from three or more biological replicates. Arrows indicate the targeted editing site, and gray dots indicate the editing efficiency of adenine or cytosine within the editing window. [Figure 7O] Figure 7O shows mitochondrial DNA copy number detected by qPCR. All data points from n=3 biologically independent experiments are shown. [Figure 7P] Figures 7P–7W show the monomeric mitoABEMutH (TALE-MutH-TadA8e(V106W)) targeting MT-ND1 (Figure 7P), the monomeric mitoABEMutH (TALE-MutH-TadA8e(V106W)) targeting MT-ND4 (Figure 7Q), the monomeric mitoABEMutH (TALE-TadA8e(V106W)-MutH) targeting MT-ND1 (Figure 7R), the monomeric mitoABEMutH (TALE-TadA8e(V106W)-MutH) targeting MT-ND4 (Figure 7S), and the monomeric mitoABEMutH (TALE-Nt.BspD6I(C)) targeting MT-ND1 (TALE-Nt.BspD6I(C)). )-TadA8e(V106W)) (Figure 7T), monomeric mitoABENt.BspD6I(C) targeting MT-ND4 (TALE-Nt.BspD6I(C)-TadA8e(V106W)) (Figure 7U), monomeric mitoABENt.BspD6I(C) targeting MT-ND1 (TALE-TadA8e(V106W)-Nt.BspD6I(C)) (Figure 7V), and monomeric mitoABENt.BspD6I(C) targeting MT-ND4 (TALE-TadA8e(V106W)-Nt.BspD6I(C)) (Figure 7W). Figures 7P-7W show three or more biological replicates. Arrows point to the target editing site, and grey dots indicate the editing efficiency of adenines within the editing window. [Figure 7Q]Figures 7P–7W show the monomeric mitoABEMutH (TALE-MutH-TadA8e(V106W)) targeting MT-ND1 (Figure 7P), the monomeric mitoABEMutH (TALE-MutH-TadA8e(V106W)) targeting MT-ND4 (Figure 7Q), the monomeric mitoABEMutH (TALE-TadA8e(V106W)-MutH) targeting MT-ND1 (Figure 7R), the monomeric mitoABEMutH (TALE-TadA8e(V106W)-MutH) targeting MT-ND4 (Figure 7S), and the monomeric mitoABEMutH (TALE-Nt.BspD6I(C)) targeting MT-ND1 (TALE-Nt.BspD6I(C)). )-TadA8e(V106W)) (Figure 7T), monomeric mitoABENt.BspD6I(C) targeting MT-ND4 (TALE-Nt.BspD6I(C)-TadA8e(V106W)) (Figure 7U), monomeric mitoABENt.BspD6I(C) targeting MT-ND1 (TALE-TadA8e(V106W)-Nt.BspD6I(C)) (Figure 7V), and monomeric mitoABENt.BspD6I(C) targeting MT-ND4 (TALE-TadA8e(V106W)-Nt.BspD6I(C)) (Figure 7W). Figures 7P-7W show three or more biological replicates. Arrows point to the target editing site, and grey dots indicate the editing efficiency of adenines within the editing window. [Figure 7R]Figures 7P–7W show the monomeric mitoABEMutH (TALE-MutH-TadA8e(V106W)) targeting MT-ND1 (Figure 7P), the monomeric mitoABEMutH (TALE-MutH-TadA8e(V106W)) targeting MT-ND4 (Figure 7Q), the monomeric mitoABEMutH (TALE-TadA8e(V106W)-MutH) targeting MT-ND1 (Figure 7R), the monomeric mitoABEMutH (TALE-TadA8e(V106W)-MutH) targeting MT-ND4 (Figure 7S), and the monomeric mitoABEMutH (TALE-Nt.BspD6I(C)) targeting MT-ND1 (TALE-Nt.BspD6I(C)). )-TadA8e(V106W)) (Figure 7T), monomeric mitoABENt.BspD6I(C) targeting MT-ND4 (TALE-Nt.BspD6I(C)-TadA8e(V106W)) (Figure 7U), monomeric mitoABENt.BspD6I(C) targeting MT-ND1 (TALE-TadA8e(V106W)-Nt.BspD6I(C)) (Figure 7V), and monomeric mitoABENt.BspD6I(C) targeting MT-ND4 (TALE-TadA8e(V106W)-Nt.BspD6I(C)) (Figure 7W). Figures 7P-7W show three or more biological replicates. Arrows point to the target editing site, and grey dots indicate the editing efficiency of adenines within the editing window. [Figure 7S]Figures 7P–7W show the monomeric mitoABEMutH (TALE-MutH-TadA8e(V106W)) targeting MT-ND1 (Figure 7P), the monomeric mitoABEMutH (TALE-MutH-TadA8e(V106W)) targeting MT-ND4 (Figure 7Q), the monomeric mitoABEMutH (TALE-TadA8e(V106W)-MutH) targeting MT-ND1 (Figure 7R), the monomeric mitoABEMutH (TALE-TadA8e(V106W)-MutH) targeting MT-ND4 (Figure 7S), and the monomeric mitoABEMutH (TALE-Nt.BspD6I(C)) targeting MT-ND1 (TALE-Nt.BspD6I(C)). )-TadA8e(V106W)) (Figure 7T), monomeric mitoABENt.BspD6I(C) targeting MT-ND4 (TALE-Nt.BspD6I(C)-TadA8e(V106W)) (Figure 7U), monomeric mitoABENt.BspD6I(C) targeting MT-ND1 (TALE-TadA8e(V106W)-Nt.BspD6I(C)) (Figure 7V), and monomeric mitoABENt.BspD6I(C) targeting MT-ND4 (TALE-TadA8e(V106W)-Nt.BspD6I(C)) (Figure 7W). Figures 7P-7W show three or more biological replicates. Arrows point to the target editing site, and grey dots indicate the editing efficiency of adenines within the editing window. [Figure 7T]Figures 7P–7W show the monomeric mitoABEMutH (TALE-MutH-TadA8e(V106W)) targeting MT-ND1 (Figure 7P), the monomeric mitoABEMutH (TALE-MutH-TadA8e(V106W)) targeting MT-ND4 (Figure 7Q), the monomeric mitoABEMutH (TALE-TadA8e(V106W)-MutH) targeting MT-ND1 (Figure 7R), the monomeric mitoABEMutH (TALE-TadA8e(V106W)-MutH) targeting MT-ND4 (Figure 7S), and the monomeric mitoABEMutH (TALE-Nt.BspD6I(C)) targeting MT-ND1 (TALE-Nt.BspD6I(C)). )-TadA8e(V106W)) (Figure 7T), monomeric mitoABENt.BspD6I(C) targeting MT-ND4 (TALE-Nt.BspD6I(C)-TadA8e(V106W)) (Figure 7U), monomeric mitoABENt.BspD6I(C) targeting MT-ND1 (TALE-TadA8e(V106W)-Nt.BspD6I(C)) (Figure 7V), and monomeric mitoABENt.BspD6I(C) targeting MT-ND4 (TALE-TadA8e(V106W)-Nt.BspD6I(C)) (Figure 7W). Figures 7P-7W show three or more biological replicates. Arrows point to the target editing site, and grey dots indicate the editing efficiency of adenines within the editing window. [Figure 7U]Figures 7P–7W show the monomeric mitoABEMutH (TALE-MutH-TadA8e(V106W)) targeting MT-ND1 (Figure 7P), the monomeric mitoABEMutH (TALE-MutH-TadA8e(V106W)) targeting MT-ND4 (Figure 7Q), the monomeric mitoABEMutH (TALE-TadA8e(V106W)-MutH) targeting MT-ND1 (Figure 7R), the monomeric mitoABEMutH (TALE-TadA8e(V106W)-MutH) targeting MT-ND4 (Figure 7S), and the monomeric mitoABEMutH (TALE-Nt.BspD6I(C)) targeting MT-ND1 (TALE-Nt.BspD6I(C)). )-TadA8e(V106W)) (Figure 7T), monomeric mitoABENt.BspD6I(C) targeting MT-ND4 (TALE-Nt.BspD6I(C)-TadA8e(V106W)) (Figure 7U), monomeric mitoABENt.BspD6I(C) targeting MT-ND1 (TALE-TadA8e(V106W)-Nt.BspD6I(C)) (Figure 7V), and monomeric mitoABENt.BspD6I(C) targeting MT-ND4 (TALE-TadA8e(V106W)-Nt.BspD6I(C)) (Figure 7W). Figures 7P-7W show three or more biological replicates. Arrows point to the target editing site, and grey dots indicate the editing efficiency of adenines within the editing window. [Figure 7V]Figures 7P–7W show the monomeric mitoABEMutH (TALE-MutH-TadA8e(V106W)) targeting MT-ND1 (Figure 7P), the monomeric mitoABEMutH (TALE-MutH-TadA8e(V106W)) targeting MT-ND4 (Figure 7Q), the monomeric mitoABEMutH (TALE-TadA8e(V106W)-MutH) targeting MT-ND1 (Figure 7R), the monomeric mitoABEMutH (TALE-TadA8e(V106W)-MutH) targeting MT-ND4 (Figure 7S), and the monomeric mitoABEMutH (TALE-Nt.BspD6I(C)) targeting MT-ND1 (TALE-Nt.BspD6I(C)). )-TadA8e(V106W)) (Figure 7T), monomeric mitoABENt.BspD6I(C) targeting MT-ND4 (TALE-Nt.BspD6I(C)-TadA8e(V106W)) (Figure 7U), monomeric mitoABENt.BspD6I(C) targeting MT-ND1 (TALE-TadA8e(V106W)-Nt.BspD6I(C)) (Figure 7V), and monomeric mitoABENt.BspD6I(C) targeting MT-ND4 (TALE-TadA8e(V106W)-Nt.BspD6I(C)) (Figure 7W). Figures 7P-7W show three or more biological replicates. Arrows point to the target editing site, and grey dots indicate the editing efficiency of adenines within the editing window. [Figure 7W]Figures 7P–7W show the monomeric mitoABEMutH (TALE-MutH-TadA8e(V106W)) targeting MT-ND1 (Figure 7P), the monomeric mitoABEMutH (TALE-MutH-TadA8e(V106W)) targeting MT-ND4 (Figure 7Q), the monomeric mitoABEMutH (TALE-TadA8e(V106W)-MutH) targeting MT-ND1 (Figure 7R), the monomeric mitoABEMutH (TALE-TadA8e(V106W)-MutH) targeting MT-ND4 (Figure 7S), and the monomeric mitoABEMutH (TALE-Nt.BspD6I(C)) targeting MT-ND1 (TALE-Nt.BspD6I(C)). )-TadA8e(V106W)) (Figure 7T), monomeric mitoABENt.BspD6I(C) targeting MT-ND4 (TALE-Nt.BspD6I(C)-TadA8e(V106W)) (Figure 7U), monomeric mitoABENt.BspD6I(C) targeting MT-ND1 (TALE-TadA8e(V106W)-Nt.BspD6I(C)) (Figure 7V), and monomeric mitoABENt.BspD6I(C) targeting MT-ND4 (TALE-TadA8e(V106W)-Nt.BspD6I(C)) (Figure 7W). Figures 7P-7W show three or more biological replicates. Arrows point to the target editing site, and grey dots indicate the editing efficiency of adenines within the editing window. [Figure 7X] Figure 7X shows the average frequency and mitochondrial genome location of each unique single nucleotide variation (SNV) in the DdCBEs targeting MT-ND4 (left TALE-DddA-G1397-N and left TALE-DddA-G1397-C). [Figure 7Y] Figure 7Y shows the average frequency and mitochondrial genome location of each unique single nucleotide variation (SNV) in the DdCBEs targeting MT-RNR2 (left TALE-DddA-G1397-N and left TALE-DddA-G1397-C). [Figure 7Z]Figures 7Z–HH show the results of untreated HEK293T cells (Figure 7Z) and those treated with non-targeting mitoABEMutH (Figure 7AA), non-targeting mitoABENt.BspD6I(C) (Figure 7BB), MT-ND4-targeting mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7CC), MT-ND4-targeting mitoABEMutH (left TALE-TadA8e(V106W) and right TALE-MutH) (Figure 7DD), MT-RNR2-targeting mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7EE), and MT-RNR2-targeting mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7EE). Figure 7F-F shows indel analysis performed on HEK293T cells treated with mitoABEN MutH (left TALE-TadA8e(V106W) and right TALE-MutH) targeting MT-ND1, mitoABENt.BspD6I(C) (left TALE-Nt.BspD6I(copyright) and TALE-TadA8e(V106W) and right TALE-TadA8e(V106W)) targeting MT-ND1, and mitoABENt.BspD6I(C) (left TALE-TadA8e(V106W) and right TALE-Nt.BspD6I(C)) targeting MT-ND1. [Figure 7AA]Figures 7Z–HH show the results of untreated HEK293T cells (Figure 7Z) and those treated with non-targeting mitoABEMutH (Figure 7AA), non-targeting mitoABENt.BspD6I(C) (Figure 7BB), MT-ND4-targeting mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7CC), MT-ND4-targeting mitoABEMutH (left TALE-TadA8e(V106W) and right TALE-MutH) (Figure 7DD), MT-RNR2-targeting mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7EE), and MT-RNR2-targeting mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7EE). Figure 7F-F shows indel analysis performed on HEK293T cells treated with mitoABEN MutH (left TALE-TadA8e(V106W) and right TALE-MutH) targeting MT-ND1, mitoABENt.BspD6I(C) (left TALE-Nt.BspD6I(copyright) and TALE-TadA8e(V106W) and right TALE-TadA8e(V106W)) targeting MT-ND1, and mitoABENt.BspD6I(C) (left TALE-TadA8e(V106W) and right TALE-Nt.BspD6I(C)) targeting MT-ND1. [Figure 7BB]Figures 7Z–HH show the results of untreated HEK293T cells (Figure 7Z) and those treated with non-targeting mitoABEMutH (Figure 7AA), non-targeting mitoABENt.BspD6I(C) (Figure 7BB), MT-ND4-targeting mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7CC), MT-ND4-targeting mitoABEMutH (left TALE-TadA8e(V106W) and right TALE-MutH) (Figure 7DD), MT-RNR2-targeting mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7EE), and MT-RNR2-targeting mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7EE). Figure 7F-F shows indel analysis performed on HEK293T cells treated with mitoABEN MutH (left TALE-TadA8e(V106W) and right TALE-MutH) targeting MT-ND1, mitoABENt.BspD6I(C) (left TALE-Nt.BspD6I(copyright) and TALE-TadA8e(V106W) and right TALE-TadA8e(V106W)) targeting MT-ND1, and mitoABENt.BspD6I(C) (left TALE-TadA8e(V106W) and right TALE-Nt.BspD6I(C)) targeting MT-ND1. [Figure 7CC]Figures 7Z–HH show the results of untreated HEK293T cells (Figure 7Z) and those treated with non-targeting mitoABEMutH (Figure 7AA), non-targeting mitoABENt.BspD6I(C) (Figure 7BB), MT-ND4-targeting mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7CC), MT-ND4-targeting mitoABEMutH (left TALE-TadA8e(V106W) and right TALE-MutH) (Figure 7DD), MT-RNR2-targeting mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7EE), and MT-RNR2-targeting mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7EE). Figure 7F-F shows indel analysis performed on HEK293T cells treated with mitoABEN MutH (left TALE-TadA8e(V106W) and right TALE-MutH) targeting MT-ND1, mitoABENt.BspD6I(C) (left TALE-Nt.BspD6I(copyright) and TALE-TadA8e(V106W) and right TALE-TadA8e(V106W)) targeting MT-ND1, and mitoABENt.BspD6I(C) (left TALE-TadA8e(V106W) and right TALE-Nt.BspD6I(C)) targeting MT-ND1. [Figure 7DD]Figures 7Z–HH show the results of untreated HEK293T cells (Figure 7Z) and those treated with non-targeting mitoABEMutH (Figure 7AA), non-targeting mitoABENt.BspD6I(C) (Figure 7BB), MT-ND4-targeting mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7CC), MT-ND4-targeting mitoABEMutH (left TALE-TadA8e(V106W) and right TALE-MutH) (Figure 7DD), MT-RNR2-targeting mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7EE), and MT-RNR2-targeting mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7EE). Figure 7F-F shows indel analysis performed on HEK293T cells treated with mitoABEN MutH (left TALE-TadA8e(V106W) and right TALE-MutH) targeting MT-ND1, mitoABENt.BspD6I(C) (left TALE-Nt.BspD6I(copyright) and TALE-TadA8e(V106W) and right TALE-TadA8e(V106W)) targeting MT-ND1, and mitoABENt.BspD6I(C) (left TALE-TadA8e(V106W) and right TALE-Nt.BspD6I(C)) targeting MT-ND1. [Figure 7EE]Figures 7Z–HH show the results of untreated HEK293T cells (Figure 7Z) and those treated with non-targeting mitoABEMutH (Figure 7AA), non-targeting mitoABENt.BspD6I(C) (Figure 7BB), MT-ND4-targeting mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7CC), MT-ND4-targeting mitoABEMutH (left TALE-TadA8e(V106W) and right TALE-MutH) (Figure 7DD), MT-RNR2-targeting mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7EE), and MT-RNR2-targeting mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7EE). Figure 7F-F shows indel analysis performed on HEK293T cells treated with mitoABEN MutH (left TALE-TadA8e(V106W) and right TALE-MutH) targeting MT-ND1, mitoABENt.BspD6I(C) (left TALE-Nt.BspD6I(copyright) and TALE-TadA8e(V106W) and right TALE-TadA8e(V106W)) targeting MT-ND1, and mitoABENt.BspD6I(C) (left TALE-TadA8e(V106W) and right TALE-Nt.BspD6I(C)) targeting MT-ND1. [Figure 7FF]Figures 7Z–HH show the results of untreated HEK293T cells (Figure 7Z) and those treated with non-targeting mitoABEMutH (Figure 7AA), non-targeting mitoABENt.BspD6I(C) (Figure 7BB), MT-ND4-targeting mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7CC), MT-ND4-targeting mitoABEMutH (left TALE-TadA8e(V106W) and right TALE-MutH) (Figure 7DD), MT-RNR2-targeting mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7EE), and MT-RNR2-targeting mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7EE). Figure 7F-F shows indel analysis performed on HEK293T cells treated with mitoABEN MutH (left TALE-TadA8e(V106W) and right TALE-MutH) targeting MT-ND1, mitoABENt.BspD6I(C) (left TALE-Nt.BspD6I(copyright) and TALE-TadA8e(V106W) and right TALE-TadA8e(V106W)) targeting MT-ND1, and mitoABENt.BspD6I(C) (left TALE-TadA8e(V106W) and right TALE-Nt.BspD6I(C)) targeting MT-ND1. [Figure 7GG]Figures 7Z–HH show the results of untreated HEK293T cells (Figure 7Z) and those treated with non-targeting mitoABEMutH (Figure 7AA), non-targeting mitoABENt.BspD6I(C) (Figure 7BB), MT-ND4-targeting mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7CC), MT-ND4-targeting mitoABEMutH (left TALE-TadA8e(V106W) and right TALE-MutH) (Figure 7DD), MT-RNR2-targeting mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7EE), and MT-RNR2-targeting mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7EE). Figure 7F-F shows indel analysis performed on HEK293T cells treated with mitoABEN MutH (left TALE-TadA8e(V106W) and right TALE-MutH) targeting MT-ND1, mitoABENt.BspD6I(C) (left TALE-Nt.BspD6I(copyright) and TALE-TadA8e(V106W) and right TALE-TadA8e(V106W)) targeting MT-ND1, and mitoABENt.BspD6I(C) (left TALE-TadA8e(V106W) and right TALE-Nt.BspD6I(C)) targeting MT-ND1. [Figure 7HH]Figures 7Z–HH show the results of untreated HEK293T cells (Figure 7Z) and those treated with non-targeting mitoABEMutH (Figure 7AA), non-targeting mitoABENt.BspD6I(C) (Figure 7BB), MT-ND4-targeting mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7CC), MT-ND4-targeting mitoABEMutH (left TALE-TadA8e(V106W) and right TALE-MutH) (Figure 7DD), MT-RNR2-targeting mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7EE), and MT-RNR2-targeting mitoABEMutH (left TALE-MutH and right TALE-TadA8e(V106W)) (Figure 7EE). Figure 7F-F shows indel analysis performed on HEK293T cells treated with mitoABEN MutH (left TALE-TadA8e(V106W) and right TALE-MutH) targeting MT-ND1, mitoABENt.BspD6I(C) (left TALE-Nt.BspD6I(copyright) and TALE-TadA8e(V106W) and right TALE-TadA8e(V106W)) targeting MT-ND1, and mitoABENt.BspD6I(C) (left TALE-TadA8e(V106W) and right TALE-Nt.BspD6I(C)) targeting MT-ND1. [Figure 7II] FIG. 7II shows detection of insertion or deletion changes (indels) in mtDNA deletions by long-range PCR. [Figure 8A] Figure 8A shows an overview of cells transfected with circRNA-encoded mitoABEMutH. [Figure 8B] Figure 8B shows the editing efficiency of mitoABEMutH encoded by two circular RNAs, which were transfected into different cell lines to achieve top-strand-specific editing. Genomic DNA was harvested two days after transfection. All data points are shown from n=3 biologically independent experiments. [Figure 8C]Figure 8C shows the editing efficiency of mitoABEMutH encoded by two circular RNAs transfected into different cell lines to achieve bottom-strand-specific editing. Genomic DNA was harvested 2 days after transfection. All data points are shown from n=3 biologically independent experiments. [Figure 8D] Figure 8D shows an overview of genomic DNA collected 2 days after transfection of HEK293T cells with mitoABENt.BspD6I(C), encoded by circRNA. [Figure 8E] Figure 8E shows the editing efficiency of the circRNA-encoded mitoABENt.BspD6I(C) targeting the start codon of MT-ND4. All data points from n=3 biologically independent experiments are shown. [Figure 8F] Figure 8F shows the editing efficiency of the circRNA-encoded mitoABENt.BspD6I(C), which targets the start codons of MT-CYB and MT-CO1. All data points from n=3 biologically independent experiments are shown. [Figure 8G] Figure 8G shows ATP levels in cells transfected with mitoABENt.BspD6I(C), a circRNA-encoded targeting the start codon of MT-ND4. All data points from n=3 biologically independent experiments are shown. [Figure 8H] Figure 8H shows ATP levels in cells transfected with mitoABENt.BspD6I(C), a circRNA-encoded gene targeting the initiation codons of MT-CYB and MT-CO1. All data points from n=3 biologically independent experiments are shown. [Figure 8I]Figure 8I shows the oxygen consumption rate (OCR) of HEK293T cells treated for 2 days with the circRNA-encoded mitoABENt.BspD6I(C) targeting the start codon of MT-ND4. All data points from n=3 biologically independent experiments are shown. [Figure 8J] Figure 8J shows an overview of the circRNA-encoded mitoABENt.BspD6I(C) system. GM10742 LHON disease cells were transfected with mitoABENt.BspD6I(C), and genomic DNA was collected and analyzed 3 days after transfection. [Figure 8K] Figure 8K shows the editing efficiency of mitoABENt.BspD6I(C) in correcting the LHON disease-causing 11778G>A mutation in GM10742 cells. All data points from n=3 biologically independent experiments are shown. [Figure 8L] Figure 8L shows ATP levels in GM10742 cells transfected with mitoABENt.BspD6I(C), a circRNA encoding the 11778G>A mutation that causes LHON disease. All data points from n=3 biologically independent experiments are shown. [Figure 8M] Figure 8M shows the OCR of the LHON disease cell line GM10742 treated for 2 days with mitoABENt.BspD6I(C), a circRNA encoding the LHON disease-causing 11778G>A mutation. All data points from n=3 biologically independent experiments are shown. [Figure 8N] Figure 8N shows the types of mitochondrial diseases (MITOMAP) and the proportion of diseases that can theoretically be treated by mitoBE. DETAILED DESCRIPTION OF THE INVENTION

[0029] In some aspects, the present disclosure provides systems for editing dsDNA, including strand-specific editing and base-specific editing of human double-stranded mitochondrial DNA. In certain aspects, the DNA editing systems taught herein can use a single-stranded (ss-) nickase to introduce a nick into double-stranded (ds) DNA, followed by a deaminase to catalyze the creation of a desired base change that is retained on the unnicked strand of the dsDNA. The systems described herein can have various configurations. For example, some embodiments relate to a base editor system that includes a first unit comprising a first dsDNA-binding polypeptide bound to the ss-nickase and a second unit comprising a second dsDNA-binding polypeptide bound to the deaminase, wherein the base editor system is configured such that when the first dsDNA-binding polypeptide of the first unit and the second dsDNA-binding polypeptide of the second unit are simultaneously bound to dsDNA, the ss-nickase and deaminase can be precisely localized to the target editing region to achieve the desired base edit. In some embodiments, the base editor system comprises a first unit comprising a first dsDNA-binding polypeptide bound to an ss-nickase and a second unit comprising a second dsDNA-binding polypeptide bound to a deaminase, wherein the base editing system is configured such that when the first dsDNA-binding polypeptide of the first unit and the second dsDNA-binding polypeptide of the second unit are simultaneously bound to dsDNA, the ss-nickase and the deaminase can be precisely localized to their respective sites of action within the same edited region of dsDNA. In other aspects, the description provides components of the systems for editing DNA described herein, such as systems encoding polynucleotides and novel deaminases, and methods of use thereof, such as therapeutic methods.

[0030] The present disclosure is based on the inventors' unique insights and unexpected discoveries regarding base editor systems comprising sequence-specific single-stranded DNA nickases and deaminases (including base-specific deaminases) that can provide efficient and specific DNA base editing. Such nucleic acid base editing systems can be delivered to and function in mitochondria, thereby enabling the editing of mitochondrial DNA, including human mitochondrial DNA. As described herein, the nucleic acid base editing systems described herein are sufficiently specific to target a single site within the mitochondrial genome. In certain aspects demonstrated herein, the base editor systems were unexpectedly shown to enable precise, strand-specific A-to-G or C-to-T assembly, such as mitochondrial DNA editing systems. In some embodiments, strand specificity is based on the unexpected discovery that the distance between the dsDNA-binding polypeptide binding sequence and the nickase target sequence can control specific DNA single-strand cleavage. Studies have shown that deamination events following DNA repair and mitochondrial DNA replication are retained only in unnicked single-stranded DNA. Furthermore, the base editor system described herein can be configured in multiple configurations (e.g., monomers and dimers) for various applications and editing targets. This system is expected to be efficiently delivered to cells and subcellular structures such as mitochondria (e.g., the dimeric configuration generally results in a smaller unit size and therefore a smaller delivered payload), and to correct all known disease-associated mtDNA mutation types (e.g., A·T to G·C and C·G to T·A). These capabilities significantly expand the applicability and safety of existing DNA editing technologies and provide new avenues for designable and precise mitochondrial DNA editing.

[0031] As demonstrated herein, the base editor system for strand-specific mitochondrial DNA editing significantly reduces the incidence of off-target editing events compared to previous systems, significantly improving safety in clinical settings. Furthermore, by avoiding the generation of double-stranded mitochondrial DNA breaks, the base editor system provided herein avoids rapid degradation of mitochondrial DNA and large-scale deletions and rearrangements within the mitochondrial genome. Furthermore, many human diseases are characterized by mitochondrial damage due to mutations within mitochondrial DNA. Currently, several molecular characteristics of mitochondria limit the development of therapeutics targeting mitochondrial damage. For example, each cell in an organism contains multiple copies of mitochondrial DNA, which may have identical sequences or may contain a mixture of different mutations. The effectiveness of drug therapies for mitochondrial diseases is limited by the proportion of mitochondrial targets. The currently disclosed method uses a mitochondrial targeting signal to uniformly localize the mitochondrial DNA editing system throughout the cell. These unexpected safety improvements result in a mitochondrial DNA editing system suitable for precise targeting of the human genome. Furthermore, the base editor system provided herein can also be used to treat mitochondrial diseases associated with homozygous mutations that do not contain wild-type mtDNA.

[0032] Furthermore, the nucleobase editing system taught herein provides a tool for generating new mitochondrial disease models, which will greatly advance clinical research into human mitochondrial diseases, as the field lacks sophisticated tools for assessing the impact of specific mitochondrial DNA mutations on cellular function and organismal development.

[0033] Thus, in some embodiments, the present disclosure provides a base editor system comprising a first unit comprising a first dsDNA-binding polypeptide bound to an ss-nickase and a second unit comprising a second dsDNA-binding polypeptide bound to a deaminase, wherein the base editor system is configured such that when the first dsDNA-binding polypeptide of the first unit and the second dsDNA-binding polypeptide of the second unit are simultaneously bound to dsDNA, the ss-nickase and the deaminase can be precisely localized to their respective sites of action within the same editing region of the dsDNA.

[0034] In another aspect, the present disclosure includes an ss-nickase, a deaminase, and a dsDNA-binding polypeptide, wherein the ss-nickase, deaminase, and dsDNA-binding polypeptide form a complex that, when the dsDNA-binding polypeptide is bound to dsDNA, enables the ss-nickase and deaminase to precisely localize to their respective sites of action within the same editing region of the dsDNA.

[0035] In other aspects, the description provides non-naturally occurring polynucleotides encoding a first unit comprising a first dsDNA-binding polypeptide bound to a ss-nickase, and / or a second unit comprising a second dsDNA-binding polypeptide bound to a deaminase.

[0036] In another aspect, the description provides a non-naturally occurring polynucleotide encoding a first unit comprising a first dsDNA-binding polypeptide bound to an ss-nickase and a second unit comprising a second dsDNA-binding polypeptide bound to a deaminase, wherein the first and second units are configured, when expressed, such that when bound to dsDNA, the first dsDNA-binding polypeptide of the first unit and the second dsDNA-binding polypeptide of the second unit are capable of precisely localizing the ss-nickase and deaminase to their respective sites of action within the same editing region of dsDNA.

[0037] In another aspect, the description provides a non-naturally occurring polynucleotide that encodes an ss-nickase, a deaminase, and a dsDNA-binding polypeptide, wherein the ss-nickase, deaminase, and dsDNA-binding polypeptide, when expressed, form a complex that is configured such that, when the dsDNA-binding polypeptide is bound to dsDNA, the ss-nickase and deaminase can be precisely localized to their respective sites of action within the same editing region of dsDNA.

[0038] In another aspect, the description provides a method of editing a target nucleotide in an edited region in a cell, the method comprising delivering a base editor system described herein or a polynucleotide encoding a base editor system described herein to the cell.

[0039] In another aspect, the description provides a method of treating an individual suffering from a disease associated with a DNA mutation, the method comprising administering to the individual one or more polynucleotides encoding a first unit comprising a first double-stranded (ds) DNA-binding polypeptide bound to an ss-nickase and a second unit comprising a second dsDNA-binding polypeptide bound to a deaminase, wherein the first and second units are configured, when expressed in the individual, such that when bound to dsDNA, the first dsDNA-binding polypeptide of the first unit and the second dsDNA-binding polypeptide of the second unit are capable of precisely localizing the ss-nickase and the deaminase to their respective sites of action within the same editing region of dsDNA.

[0040] In another aspect, the description provides a kit for a base editor system, the kit comprising: a first unit comprising a first dsDNA-binding polypeptide bound to an ss-nickase; and a second unit comprising a second dsDNA-binding polypeptide bound to a deaminase, wherein the base editor system is configured such that when the first dsDNA-binding polypeptide of the first unit and the second dsDNA-binding polypeptide of the second unit are simultaneously bound to dsDNA, the ss-nickase and the deaminase can be precisely localized to their respective sites of action within the same editing region of dsDNA.

[0041] In other aspects, the description provides kits for base editor systems, the kits comprising one or more polynucleotides encoding a first unit comprising a first dsDNA-binding polypeptide bound to an ss-nickase and a second unit comprising a second dsDNA-binding polypeptide bound to a deaminase, wherein the base editor system is configured, when expressed, such that when the first dsDNA-binding polypeptide of the first unit and the second dsDNA-binding polypeptide of the second unit are simultaneously bound to dsDNA, the ss-nickase and the deaminase can be precisely localized to their respective sites of action within the same edited region of dsDNA.

[0042] In another aspect, the present specification provides a non-naturally occurring polypeptide having nickase activity, wherein the non-naturally occurring polypeptide comprises a variant of the amino acid sequence of SEQ ID NO:1 with E91A and F94A mutations (SEQ ID NO:2).

[0043] In another aspect, the present disclosure provides a non-naturally occurring polypeptide comprising the amino acid sequence of SEQ ID NO:2.

[0044] In another aspect, the present specification provides a polynucleotide encoding the non-naturally occurring polypeptide of SEQ ID NO:2.

[0045] I. Definition For purposes of interpreting this specification, the following definitions shall apply, and where appropriate, terms used in the singular shall include the plural and vice versa. In the event that any definition below conflicts with any document referenced herein, the definition below shall control.

[0046] As used herein, the terms "polypeptide" and "protein" are used interchangeably to refer to polymers comprising amino acid residues, with no minimum length restriction. Such polymers may contain natural or non-natural amino acid residues, or combinations thereof, and include, but are not limited to, peptides, polypeptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. This definition includes not only full-length polypeptides or proteins, but also fragments thereof. The term also includes the type of modification, such as post-translational modification of one or more residues, such as methylation, phosphorylation, glycosylation, sialylation, acetylation, etc.

[0047] As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). Therefore, unless otherwise specified, the term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, mitochondrial DNA (mtDNA), cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of a polynucleotide may contain sugars and phosphate groups (as commonly found in RNA and DNA), or modified or substituted sugars or phosphate groups. Alternatively, the backbone of a polynucleotide may comprise a polymer of synthetic subunits, such as phosphoramidates or thiophosphates, thus resulting in oligodeoxynucleoside phosphoramidate (P-NH2) or mixed phosphoramidate-phosphodiester oligomers. Additionally, double-stranded polynucleotides can be obtained from chemically synthesized single-stranded polynucleotide products by synthesizing the complementary strand and annealing the strands under appropriate conditions, or by synthesizing the complementary strand de novo using DNA polymerase and an appropriate primer.

[0048] As used herein, "treatment" refers to a method for obtaining beneficial or desired results (including clinical results). For purposes of this application, beneficial or desired clinical results include, but are not limited to, alleviating one or more symptoms of a disease associated with a DNA mutation (e.g., a mitochondrial disease), reducing one or more symptoms of a disease, preventing one or more symptoms of a disease, treating one or more symptoms of a disease, ameliorating one or more symptoms of a disease, delaying the onset of one or more symptoms associated with a disease, alleviating the severity of one or more symptoms of a disease, stabilizing the disease (e.g., preventing or delaying worsening of the disease), delaying or slowing the progression of the disease, ameliorating one or more symptoms of the disease, reducing the dosage of one or more other drugs and / or treatments required to treat the disease, improving the quality of life of an individual, and / or extending the survival of an individual. "Treatment" also includes reducing the pathological effects of a disease associated with a DNA mutation (e.g., a mitochondrial disease). The methods of this application encompass one or more of these therapeutic aspects.

[0049] The term "individual" refers to a mammal, including, but not limited to, a human, bovine, equine, feline, canine, rodent, or primate. In some embodiments, the individual is a human.

[0050] As used herein, the terms "comprise," "have," "contain," "comprise," and other similar expressions and their grammatical equivalents are intended to convey the same meaning and be open-ended in nature. That is, the items listed after these terms are not exhaustive and are not limited to the listed items. For example, an article "comprising" components A, B, and C may consist of (or contain only) components A, B, and C, or may include not only components A, B, and C but also one or more other components. Thus, "comprising" and similar expressions and their grammatical equivalents should be understood to include disclosure of embodiments "consisting essentially of" or "consisting of."

[0051] Where a range of values ​​is presented, unless the context clearly dictates otherwise, it is understood that every intervening value (to the nearest tenth of the lower limit) between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is included within the scope of the disclosure, except for any expressly excluded limit within that range. Where the stated range includes one or both of the limits, ranges excluding either or both of those limits are also included in the disclosure.

[0052] As used herein, reference to "about" a value or parameter includes (and describes) variations about that value or parameter itself. For example, a statement of "about X" includes a statement of "X."

[0053] As used in this specification, including the appended claims, the singular forms "a," "an," "or," and "said" include plural referents unless the context clearly dictates otherwise.

[0054] II. Base Editor Systems In certain aspects, provided herein are base editor systems for editing dsDNA, including strand-specific editing and base-specific editing of human mitochondrial DNA. In some embodiments, the base editor systems taught herein include a single-stranded (ss-) nickase, a deaminase, and one or more double-stranded (ds) DNA-binding polypeptides, where the system components are configured to bring the ss-nickase and deaminase into close proximity with an editing region and at least partially catalyze a desired nucleotide base edit. In some embodiments, the base editor system is configured to edit dsDNA in a strand-specific manner. In some embodiments, the base editor system is configured as a dual-polypeptide system, e.g., including a first polypeptide comprising a first double-stranded (ds) DNA-binding polypeptide and a single-stranded (ss-) nickase, and a second polypeptide comprising a second dsDNA-binding polypeptide and a deaminase. In some embodiments, the base editor system is configured as an adenine base editing system, e.g., where the deaminase converts adenine to guanine. In some embodiments, the base editor system is configured as a cytosine base editing system, e.g., the deaminase converts cytosine to thymine. In some embodiments, the dsDNA-binding polypeptide is a transcription activator-like (TAL) effector. In some embodiments, the ss-nickase recognizes a palindromic recognition sequence. In some embodiments, the ss-nickase recognizes a non-palindromic recognition sequence. In some embodiments, the ss-nickase is selected from MutH, MutH* (SEQ ID NO:2), and derivatives thereof, such as BspD6I or BspD6I(C). In some embodiments, the dsDNA is mitochondrial DNA. In some embodiments, the dsDNA is mitochondrial genomic DNA.

[0055] Certain aspects of the base editor systems taught herein are described in further detail below in a modular manner. One of skill in the art can readily understand how to combine various aspects of the present specification to obtain any base editor system encompassed by the teachings provided herein. The modular description of the base editor system (including its components and configuration) does not limit the scope of the description herein.

[0056] A. Single-stranded nickase The base editor systems provided herein include one or more single-stranded (ss-) nickases. As described herein, an ss-nickase is a polypeptide with single-stranded DNA nickase activity, thereby nicking a single strand of a dsDNA molecule. The ss-nickase may be a full-length nickase or a portion of an ss-nickase that includes a functional domain that catalyzes DNA nicking, such as a portion of a naturally occurring nickase, or a derivative thereof that has nickase activity. In some embodiments, the ss-nickase is highly specific for nicking at a specific position relative to the recognition sequence (e.g., at or near the recognition sequence). In some embodiments, the ss-nickase does not nick at a specific position relative to the recognition sequence. For example, the ss-nickase includes a domain for cleavage but does not include a domain for recognizing a DNA sequence. In some embodiments, the ss-nickase retains nickase activity but does not bind to the recognition sequence with high specificity. In some embodiments, when the ss-nickase is not bound to a recognition sequence, the site of action of the nickase depends on a dsDNA-binding polypeptide that localizes the ss-nickase to the correct DNA location. In certain aspects herein, the site of the nick generated by the ss-nickase is referred to as the site of action of the ss-nickase.

[0057] In some embodiments, the single-stranded nickase recognizes a recognition sequence present in the dsDNA editing region. In some embodiments, the single-stranded nickase recognizes a recognition sequence near the dsDNA editing region, for example, the single-stranded nickase recognizes the recognition sequence and then nicks at a specific distance from the recognition sequence. In some embodiments, the recognition sequence is present on both strands of the dsDNA, for example, as a palindromic recognition sequence. In some embodiments, the recognition sequence is present on one strand of the dsDNA. In some embodiments, the recognition sequence of the ss-nickase is a palindromic recognition sequence. In some embodiments, the recognition sequence of the ss-nickase is 5'-GATC-3', where the ss-nickase cleaves on the 5' side of guanine. In some embodiments, the recognition sequence of the ss-nickase is a non-palindromic recognition sequence. In some embodiments, the recognition sequence for the ss-nickase is 5'-GATD-3', where D is a base selected from G, A, and T, and further wherein the ss-nickase cleaves 5' to the guanine. In some embodiments, the recognition sequence for the ss-nickase is 5'-GAGTC-3', where the ss-nickase cleaves only the strand with the recognition site at the fourth nucleotide downstream toward the 3' end of the recognition site.

[0058] In some embodiments, the recognition sequence for the ss-nickase is unmethylated. In some embodiments, the recognition sequence for the ss-nickase is methylated. In some embodiments, the recognition sequence for the ss-nickase is hemimethylated.

[0059] The base editor systems described herein may be configured to have strand specificity. In some embodiments, strand specificity is conferred based on the localization of the ss-nickase relative to the ss-nickase recognition site, such as by designing the binding site of the dsDNA-binding polypeptide and the linkage (e.g., a linker) between the dsDNA-binding polypeptide and the ss-nickase. In some embodiments, the site of action of the ss-nickase is located on the same strand of dsDNA as the dsDNA-binding polypeptide bound to the ss-nickase. In some embodiments, the dsDNA-binding polypeptide bound to the ss-nickase is bound to the dsDNA 5 to 9 bases away from the site of action of the ss-nickase. In some embodiments, the site of action of the ss-nickase is located on the complementary strand of dsDNA as the dsDNA-binding polypeptide bound to the ss-nickase. In some embodiments, the dsDNA-binding polypeptide bound to the ss-nickase is bound to the dsDNA 0 to 4 bases away from the site of action of the ss-nickase.

[0060] In some embodiments, the ss-nickases are heterologous. In some embodiments, the ss-nickases of the first unit are type I nickase, type II nickase, type III nickase, or type IV nickase. In some embodiments, the ss-nickases are MutH or Nt.BspD6I, or nickases derived therefrom. In some embodiments, the ss-nickases are MutH* (SEQ ID NO:2). In some embodiments, the ss-nickases are Nt.BspD6I(C) (SEQ ID NO:3). In some embodiments, the ss-nickase is selected from the group consisting of FokI-FokI (D450A) (SEQ ID NO:4), Nb. BsaI (C, N441D / R442G) (SEQ ID NO:5), Nt. BsaI (C, R236D) (SEQ ID NO:6), Nb. BsmBI (C, R438D) (SEQ ID NO:7), Nt. BsmAI (C, R221D) (SEQ ID NO:8), Nb. BsrDI (C) (SEQ ID NO:9), Nt. CviPII (SEQ ID NO:10), BspQI (C) (SEQ ID NO:11), N. AlwI (C) (SEQ ID NO:12), Nt. BsrDI (SEQ ID NO:13), Nt. BtsI (SEQ ID NO:14), Nt. In some embodiments, the nickase comprises (e.g., is) the small subunit of BspD6I (ss.BspD6I) (SEQ ID NO:16), the small subunit of BsrDI (ss.BsrDI) (SEQ ID NO:17), or the small subunit of BtsI (ss.BtsI) (SEQ ID NO:18).In some embodiments, the ss-nickases are those reported in Desai & Shankar, FEMS Microbiology Reviews, 26, 2003 (incorporated herein by reference in its entirety), such as S1 nuclease, P1 nuclease, mycelium, conidia, slow (S)BAL 31 nuclease, fast (F)BAL 31 nuclease, and the like. 31 nuclease, alpha smut nuclease, beta smut nuclease, nuclease Bhl, Aspergillus nuclease, Physarum nuclease, SP nuclease, mung bean nuclease, wheat chloroplast nuclease, nuclease I, pea seed nuclease, tobacco nuclease I, alfalfa seedling acid nuclease, alfalfa seedling neutral nuclease, SK nuclease, chicken liver nuclease, rat hepatocyte nuclear nuclease, and mouse mitochondrial nuclease.

[0061] B. Deaminase The base editor systems provided herein include one or more deaminases. As described herein, a deaminase is a polypeptide having nucleotide base transferase activity, e.g., converting one nucleotide base to another, such as converting adenine (A) to guanine (G). The deaminase may be a full-length deaminase, or a portion of a deaminase that includes a functional domain that catalyzes nucleotide base conversion, such as a portion of a naturally occurring deaminase, or a derivative thereof that has nucleotide base conversion activity. In some embodiments, the deaminase is highly specific for converting one type of nucleotide base. In certain aspects herein, the desired converted nucleotide base that remains after editing (e.g., the target nucleotide base to be edited) is referred to as the site of action of the deaminase.

[0062] In some embodiments, the deaminase's site of action is located in the unnicked strand of dsDNA. For example, as described herein, the unnicked dsDNA strand retains the edited nucleotide base, thereby causing the unnicked dsDNA strand to contain the deaminase's site of action. In some embodiments, the deaminase is heterologous. In some embodiments, the deaminase is a single-stranded deaminase (ss-deaminase). In some embodiments, the deaminase is a cytosine to uracil deaminase, a 5-methylcytosine to thymine deaminase, a guanine to xanthine deaminase, an adenine to hypoxanthine deaminase, or an adenine to inosine deaminase.

[0063] Numerous deaminases suitable for the base editor systems described herein are known in the art. In some embodiments, the deaminase is TadA8e, APOBEC, AID, or a derivative thereof. In some embodiments, the deaminase is TadA8e(V106W) (SEQ ID NO:19). In some embodiments, the deaminase includes (or is), e.g., TadA-DE, TadA-CDa, TadA-CDa (V106W), evoAPOBEC1, evoCDA1, or evoFERNY. In some embodiments, the deaminase is APOBEC-1, APOBEC-2, APOBEC-3A, APOBEC-3B, APOBEC-3C, APOBEC-3E, APOBEC-3F, APOBEC-3G, APOBEC-3H, or APOBEC-4. In some embodiments, the deaminase is AICDA, CDA, DCTD, AMPD1, ADAT, ADAR, ADARB1, ADA, GDA, TadA, ecTadA, pCDM, or ABE8, or a derivative thereof. In some embodiments, the deaminase is paired with another functional component to perform a desired nucleotide base conversion. For example, in some embodiments, a base editor system comprises a deaminase (e.g., APOBEC1) and a uracil glycosylase inhibitor (UGI) to perform a C to T edit. For example, in some embodiments, a base editor system comprises a deaminase such as APOBEC1 that performs a C to G or a C to A edit together with uracil DNA glycosylase (UNG) (rAPOBEC1-2×UGI; SEQ ID NO:20). For example, in some embodiments, a base editor system comprises a deaminase such as APOBEC1 to perform a C to G or a C to A edit.

[0064] C. dsDNA-binding polypeptides The base editor systems provided herein include one or more double-stranded (ds) DNA-binding polypeptides. As described herein, dsDNA-binding polypeptides include, for example, polypeptides configured to bind to dsDNA at a specific position. As described in more detail below, dsDNA-binding polypeptides can be configured (e.g., designed) to bind to a specific position of dsDNA, including a specific strand associated with a desired editing region that contains nucleotide bases for editing. In some embodiments, the dsDNA-binding polypeptide is a TALE or zinc finger, or a portion thereof, capable of binding to dsDNA. In other embodiments, the dsDNA-binding polypeptide comprises a region of a CRISPR-Cas protein (e.g., Cas9) that binds to DNA. In some embodiments, the dsDNA-binding polypeptide is a catalytically inactive form of a nickase (e.g., an endonuclease).

[0065] In some embodiments, the base editor system comprises multiple dsDNA binding polypeptides. In such embodiments, the base editor system can comprise the same type of dsDNA binding polypeptide (e.g., TALEs) or different types of dsDNA binding polypeptides (e.g., TALEs and zinc fingers).

[0066] The base editor systems described herein can employ multiple configurations in which the ss-nickase and / or deaminase approaches the edited region from different directions. For example, in some embodiments, a base editor system includes a first unit, the first unit comprising a first dsDNA-binding polypeptide bound to an ss-nickase, wherein the first dsDNA-binding polypeptide of the first unit specifically binds to a portion of dsDNA upstream of the edited region in a dsDNA sequence. In some embodiments, such a base editor system includes a second dsDNA-binding polypeptide bound to a deaminase, wherein the second dsDNA-binding polypeptide of the second unit specifically binds to a portion of dsDNA downstream of the edited region in a dsDNA sequence. In some embodiments, the second dsDNA-binding polypeptide is bound to the dsDNA strand of the first unit to which the first dsDNA-binding polypeptide is not bound.

[0067] In some embodiments, a base editor system includes a first unit, the first unit comprising a first dsDNA-binding polypeptide linked to a ss-nickase, wherein the first dsDNA-binding polypeptide of the first unit specifically binds to a portion of dsDNA downstream of an edited region in a dsDNA sequence. In some embodiments, such a base editor system includes a second dsDNA-binding polypeptide linked to a deaminase, wherein the second dsDNA-binding polypeptide of the second unit specifically binds to a portion of dsDNA upstream of an edited region in a dsDNA sequence. In some embodiments, the second dsDNA-binding polypeptide binds to a dsDNA strand not bound to the first dsDNA-binding polypeptide of the first unit.

[0068] As described elsewhere herein, the dsDNA-binding polypeptide of a base editor system may be configured to localize the associated β-nickase and / or deaminase relative to the editing region and / or site of action. In some embodiments, such localization can alter the editing ability of the nucleic acid base editor system. In some embodiments, the localization of the ss-nickase and / or deaminase can be further adjusted, such as by adjusting the length of the linker connecting the dsDNA-binding polypeptide and the ss-nickase. The present disclosure encompasses such variants of the base editor systems described herein. For example, multiple variants of base editor systems that can achieve the same DNA editing effect can be designed based on the technical teachings provided herein. In some embodiments, the dsDNA-binding polypeptide is configured to bind to dsDNA within 0 to 25 base pairs of the editing region and / or site of action of the ss-nickase or deaminase.

[0069] In some embodiments, the dsDNA-binding polypeptide of the base editor system may be configured to localize the associated ss-nickases and / or deaminases to specific locations in the mitochondrial genome. In some embodiments, the specific locations in the mitochondrial genome are within the MT-ATP6, MT-ATP8, MT-CO1, MT-CO2, MT-CO3, MT-CYB, MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-ND4L, MT-ND5, or MT-ND6 genes. In some embodiments, the specific locations in the mitochondrial genome are within mitochondrial ribosomal RNA (rRNA) or transfer RNA (tRNA) genes. In some embodiments, the specific locations in the mitochondrial genome are within mitochondrial short peptide genes, such as MT-RNR2 (human), MOTS-c, or gau.

[0070] D. Construction of a Unit Containing Single-Stranded Nickase and / or Deaminase As described herein, a base editor system is configured to position an ss-nickase and a deaminase in proximity to an editing region and at least partially catalyze a desired nucleotide base edit. In some embodiments, the base editor system includes a single-stranded (ss-) nickase, a deaminase, and one or more double-stranded (ds) DNA-binding polypeptides, and such components can be configured in a variety of different configurations (e.g., monomeric or dimeric) capable of performing the desired nucleotide base edit, all of which are encompassed by the description provided herein. As described herein, after nicking and nucleotide base conversion, DNA repair mechanisms (e.g., mechanisms carried out by endogenous DNA repair proteins) can be involved in the formation of the final edited dsDNA.

[0071] In some embodiments, provided herein are base editor systems comprising a first unit comprising a first double-stranded (ds) DNA-binding polypeptide bound to a single-stranded (ss-) nickase and a second unit comprising a second dsDNA-binding polypeptide bound to a deaminase, wherein the base editor system is configured such that when the first dsDNA-binding polypeptide of the first unit and the second dsDNA-binding polypeptide of the second unit are bound to dsDNA, the ss-nickase and deaminase can be precisely localized to their respective sites of action within the same editing region of the dsDNA. Such an embodiment is an example of a dimeric configuration. In some embodiments, the dimeric base editor system comprises the ss-nickase and deaminase on separate molecules.

[0072] In some embodiments, a base editing system is provided that includes a single-stranded (ss-) nickase, a deaminase, and a double-stranded (ds) DNA-binding polypeptide, wherein the ss-nickase, the deaminase, and the dsDNA-binding polypeptide form a complex that, when the dsDNA-binding polypeptide is bound to the dsDNA, enables the ss-nickase and the deaminase to precisely localize to their respective sites of action within the same edited region of the dsDNA. Such an embodiment is an example of a monomeric configuration. In some embodiments, the monomeric base editing system includes the ss-nickase and the deaminase on a single molecule.

[0073] In some embodiments, a base editor system configured to edit two or more nucleotide bases, e.g., nucleotide bases at different DNA positions, is provided. In some embodiments, when a base editor system is configured to edit a plurality of nucleotide bases, at least two or more of the plurality of nucleotide bases are located in different edited regions. Such editing can be performed using various configurations of the base editor systems described herein. For example, in some embodiments, a base editor system configured to edit a plurality of nucleotide bases includes a first unit comprising a first double-stranded (ds)DNA-binding polypeptide bound to a first single-stranded (ss-)nickase; a second unit comprising a second dsDNA-binding polypeptide bound to a second ss-nickase, where the first dsDNA-binding polypeptide and the second dsDNA-binding polypeptide recognize different recognition sequences; a third unit comprising a third dsDNA-binding polypeptide bound to a first deaminase; and a fourth unit comprising a fourth dsDNA-binding polypeptide bound to the second deaminase. The base editor system is configured such that when the first dsDNA-binding polypeptide of the first unit and the third dsDNA-binding polypeptide of the third unit are bound to dsDNA, the first ss-nickase and the first deaminase can be precisely localized to their respective sites of action within a first editing region in the dsDNA, and when the third dsDNA-binding polypeptide of the third unit and the fourth dsDNA-binding polypeptide of the fourth unit are bound to dsDNA, the second ss-nickase and the second deaminase can be precisely localized to their respective sites of action within a second editing region in the dsDNA. In some embodiments, the first ss-nickase and the second ss-nickase recognize different recognition sequences. In some embodiments, the first deaminase and the second deaminase are the same. In some embodiments, the first deaminase and the second deaminase catalyze the conversion of the same nucleotide base. In some embodiments, the first deaminase and the second deaminase catalyze the conversion of different nucleotide bases.

[0074] In some embodiments, the base editor system includes multiple components, such as a dsDNA-binding polypeptide and an ss-nickase, which may be described individually herein but are configured as a unit to perform the desired edit. Such components can be linked in a variety of ways, including direct fusion (e.g., as a single expressed polypeptide), non-covalent interactions, or covalent linkage (e.g., via a polypeptide linker). In some embodiments, the base editor system includes a first unit including a first dsDNA-binding polypeptide and an ss-nickase, wherein the first unit is a fusion polypeptide, and the first dsDNA-binding polypeptide of the first unit is fused to the ss-nickase of the first unit. In some embodiments, the first dsDNA-binding polypeptide of the first unit is fused to the C-terminus of the ss-nickase of the first unit. In some embodiments, the first dsDNA-binding polypeptide of the first unit is fused to the N-terminus of the ss-nickase of the first unit. In some embodiments, the first unit further includes a linker connecting the first dsDNA-binding polypeptide and the ss-nickase. In some embodiments, the base editor system includes a second unit comprising a second dsDNA-binding polypeptide and a deaminase, wherein the second unit is a fusion polypeptide, and the second dsDNA-binding polypeptide of the second unit is fused to the deaminase of the second unit. In some embodiments, the second dsDNA-binding polypeptide of the second unit is fused to the C-terminus of the deaminase of the second unit. In some embodiments, the second dsDNA-binding polypeptide of the second unit is fused to the N-terminus of the deaminase of the second unit. In some embodiments, the second unit further comprises a linker connecting the second dsDNA-binding polypeptide and the deaminase. In some embodiments, the linker of the first unit and / or the linker of the second unit comprises a polypeptide linker. In some embodiments, the length of the polypeptide linker is 1 to 100 amino acids, e.g., 1 to 60 amino acids, 1 to 50 amino acids, 1 to 40 amino acids, or 2 to 32 amino acids.In some embodiments, the polypeptide linker is any of the following amino acids in length: 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, or 40. In some embodiments, the linker comprises, consists essentially of, or consists of GS, AEAAAKEAAAKEAAAAKEAAAKA (SEQ ID NO:25), or GSGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO:26).

[0075] In some embodiments, the dsDNA binding polypeptide is non-covalently bound to the ss-nickase. In some embodiments, the dsDNA binding polypeptide is non-covalently bound to the deaminase.

[0076] In some embodiments, the base editor system comprises a first unit comprising, from N- to C-terminus, a dsDNA binding polypeptide (e.g., a TALE), e.g., from an array (e.g., a TALE N-terminal non-repeated-TALE repeat array), a linker (e.g., a two amino acid linker), and an ss-nickase; and a second unit comprising, from N- to C-terminus, a dsDNA binding polypeptide (e.g., a TALE), e.g., from an array (e.g., a TALE N-terminal non-repeated-TALE repeat array), a linker (e.g., a two amino acid linker), and a deaminase. Such an embodiment is an example of a dimeric configuration.

[0077] In some embodiments, the base editor system comprises, from N-terminus to C-terminus, a dsDNA binding polypeptide (e.g., a TALE), e.g., one from an array (e.g., a TALE N-terminal non-repeated-TALE repeat array), a linker (e.g., a two amino acid linker), a ss-nickase, a linker (e.g., a two amino acid linker), and a deaminase. In some embodiments, the base editor system comprises, from N-terminus to C-terminus, a dsDNA binding polypeptide (e.g., a TALE), e.g., one from an array (e.g., a TALE N-terminal non-repeated-TALE repeat array), a linker (e.g., a two amino acid linker), a deaminase, a linker (e.g., a two amino acid linker), and a ss-nickase. Such an embodiment is an example of a monomeric configuration.

[0078] E. Edited Regions and Types of dsDNA For ease of description of the nucleobase editor systems described herein, the term "edited region" is used to refer to the region of dsDNA where ss-nickases and deaminases exhibit their targeted (expected) activity (i.e., the target site of action of the nickases and deaminases). Nucleotide editing can occur outside the editing region, such as off-target editing. In some embodiments, the subject matter of the related art is described herein in terms of base pair length. It should be noted that descriptions such as the site of action appearing on the complementary strand (e.g., the site of action of ss-nickases or deaminases) and base pair distance can be assessed based on the complementary dsDNA form of the DNA.

[0079] In some embodiments, the length of the edited region of the dsDNA is 0 to 24 base pairs, including 1 to 20 base pairs, 1 to 10 base pairs, 10 to 20 base pairs, 10 to 16 base pairs, 14 to 20 base pairs, or 14 to 16 base pairs. In some embodiments, the site of action of the ss-nickase is 10 base pairs or less, e.g., 9 base pairs or less, 8 base pairs or less, 7 base pairs or less, 6 base pairs or less, 5 base pairs or less, 4 base pairs or less, 3 base pairs or less, or 1 base pair or less, from the site of action of the deaminase.

[0080] In some embodiments, the nucleobase editor system has strand bias, e.g., at least about 65% (e.g., at least about 70%, 75%, 80%, 85%, 90%, or 95%) of the editing is directed to the desired strand of the dsDNA. In some embodiments, strand specificity is equivalent to strand bias.

[0081] In some embodiments, the dsDNA to be edited by the base editor systems described herein can be any type of dsDNA. In some embodiments, the dsDNA is circularized dsDNA. In some embodiments, the dsDNA is mitochondrial DNA (mtDNA). In some embodiments, the mtDNA is located in mitochondria, e.g., mitochondria within the cells of an individual. In some embodiments, the dsDNA is mitochondrial genomic DNA. In some embodiments, the dsDNA is in a B-DNA structure. In some embodiments, the dsDNA is in an A-DNA structure. In some embodiments, the dsDNA is in a Z-DNA structure.

[0082] F. Additional Features In certain embodiments, the nucleotide editor systems provided herein may include one or more additional features to contribute to the delivery and / or function of the nucleotide editor system.

[0083] Mitochondria are unique organelles with their own DNA, RNA, and translation machinery, yet express only 10% of their proteins. Mitochondria, however, rely in part on the translation products of nuclear genes. These products pass through the cytoplasm and are "imported" into the mitochondria by a system of protein complexes bound to the inner and outer membranes, where they are transported to the appropriate mitochondrial compartment for activation. The mitochondrial import process is controlled by N-terminal presequences of nuclear gene proteins. These sequences tag proteins with specific peptide segments that instruct the import machinery where to deliver the protein. These are called mitochondrial localization signals (MLS) or mitochondrial targeting signals (MTS). Once the protein is transported to the target compartment, the MTS portion of the protein is removed by mitochondrial peptidases, allowing the protein to fold into a functional state and become activated.

[0084] In some embodiments, the base editor system, or one or more components thereof, comprises a mitochondrial localization signal (MLS), also referred to as a mitochondrial targeting signal (MTS). In some embodiments, the ss-nickase is linked to a localization signal (e.g., a mitochondrial localization signal (MLS)). In some embodiments, the deaminase is linked to a localization signal (e.g., a mitochondrial localization signal (MLS)). In some embodiments, when the base editor system comprises two or more units (e.g., a first unit comprising a dsDNA binding polypeptide and a ss-nickase and a second unit comprising a dsDNA binding polypeptide and a deaminase), any one or more (including all) units of the base editor system may comprise an MLS.

[0085] In some embodiments, the base editor system comprises a first unit comprising an ss-nickase and a second unit comprising a deaminase, wherein the first unit further comprises a mitochondrial localization signal (MLS). In some embodiments, the MLS is located at the N-terminus of the first unit. In some embodiments, the base editor system comprises a first unit comprising an ss-nickase and a second unit comprising a deaminase, wherein the second unit further comprises a mitochondrial localization signal (MLS). In some embodiments, the MLS is located at the N-terminus of the second unit.

[0086] In some embodiments, the length of the MLS is about 10 to about 80 amino acids, for example, about 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, 35 to 40, 40 to 45, 45 to 50, 50 to 55, 55 to 60, 60 to 65, 65 to 70, 70 to 75, or 75 to 80 amino acids.

[0087] In some embodiments, the MLS comprises an amphipathic helical motif. To adopt the amphipathic helical motif, the MLS may be rich in basic residues (e.g., Arg, Lys), hydroxylated residues (e.g., Ser, Thr), and / or hydrophobic residues (e.g., Ala, Leu, Ile). In some embodiments, an MLS comprising an amphipathic helical motif has alternating hydrophobic and hydrophilic segments. In some embodiments, at least about 20% (e.g., at least about 30%, 40%, 50%, or 60%) of the amino acid residues in the MLS are basic amino acid residues. In some embodiments, at least about 20% (e.g., at least about 30%, 40%, 50%, or 60%) of the amino acid residues in the MLS are hydrophobic amino acid residues. In some embodiments, the MLS is amphipathic, e.g., forms an amphipathic helix. In some embodiments, the MLS comprises an alternating pattern of hydrophobic and basic residues. In some embodiments, the MLS is derived from a protein selected from ATP synthase, cytochrome C oxidase peptide VIII, Su9, and HSP60. In some embodiments, the MLS is capable of selectively targeting a compound to the outer membrane, the inner membrane and intermembrane space, or the mitochondrial matrix.

[0088] In some embodiments, the base editor system, when expressed as one or more polypeptides (e.g., monomeric and dimeric forms of the base editor systems described herein), has a molecular weight of less than about 150 kDa, e.g., less than about 145 kDa, 140 kDa, 135 kDa, 130 kDa, 125 kDa, 120 kDa, 115 kDa, 110 kDa, 105 kDa, 100 kDa, 95 kDa, 90 kDa, 85 kDa, 80 kDa, 75 kDa, 70 kDa, 65 kDa, 60 kDa, 55 kDa, 50 kDa, 45 kDa, or 40 kDa. In some embodiments, a unit of a base editor system, e.g., a first unit comprising a first double-stranded (ds) DNA binding polypeptide linked to a single-stranded (ss-) nickase, or a second unit comprising a second dsDNA binding polypeptide linked to a deaminase, when expressed as one or more polypeptides, has a molecular weight of less than about 150 kDa, e.g., less than any of about 145 kDa, 140 kDa, 135 kDa, 130 kDa, 125 kDa, 120 kDa, 115 kDa, 110 kDa, 105 kDa, 100 kDa, 95 kDa, 90 kDa, 85 kDa, 80 kDa, 75 kDa, 70 kDa, 65 kDa, 60 kDa, 55 kDa, 50 kDa, 45 kDa, or 40 kDa.

[0089] G. Polynucleotide Forms of Base Editor Systems In certain aspects, provided herein are polynucleotide forms of the base editor systems described herein. The disclosure provided herein encompasses a variety of forms that can be used to introduce the functional base editor systems described herein into cells, including different types of polynucleotides (e.g., DNA or RNA, e.g., circular RNA) and polynucleotides of different designs.

[0090] As used herein, "introducing" or "introduction," with respect to delivery of a base editor system, refers to delivering one or more components of a base editor system or precursors thereof (e.g., one or more polynucleotides encoding a base editor system, or a component of a base editor system including an MLS) to a cell. The methods of the present application can use a variety of delivery systems, including, but not limited to, viruses, liposomes, electroporation, microinjection, and conjugation, to introduce the constructs described herein into cells. Conventional viral and non-viral-based gene transfer methods can be used to introduce nucleic acids into mammalian cells or target tissues. Such methods can be used to administer the nucleic acids of the present application to cultured cells or host organisms. Non-viral vector delivery systems include DNA plasmids, RNA (e.g., transcripts of the constructs described herein), naked nucleic acid, and nucleic acid complexed with a delivery vehicle (e.g., liposomes). Viral vector delivery systems include DNA viruses and RNA viruses, which achieve delivery to cells via episomal or integrated genomes. As described herein, a polynucleotide encoding a base editor system taught herein can be one or more polynucleotides. In some embodiments, introducing the base editor system can comprise introducing two or more different polynucleotides, including introducing the two or more different polynucleotides simultaneously, sequentially, or in parallel, into the cell.

[0091] Non-viral methods of delivering one or more components (including nucleic acids) of the base editor system include lipofection, nucleofection, microinjection, gene gun technology, virus-like particles, liposomes, immunoliposomes, polycation or lipid-nucleic acid complexes, electroporation, nanoparticles, exosomes, microvesicles, or gene gun technology, naked DNA, and artificial virions.

[0092] The use of RNA or DNA virus-based systems is highly effective in achieving nucleic acid delivery, allowing for specific targeting of specific cells and transport of the virally carried payload to the cell nucleus. In some embodiments, delivery involves introducing a viral vector (e.g., a lentiviral vector) encoding the nucleic acid into the cell. In some embodiments, the viral vector is AAV, e.g., AAV8. In some embodiments, such as delivery using AAV, the inventors believe that the monomeric form of the base editor system described herein may be more suitable for packaging and as a vector, for example, due to the smaller overall size of the monomers of the base editor systems compared to the dimers of the base editor systems. In some embodiments, delivery involves introducing a plasmid encoding one or more components of the nucleobase editor system into the cell. In some embodiments, delivery involves introducing one or more components of the nucleic acid editor editing system into the cell (e.g., by electroporation). In some embodiments, delivery involves transfecting one or more components of the nucleobase editor system into the cell.

[0093] In some embodiments, the polynucleotide (e.g., a polynucleotide introduced into a cell) is DNA or RNA. In some embodiments, the RNA is linear RNA. In some embodiments, the RNA is circular RNA. In some embodiments, the linear RNA can form a circular RNA. For example, circularization can be performed using the Tornado expression system ("Twister-optimized RNA for durable overexpression"), as described in Litke, JL & Jaffrey, SR "Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts." Nat Biotechnol 37, 667-675 (2019), the entire contents of which are incorporated herein by reference. Briefly, Tornado expresses a transcript containing an RNA of interest flanked by a Twister ribozyme. A Twister ribozyme is any catalytic RNA sequence capable of self-cleavage. The ribozyme undergoes rapid autocatalytic cleavage, leaving ends joined by an RNA ligase. Non-limiting examples of RNA ligases include RtcB, T4 RNA ligase 1, T4 RNA ligase 2, Rnl3, and Trl1. In some embodiments, the RNA ligase is endogenously expressed in the cell. In some embodiments, the RNA ligase is RNA ligase RtcB. In some embodiments, the method further includes introducing an RNA ligase (e.g., RtcB) into the cell. In some embodiments, the RNA is circularized before being introduced into the cell. In some embodiments, the RNA is chemically synthesized. In some embodiments, the RNA is circularized by in vitro enzymatic ligation (e.g., using an RNA or DNA ligase) or chemical ligation (e.g., using cyanogen bromide or a similar condensing agent).

[0094] In some embodiments, the polynucleotides described herein comprise additional features useful for expressing the base editor system in a cell, such as a promoter sequence.

[0095] In some embodiments, provided herein are non-naturally occurring polynucleotides comprising one or more polynucleotides encoding the base editor systems described herein. In some embodiments, provided herein are non-naturally occurring polynucleotides comprising one or more polynucleotides encoding a first unit comprising a first double-stranded (ds)DNA-binding polypeptide bound to a single-stranded (ss-) nickase, and / or a second unit comprising a second dsDNA-binding polypeptide bound to a deaminase.

[0096] In some embodiments, provided herein are non-naturally occurring polynucleotides comprising one or more polynucleotides encoding a first unit comprising a first double-stranded (ds) DNA-binding polypeptide bound to a single-stranded (ss-) nickase and a second unit comprising a second dsDNA-binding polypeptide bound to a deaminase, wherein the first and second units are configured, when expressed, such that when the first dsDNA-binding polypeptide of the first unit and the second dsDNA-binding polypeptide of the second unit are simultaneously bound to dsDNA, the ss-nickase and deaminase can be precisely localized to their respective sites of action within the same editing region of dsDNA.

[0097] In some embodiments, provided herein are non-naturally occurring polynucleotides comprising one or more polynucleotides encoding a single-stranded (ss-) nickase, a deaminase, and a double-stranded (ds) DNA-binding polypeptide, wherein the ss-nickase, deaminase, and dsDNA-binding polypeptide, when expressed, form a complex that is configured such that, when the dsDNA-binding polypeptide is bound to dsDNA, the ss-nickase and deaminase can be precisely localized to their respective sites of action within the same editing region of dsDNA.

[0098] H. Example of a base editor system In some embodiments, a base editing system is provided, comprising: a first unit comprising a TALE bound to a single-stranded (ss-) nickase, where the ss-nickase is MutH; and a second unit comprising a second TALE bound to an engineered deoxyadenosine deaminase, where the deaminase is TadA8e(V106W). In some embodiments, the base editor system is configured such that the TALE-associated MutH unit and the TALE-associated TadA8e(V106W) unit, when bound to dsDNA, precisely localize the ss-nickase and deaminase to their respective sites of action within the same editing region of the dsDNA, where the editing region is 1-24 bases in length. In some embodiments, the TALE of the first unit is fused to the N-terminus of MutH. In some embodiments, the TALE of the second unit is fused to the C-terminus of TadA8e(V106W). In some embodiments, the first unit further comprises a linker connecting the TALE and MutH. In some embodiments, the second unit further comprises a linker connecting the TALE and TadA8e(V106W). In some embodiments, the TALE of the first unit is bound to a DNA region upstream of the 5'-GATC-3' MutH recognition sequence. In some embodiments, the TALE of the second unit is bound to a DNA region downstream of the 5'-GATC-3' MutH recognition sequence. In some embodiments, provided herein are polynucleotides encoding the base editor system. In some embodiments, the first and second units of the base editor system are separate single polypeptides.

[0099] In some embodiments, a base editor system is provided that includes a first unit comprising a TALE bound to an engineered deoxyadenosine deaminase, where the deaminase is TadA8e(V106W), and a second unit comprising a second TALE bound to a single-stranded (ss-) nickase, where the ss-nickase is MutH. In some embodiments, the base editor system is configured such that the TALE-associated MutH unit and the TALE-associated TadA8e(V106W) unit, when bound to dsDNA, precisely localize the ss-nickase and deaminase to their respective sites of action within the same editing region of the dsDNA, where the editing region is 1-24 bases long. In some embodiments, the TALE of the first unit is fused to the N-terminus of TadA8e(V106W). In some embodiments, the TALE of the second unit is fused to the C-terminus of MutH. In some embodiments, the first unit further comprises a linker linking the TALE and TadA8e(V106W). In some embodiments, the second unit further comprises a linker linking the TALE and MutH. In some embodiments, the TALE of the first unit is bound to a DNA region upstream of the 5'-GATC-3' MutH recognition sequence. In some embodiments, the TALE of the second unit is bound to a DNA region downstream of the 5'-GATC-3' MutH recognition sequence. In some embodiments, provided herein are polynucleotides encoding the base editor system. In some embodiments, the first and second units of the base editor system are separate single polypeptides.

[0100] In some embodiments, the first unit comprises a TALE bound to a single-stranded (ss-) nickase, wherein the ss-nickase is MutH. *and a second unit comprising a second TALE bound to an engineered deoxyadenosine deaminase, wherein the deaminase is TadA8e(V106W). In some embodiments, the base editor system is configured such that the TALE-associated MutH* unit and the TALE-associated TadA8e(V106W) unit, when bound to dsDNA, precisely localize the ss-nickase and deaminase to their respective sites of action within the same editing region of the dsDNA, the editing region being 1-24 bases in length. In some embodiments, the TALE of the first unit is fused to the N-terminus of MutH*. In some embodiments, the TALE of the second unit is fused to the C-terminus of TadA8e(V106W). In some embodiments, the first unit further comprises a linker connecting the TALE and MutH*. In some embodiments, the second unit further comprises a linker connecting the TALE and TadA8e(V106W). In some embodiments, the TALE of the first unit is bound to a DNA region upstream of the MutH* recognition sequence of 5'-GATD-3', where D is a base selected from G, A, and T. In some embodiments, the TALE of the second unit is bound to a DNA region downstream of the MutH* recognition sequence of 5'-GATD-3', where D is a base selected from G, A, and T. In some embodiments, provided herein are polynucleotides encoding the base editor system. In some embodiments, the first and second units of the base editor system are separate single polypeptides.

[0101] In some embodiments, a base editor system is provided that includes a first unit comprising a TALE bound to an engineered deoxyadenosine deaminase, where the deaminase is TadA8e(V106W), and a second unit comprising a second TALE bound to a single-stranded (ss-) nickase, where the ss-nickase is MutH*. In some embodiments, the base editor system is configured such that the TALE-associated MutH* unit and the TALE-associated TadA8e(V106W) unit, when bound to dsDNA, precisely localize the ss-nickase and deaminase to their respective sites of action within the same editing region of the dsDNA, where the editing region is 1-24 bases long. In some embodiments, the TALE of the first unit is fused to the N-terminus of TadA8e(V106W). In some embodiments, the TALE of the second unit is fused to the C-terminus of MutH*. In some embodiments, the first unit further comprises a linker connecting the TALE and TadA8e(V106W). In some embodiments, the second unit further comprises a linker connecting the TALE and MutH*. In some embodiments, the TALE of the first unit is bound to a DNA region upstream of the MutH* recognition sequence of 5'-GATD-3', where D is a base selected from G, A, and T. In some embodiments, the TALE of the second unit is bound to a DNA region downstream of the MutH* recognition sequence of 5'-GATD-3', where D is a base selected from G, A, and T. In some embodiments, a polynucleotide encoding the base editor system is provided herein. In some embodiments, the first and second units of the base editor system are separate single polypeptides.

[0102] In some embodiments, a base editor system is provided that includes a first unit comprising a TALE bound to a single-stranded (ss-) nickase, where the ss-nickase is Nt.BspD6I(C), and a second unit comprising a second TALE bound to an engineered deoxyadenosine deaminase, where the deaminase is TadA8e(V106W). In some embodiments, the base editor system is configured such that the TALE-associated Nt.BspD6I(C) unit and the TALE-associated TadA8e(V106W) unit, when bound to dsDNA, precisely localize the ss-nickase and deaminase to their respective sites of action within the same editing region of the dsDNA, where the editing region is 1-24 bases long. In some embodiments, the TALE of the first unit is fused to the N-terminus of Nt.BspD6I(C). In some embodiments, the TALE of the second unit is fused to the C-terminus of TadA8e(V106W). In some embodiments, the first unit further comprises a linker connecting the TALE and Nt.BspD6I(C). In some embodiments, the second unit further comprises a linker connecting the TALE and TadA8e(V106W). In some embodiments, the TALE of the first unit is bound to a DNA region upstream of the Nt.BspD6I(C) action site. In some embodiments, the TALE of the second unit is bound to a DNA region downstream of the Nt.BspD6I(C) action site. In some embodiments, provided herein are polynucleotides encoding the base editor system. In some embodiments, the first and second units of the base editor system are separate single polypeptides.

[0103] In some embodiments, a base editor system is provided that includes a first unit comprising a TALE bound to an engineered deoxyadenosine deaminase, where the deaminase is TadA8e(V106W), and a second unit comprising a second TALE bound to a single-stranded (ss-) nickase, where the ss-nickase is Nt.BspD6I(C). In some embodiments, the base editor system is configured such that the TALE-associated Nt.BspD6I(C) unit and the TALE-associated TadA8e(V106W) unit, when bound to dsDNA, precisely localize the ss-nickase and deaminase to their respective sites of action within the same editing region of the dsDNA, where the editing region is 1-24 bases long. In some embodiments, the TALE of the first unit is fused to the N-terminus of TadA8e(V106W). In some embodiments, the TALE of the second unit is fused to the C-terminus of Nt.BspD6I(C). In some embodiments, the first unit further comprises a linker connecting the TALE and TadA8e(V106W). In some embodiments, the second unit further comprises a linker connecting the TALE and Nt.BspD6I(C). In some embodiments, the TALE of the first unit is bound to a DNA region upstream of the Nt.BspD6I(C) action site. In some embodiments, the TALE of the second unit is bound to a DNA region downstream of the Nt.BspD6I(C) action site. In some embodiments, provided herein is a polynucleotide encoding the base editor system. In some embodiments, the first and second units of the base editor system are separate single polypeptides.

[0104] In some embodiments, a base editor system is provided that includes a first unit comprising a TALE bound to a single-stranded (ss-) nickase, where the ss-nickase is MutH, and a second unit comprising a second TALE bound to an engineered deoxyadenosine deaminase, where the deaminase is rAPOBEC1-2xUGI. In some embodiments, the base editor system is configured such that the TALE-associated MutH unit and the TALE-associated rAPOBEC1-2xUGI unit, when bound to dsDNA, precisely localize the ss-nickase and deaminase to their respective sites of action within the same editing region of the dsDNA, where the editing region is 1-24 bases in length. In some embodiments, the TALE of the first unit is fused to the N-terminus of MutH. In some embodiments, the TALE of the second unit is fused to the C-terminus of rAPOBEC1-2xUGI. In some embodiments, the first unit further comprises a linker connecting the TALE and MutH. In some embodiments, the second unit further comprises a linker connecting the TALE and rAPOBEC1-2xUGI. In some embodiments, the TALE of the first unit is bound to a DNA region upstream of the 5'-GATC-3' MutH recognition sequence. In some embodiments, the TALE of the second unit is bound to a DNA region downstream of the 5'-GATC-3' MutH recognition sequence. In some embodiments, a polynucleotide encoding the base editor system is provided herein. In some embodiments, the first and second units of the base editor system are separate single polypeptides.

[0105] In some embodiments, a base editor system is provided that includes a first unit comprising a TALE bound to an engineered deoxyadenosine deaminase, where the deaminase is rAPOBEC1-2xUGI, and a second unit comprising a second TALE bound to a single-stranded (ss-) nickase, where the ss-nickase is MutH. In some embodiments, the base editor system is configured such that, when bound to dsDNA, the TALE-associated MutH unit and the TALE-associated rAPOBEC1-2xUGI unit precisely localize the ss-nickase and deaminase to their respective sites of action within the same editing region of the dsDNA, where the editing region is 1-24 bases in length. In some embodiments, the TALE of the first unit is fused to the N-terminus of rAPOBEC1-2xUGI. In some embodiments, the TALE of the second unit is fused to the C-terminus of MutH. In some embodiments, the first unit further comprises a linker connecting the TALE and rAPOBEC1-2xUGI. In some embodiments, the second unit further comprises a linker connecting the TALE and MutH. In some embodiments, the TALE of the first unit is bound to a DNA region upstream of the 5'-GATC-3' MutH recognition sequence. In some embodiments, the TALE of the second unit is bound to a DNA region downstream of the 5'-GATC-3' MutH recognition sequence. In some embodiments, provided herein is a polynucleotide encoding the base editor system. In some embodiments, the first and second units of the base editor system are separate single polypeptides.

[0106] In some embodiments, a base editing system is provided that includes a first unit comprising a TALE bound to a single-stranded (ss-) nickase, where the ss-nickase is MutH*, and a second unit comprising a second TALE bound to an engineered deoxyadenosine deaminase, where the deaminase is rAPOBEC1-2xUGI. In some embodiments, the base editor system is configured such that the TALE-associated MutH* unit and the TALE-associated rAPOBEC1-2xUGI unit, when bound to dsDNA, precisely localize the ss-nickase and deaminase to their respective sites of action within the same editing region of the dsDNA, where the editing region is 1-24 bases long. In some embodiments, the TALE of the first unit is fused to the N-terminus of MutH*. In some embodiments, the TALE of the second unit is fused to the C-terminus of rAPOBEC1-2xUGI. In some embodiments, the first unit further comprises a linker connecting the TALE and MutH*. In some embodiments, the second unit further comprises a linker connecting the TALE and rAPOBEC1-2xUGI. In some embodiments, the TALE of the first unit is bound to a DNA region upstream of the MutH* recognition sequence of 5'-GATD-3', where D is a base selected from G, A, and T. In some embodiments, the TALE of the second unit is bound to a DNA region downstream of the MutH* recognition sequence of 5'-GATD-3', where D is a base selected from G, A, and T. In some embodiments, a polynucleotide encoding the base editor system is provided herein. In some embodiments, the first and second units of the base editor system are separate single polypeptides.

[0107] In some embodiments, a base editor system is provided that includes a first unit comprising a TALE bound to an engineered deoxyadenosine deaminase, where the deaminase is rAPOBEC1-2xUGI, and a second unit comprising a second TALE bound to a single-stranded (ss-) nickase, where the ss-nickase is MutH*. In some embodiments, the base editor system is configured such that, when bound to dsDNA, the TALE-associated MutH* unit and the TALE-associated rAPOBEC1-2xUGI unit precisely localize the ss-nickase and deaminase to their respective sites of action within the same editing region of the dsDNA, where the editing region is 1-24 bases in length. In some embodiments, the TALE of the first unit is fused to the N-terminus of rAPOBEC1-2xUGI. In some embodiments, the TALE of the second unit is fused to the C-terminus of MutH*. In some embodiments, the first unit further comprises a linker connecting the TALE and rAPOBEC1-2xUGI. In some embodiments, the second unit further comprises a linker connecting the TALE and MutH*. In some embodiments, the TALE of the first unit is bound to a DNA region upstream of the MutH* recognition sequence of 5'-GATD-3', where D is a base selected from G, A, and T. In some embodiments, the TALE of the second unit is bound to a DNA region downstream of the MutH* recognition sequence of 5'-GATD-3', where D is a base selected from G, A, and T. In some embodiments, a polynucleotide encoding the base editor system is provided herein. In some embodiments, the first and second units of the base editor system are separate single polypeptides.

[0108] In some embodiments, a base editor system is provided that includes a first unit comprising a TALE bound to a single-stranded (ss-) nickase, where the ss-nickase is Nt.BspD6I(C), and a second unit comprising a second TALE bound to an engineered deoxyadenosine deaminase, where the deaminase is rAPOBEC1-2xUGI. In some embodiments, the base editor system is configured such that the TALE-associated Nt.BspD6I(C) unit and the TALE-associated rAPOBEC1-2xUGI unit, when bound to dsDNA, precisely localize the ss-nickase and deaminase to their respective sites of action within the same editing region of the dsDNA, where the editing region is 1-24 bases long. In some embodiments, the TALE of the first unit is fused to the N-terminus of Nt.BspD6I(C). In some embodiments, the TALE of the second unit is fused to the C-terminus of rAPOBEC1-2xUGI. In some embodiments, the first unit further comprises a linker connecting the TALE and Nt.BspD6I(C). In some embodiments, the second unit further comprises a linker connecting the TALE and rAPOBEC1-2xUGI. In some embodiments, the TALE of the first unit is bound to a DNA region upstream of the Nt.BspD6I(C) action site. In some embodiments, the TALE of the second unit is bound to a DNA region downstream of the Nt.BspD6I(C) action site. In some embodiments, provided herein is a polynucleotide encoding the base editor system. In some embodiments, the first and second units of the base editor system are separate single polypeptides.

[0109] In some embodiments, a base editor system is provided that includes a first unit comprising a TALE bound to an engineered deoxyadenosine deaminase, where the deaminase is rAPOBEC1-2xUGI, and a second unit comprising a second TALE bound to a single-stranded (ss-) nickase, where the ss-nickase is Nt.BspD6I(C). In some embodiments, the base editor system is configured such that the TALE-associated Nt.BspD6I(C) unit and the TALE-associated rAPOBEC1-2xUGI unit, when bound to dsDNA, precisely localize the ss-nickase and deaminase to their respective sites of action within the same editing region of the dsDNA, where the editing region is 1-24 bases in length. In some embodiments, the TALE of the first unit is fused to the N-terminus of rAPOBEC1-2xUGI. In some embodiments, the TALE of the second unit is fused to the C-terminus of Nt.BspD6I(C). In some embodiments, the first unit further comprises a linker connecting the TALE and rAPOBEC1-2xUGI. In some embodiments, the second unit further comprises a linker connecting the TALE and Nt.BspD6I(C). In some embodiments, the TALE of the first unit is bound to a DNA region upstream of the Nt.BspD6I(C) action site. In some embodiments, the TALE of the second unit is bound to a DNA region downstream of the Nt.BspD6I(C) action site. In some embodiments, provided herein is a polynucleotide encoding the base editor system. In some embodiments, the first and second units of the base editor system are separate single polypeptides.

[0110] In some embodiments, a base editor system is provided, comprising individual units comprising a TALE bound to a single-stranded (ss-) nickase, where the ss-nickase is MutH, and an engineered deoxyadenosine deaminase, where the deaminase is TadA8e(V106W). In some embodiments, the base editor system is configured such that the TALE-associated MutH and TadA8e(V106W) form a complex, where the complex is configured such that when the TALE is bound to dsDNA, the TALE can precisely localize the ss-nickase and deaminase to their respective sites of action within the same editing region of the dsDNA, where the editing region is 1-24 bases in length. In some embodiments, a single polypeptide is arranged such that the TALE is fused to the N-terminus of MutH, and then MutH is fused to the N-terminus of TadA8e(V106W). In some embodiments, a linker polypeptide separates the TALE, MutH, and TadA8e(V106W) domains of the base editor system. In some embodiments, the TALE of the first unit is attached to a DNA region upstream of the MutH recognition sequence of 5'-GATC-3'. In some embodiments, provided herein are polynucleotides encoding the base editor system.

[0111] In some embodiments, a base editor system is provided that includes individual units comprising a TALE linked to an engineered deoxyadenosine deaminase and an engineered ss-nickase, wherein the deaminase is TadA8e(V106W) and the ss-nickase is MutH. In some embodiments, the base editor system is configured such that the TALE-associated TadA8e(V106W) and MutH form a complex, wherein the complex is configured such that when the TALE is bound to dsDNA, the TALE precisely localizes the ss-nickase and deaminase to their respective sites of action within the same editing region of the dsDNA, the editing region being 1-24 bases in length. In some embodiments, a single polypeptide is arranged such that the TALE is fused to the N-terminus of TadA8e(V106W), which in turn is fused to the N-terminus of MutH. In some embodiments, a linker polypeptide separates the TALE, MutH, and TadA8e(V106W) domains of the base editor system. In some embodiments, the TALE of the first unit is bound to a DNA region upstream of the MutH recognition sequence of 5'-GATC-3'. In some embodiments, provided herein are polynucleotides encoding the base editor system.

[0112] In some embodiments, a base editor system is provided that includes separate units comprising a TALE and an engineered deoxyadenosine deaminase bound to a single-stranded (ss-) nickase, wherein the ss-nickase is Nt.BspD6I(C) and the deaminase is TadA8e(V106W). In some embodiments, the base editor system is configured such that the TALE-associated Nt.BspD6I(C) and TadA8e(V106W) form a complex, such that when the TALE is bound to dsDNA, the TALE can precisely localize the ss-nickase and deaminase to their respective sites of action within the same editing region of the dsDNA, the editing region being 1-24 bases in length. In some embodiments, a single polypeptide is arranged such that the TALE is fused to the N-terminus of Nt.BspD6I(C), which is then fused to the N-terminus of TadA8e(V106W). In some embodiments, a linker polypeptide separates the TALE, Nt.BspD6I(C), and TadA8e(V106W) domains of the base editor system. In some embodiments, the TALE of the first unit is bound to a DNA region upstream of the Nt.BspD6I(C) action site. In some embodiments, provided herein are polynucleotides encoding the base editor system.

[0113] In some embodiments, a base editor system is provided that includes separate units comprising a TALE linked to an engineered deoxyadenosine deaminase and an engineered ss-nickase, wherein the deaminase is TadA8e(V106W) and the ss-nickase is Nt.BspD6I(C). In some embodiments, the base editor system is configured such that the TALE-associated TadA8e(V106W) and Nt.BspD6I(C) form a complex, such that when the TALE is bound to dsDNA, the TALE precisely localizes the ss-nickase and deaminase to their respective sites of action within the same editing region of the dsDNA, the editing region being 1-24 bases long. In some approaches, a single polypeptide is arranged such that a TALE is fused to the N-terminus of TadA8e(V106W), which is then fused to the N-terminus of Nt.BspD6I(C). In some embodiments, a linker polypeptide separates the TALE, Nt.BspD6I(C), and TadA8e(V106W) domains of the base editor system. In some embodiments, the TALE of the first unit is bound to a DNA region upstream of the Nt.BspD6I(C) action site. In some embodiments, provided herein are polynucleotides encoding the base editor system.

[0114] III. Methods of Use and Manufacturing In certain aspects, provided herein are methods of using the base editor systems taught herein.

[0115] In certain aspects, the methods provided herein relate to editing a target nucleotide within an edited region of a cell or a particular structure thereof (e.g., mitochondria). In some embodiments, the modification resulting from the editing is not transient, e.g., remains edited for at least about 10 days, e.g., at least about 15 days, 25 days, 1 month, 3 months, 6 months, 9 months, or 1 year.

[0116] In some embodiments, a method of editing a target nucleotide in an edited region of a cell is provided, the method comprising delivering any of the base editor systems described herein (e.g., a monomeric or dimeric base editor system) to a cell. In some embodiments, the base editor system, or at least one component thereof, is in polypeptide form. In some embodiments, such polypeptide form of the base editor system, or at least one component thereof, comprises a localization signal, e.g., a mitochondrial localization signal (MLS), configured to transport the polypeptide to one or more mitochondria. In some embodiments, the base editor system, or at least one component thereof, is in the form of a polynucleotide, such as one or more polynucleotides encoding the base editor system, or at least one component thereof. In such systems, the one or more polynucleotides are configured to express an associated polypeptide of the base editor system in the cell. In some embodiments, the edited region is located in mitochondrial DNA.

[0117] In some embodiments, a method of editing a target nucleotide within an edited region in a mitochondria of a cell is provided, the method comprising delivering any of the base editor systems described herein (e.g., a monomeric or dimeric base editor system) to the cell, wherein the base editor system is delivered to the cell in the form of a polynucleotide, wherein the polynucleotide form of the base editor system is configured to express the base editor system in the cell. In some embodiments, the expressed base editor system comprises one or more mitochondrial localization signals (MLSs) such that the base editor system is transported to the mitochondria of the cell.

[0118] The purposes for editing a target nucleotide in a cell (e.g., mitochondria) using the base editor systems described herein are diverse, all of which are encompassed by the description provided herein. For example, in some embodiments, the methods for editing a target nucleotide in a cell using the base editor systems provided herein are used to create a cellular model. In some embodiments, the cellular model is a model of a mitochondrial disease. In some embodiments, the target nucleotide is a known SNP site, and the base editor system is configured to edit the target nucleotide to restore the SNP to a wild-type nucleotide, create a SNP, or adjust the disease-associated SNP to another nucleotide base.

[0119] In some embodiments, methods of treating an individual suffering from a disease associated with DNA mutation are provided, the methods comprising administering to the individual a base editor system (e.g., a monomeric or dimeric base editor system) or a precursor thereof described herein. In some embodiments, the base editor system or precursor thereof comprises one or more polynucleotides encoding a first unit comprising a first double-stranded (ds)DNA-binding polypeptide bound to a single-stranded (ss-)nickase and a second unit comprising a second dsDNA-binding polypeptide bound to a deaminase, wherein the first and second units are configured, when expressed in the individual, such that when bound to dsDNA, the first dsDNA-binding polypeptide of the first unit and the second dsDNA-binding polypeptide of the second unit are capable of precisely localizing the ss-nickase and deaminase to their respective sites of action within the same edited region of dsDNA. In some embodiments, the base editor system or precursor thereof comprises one or more polynucleotides encoding a double-stranded (ds)DNA binding polypeptide bound to a single-stranded (ss-) nickase and a deaminase, wherein, when expressed in an individual, the polynucleotides are configured such that when the dsDNA binding polypeptide is bound to dsDNA, the ss-nickase and deaminase can precisely localize to their respective sites of action within the same edited region of the dsDNA. In some embodiments, the disease is a mitochondrial disease.In some embodiments, mitochondrial diseases (including conditions) include autism spectrum disorders, 3-methylglutaric aciduria (3-MGA), Charcot-Marie-Tooth disease, mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome, epilepsy, myoclonic epilepsy, myoclonic epilepsy with ragged-red fibers (MERRF), neuropathy, ataxia syndromes, spinocerebellar ataxia, neurogenic ataxia-retinitis pigmentosa (NARP) syndrome, myotonic dystonia, and the like. Loffey's syndrome (DM), Duchenne muscular dystrophy (DMD), Leber's hereditary optic neuropathy (LHON), Leber's optic neuropathy with dystonia (LDYT), Leigh syndrome, Kearns-Sayre syndrome (KSS), Pearson syndrome, chronic progressive external ophthalmoplegia (CPEO), focal segmental glomerular sclerosis (FSGS), Gittelman-like syndrome, mitochondrial myopathy with lactic acidosis and sideroblastic anemia (MLASA), lactic acidemia, maternally inherited diabetes mellitus and deafness (MIDD), rhabdomyolysis Diabetes mellitus, Non-insulin-dependent diabetes mellitus (NIDM), Aminoglycoside-induced hearing loss, Alpers disease, Complex I deficiency, Complex II deficiency, Complex III deficiency, Complex IV deficiency, Complex V deficiency, Cardiomyopathy, Maternally inherited cardiomyopathy (MICM), Hypertrophic cardiomyopathy (HCM), Infantile cardiomyopathy, Encephalomyopathy, Progressive encephalomyopathy, Progressive mitochondrial cytopathies, Hearing impairment, Dementia, Depressive mood disorder, Dystonia, Progressive dystonia, Exercise intolerance, Hyperammonemia, IgG nephropathy, Leukoencephalopathy, Maternally inherited epilepsy, Maternal The mutated DNA may include hereditary non-syndromic hearing loss, mitochondrial tubulointerstitial kidney disease (MITKD), mitochondrial myopathy, severe multisymptomatic myopathy in adults, multiple myeloma (MM), myelomeningocele (MMC), mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) syndrome, optic nerve atrophy, myoclonus, post-exertional fatigue (PEM), ptosis, renal failure, reversible COX deficiency myopathy, septo-optic dysplasia, sensorineural hearing loss (SNHL), spastic paraplegia, stroke, or mitochondrial cell disease. In some embodiments, the patient or individual has Leber's hereditary optic neuropathy (LHON). In some embodiments, the mutated DNA is mitochondrial DNA.In some embodiments, the edited region comprises a DNA mutation, e.g., the target nucleotide edited by the base editor system is (or includes) the nucleotide of the DNA mutation. In some embodiments, the edited region comprises other DNA characteristics associated with a disease, the DNA mutation, or provides a mechanism for at least partially treating a disease. For example, in some embodiments, the edited region comprises a start codon, e.g., the nucleic acid base editing system controls (including inhibits or prohibits) expression of a gene. In some embodiments, the one or more polynucleotides administered to the individual comprise RNA, such as a circular RNA.

[0120] In some embodiments, the methods of use (e.g., therapeutic methods) provided herein involve using a base editor system configured to edit two or more nucleotide bases (e.g., nucleotide bases at different DNA positions). In some embodiments, when a base editor system is configured to edit a plurality of nucleotide bases, at least two or more of the plurality of nucleotide bases are located in different edited regions. Such editing can be performed using various configurations of the base editor systems described herein. For example, in some embodiments, a base editor system configured to edit a plurality of nucleotide bases includes a first unit comprising a first double-stranded (ds)DNA binding polypeptide bound to a first single-stranded (ss-) nickase, a second unit comprising a second dsDNA binding polypeptide bound to a second ss-nickase, where the first dsDNA binding polypeptide and the second dsDNA binding polypeptide recognize different recognition sequences, a third unit comprising a third dsDNA binding polypeptide bound to a first deaminase, and a fourth dsDNA binding polypeptide bound to the second deaminase. and a fourth unit comprising a polypeptide, wherein the base editor system is configured such that when the first dsDNA-binding polypeptide of the first unit and the third dsDNA-binding polypeptide of the third unit are bound to dsDNA, the first ss-nickase and the first deaminase can be precisely localized to their respective sites of action within a first editing region of dsDNA, and when the third dsDNA-binding polypeptide of the third unit and the fourth dsDNA-binding polypeptide of the fourth unit are bound to dsDNA, the second ss-nickase and the second deaminase can be precisely localized to their respective sites of action within a second editing region of dsDNA. In some embodiments, the first ss-nickase and the second ss-nickase recognize different recognition sequences. In some embodiments, the first deaminase and the second deaminase are the same. In some embodiments, the first deaminase and the second deaminase catalyze the same nucleotide base conversion. In some embodiments, the first deaminase and the second deaminase catalyze different nucleotide base conversions.In some embodiments, the treatment is for Leber's hereditary optic neuropathy (LHON).

[0121] In some embodiments, there is provided a use of a base editor system described herein in the manufacture of a medicament for treating a disease (e.g., a mitochondrial disease) in an individual.

[0122] In some embodiments, the base editor system has a first editing efficiency (e.g., measured as a percentage of editing) in a first cell type and a second editing efficiency in a second cell type, where the first editing efficiency is different from the second editing efficiency. For example, in some embodiments, the base editor system can be designed to have cell type or tissue specificity, where the editing efficiency of the base editor system is high in a target cell type or tissue, but low, or essentially no editing occurs (e.g., the editing rate is about 5% or less), in other cell types or tissues.

[0123] In some embodiments, the editing efficiency of the target DNA nucleotide base is at least about 10%, such as at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or more. In some embodiments, the editing efficiency is determined by Sanger sequencing. In some embodiments, the editing efficiency is determined by next-generation sequencing.

[0124] In some embodiments, the method has a low off-target editing rate. In some embodiments, the method has an editing efficiency of less than about 5% (e.g., about 4.0%, 3.0%, 2.0%, 1.0%, 0.5%, 0.1%, 0.05%, 0.01%, 0.001% or less) for non-target DNA nucleotide bases compared to target DNA nucleotide bases. In some embodiments, the method does not edit non-target DNA nucleotide bases.

[0125] In certain aspects, the present specification provides methods for producing and implementing the technical solutions. Unless otherwise specified, these production and implementation methods can be accomplished using conventional techniques of molecular biology, microbiology, and cell biology well known to those skilled in the art. See, for example, "Molecular Cloning: A Laboratory Manual," 2nd Edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Enzymatic Methods," Handbook of Experimental Immunology (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Modern Laboratory Methods in Molecular Biology" (Ausubel, 1987); "PCR: Polymerase Chain Reaction" (Mullis, 1994); and "Modern Laboratory Methods in Immunology" (Coligan, 1991). These teachings and knowledge of those skilled in the art can be applied to the production of the polynucleotides and polypeptides described herein. The examples provided herein are intended to partially illustrate certain techniques.

[0126] IV. KITS, DRUGS, AND COMPOSITIONS In certain aspects, provided herein are kits and compositions of the base editor systems taught herein that include an ss-nickase having a desired recognition sequence.

[0127] In some embodiments, provided herein is a kit for a nucleic acid base editing system, the kit comprising: a first unit comprising a first double-stranded (ds) DNA-binding polypeptide bound to a single-stranded (ss-) nickase; and a second unit comprising a second dsDNA-binding polypeptide bound to a deaminase, wherein the base editor system is configured such that when the first dsDNA-binding polypeptide of the first unit and the second dsDNA-binding polypeptide of the second unit are bound to dsDNA, the ss-nickase and the deaminase can be precisely localized to their respective sites of action within the same edited region of the dsDNA.

[0128] In some embodiments, provided herein are kits for base editing systems, the kits comprising one or more polynucleotides encoding a first unit comprising a first double-stranded (ds) DNA-binding polypeptide bound to a single-stranded (ss-) nickase and a second unit comprising a second dsDNA-binding polypeptide bound to a deaminase, wherein the base editor system is configured, when expressed, such that when the first dsDNA-binding polypeptide of the first unit and the second dsDNA-binding polypeptide of the second unit are bound to dsDNA, the ss-nickase and the deaminase can be precisely localized to their respective sites of action within the same edited region of dsDNA.

[0129] In some embodiments, provided herein are kits for base editor systems, the kits comprising one or more polynucleotides encoding a single-stranded (ss-) nickase, a deaminase, and a double-stranded (ds) DNA-binding polypeptide, wherein the ss-nickase, deaminase, and dsDNA-binding polypeptide form a complex configured such that, when the dsDNA-binding polypeptide is bound to dsDNA, the ss-nickase and deaminase can be precisely localized to their respective sites of action within the same edited region of the dsDNA.

[0130] The kits provided herein can include one or more containers and instructions for following the methods provided herein. The instructions provided in the kits of the present application are typically instructions written on a label or package insert (e.g., paper included in the kit), although machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0131] All kits provided herein are packaged in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (such as sealed Mylar or plastic bags), etc. The kits may optionally include additional components such as buffers and interpretive information. The present application also provides articles of manufacture such as vials (such as sealed vials), bottles, jars, and flexible packaging.

[0132] Also provided are medicaments, compositions, and unit dosage forms useful in the methods described herein.

[0133] In certain aspects, provided herein are one or more non-naturally occurring polypeptides that comprise the base editor systems described herein. In some embodiments, one of the one or more non-naturally occurring polypeptides that comprise the base editor systems described herein comprises a polypeptide with nickase activity and a polypeptide with dsDNA binding ability, such as a TALE. In some embodiments, one of the one or more non-naturally occurring polypeptides that comprise the base editor systems described herein comprises a polypeptide with deaminase activity and a polypeptide with dsDNA binding ability, such as a TALE. In some embodiments, one of the one or more non-naturally occurring polypeptides that comprise the base editor systems described herein comprises a polypeptide with nickase activity, a polypeptide with deaminase activity, and a polypeptide with dsDNA binding ability, such as a TALE, for example, a polypeptide comprising any one of SEQ ID NOs: 21-24.

[0134] In certain aspects, provided herein is a non-naturally occurring polypeptide having nickase activity comprising the amino acid sequence of SEQ ID NO:1 with the following mutations E91A and F94A (SEQ ID NO:2). In certain aspects, provided herein is a non-naturally occurring polypeptide having nickase activity comprising the amino acid sequence of SEQ ID NO:2 (also referred to herein as MutH*). In some embodiments, the non-naturally occurring polypeptide is isolated. In some embodiments, provided herein is a polynucleotide encoding a non-naturally occurring polypeptide comprising the amino acid sequence of SEQ ID NO:2. In some embodiments, the non-naturally occurring polypeptide having nickase activity and comprising the amino acid sequence of SEQ ID NO:2 recognizes the ss-nickase recognition sequence of 5'-GATD-3', where D is A, T, or G.

[0135] V. Sequence In certain aspects, provided herein are sequences that can be used in the nucleotide editor systems provided herein. Specifically, provided herein are ss-nickase sequences for MutH and its derivative MutH*, which has E91A and F94A mutations. MutH* has a ss-nickase recognition sequence of 5'-GATD-3', where D is A, T, or G.

[0136] MutH (E. coli); protein sequence; SEQ ID NO: 1 MSQPRPLLSPPETEEQLLAQAQQLSGYTLGELAALVGLVTPENLKRDKGWIGVLLEIWLGASAGSKPEQDFAALGVELKTIPVDSLGRPLETTFVCVAPLTGNSGVTTWETSHVR HKLKRVLWIPVEGERSIPLAQRRVGSPLLWSPNEEEDRQLREDWEELMDMIVLGQVERITARHGEYLQIRPKAANAKALTEAIGARGERILTLPRGFYLKKNFTSALLARHFLIQ

[0137] MutH*; Protein sequence; SEQ ID NO:2 MSQPRPLLSPPETEEQLLAQAQQLSGYTLGELAALVGLVTPENLKRDKGWIGVLLEIWLGASAGSKPEQDFAALGVELKTIPVDSLGRPLATTAVCVAPLTGNSGVTWETSHVR HKLKRVLWIPVEGERSIPLAQRRVGSPLLWSPNEEEDRQLREDWEELMDMIVLGQVERITARHGEYLQIRPKAANAKALTEAIGARGERILTLPRGFYLKKNFTSALLARHFLIQ

[0138] Nt.BspD6I(C); Protein sequence; SEQ ID NO:3 RQLEEVIDLLEVYHEKKNVIEEKIKARFIANKNTVFEWLTWNGFIILGNALEYKNNFVIDEELQPVTHAAGNQPDMEIIYEDFIVLGEVTTSKGATQFKMESEPVTRHYLN KKKELEKQGVEKELYCLFIAPEINKNTFEEFMKYNIVQNTRIIPLSLKQFNMLLMVQKKLIEKGRRLSSYDIKNLMVSLYRTTIECERKYTQIKAGLEETLNNWVVDKEVRF

[0139] FokI-FokI(D450A); protein sequence; SEQ ID NO:4 FKQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGIVVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVGSHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGEMIKAGTLTLEEVRRKFNNGEINFSGSGGGSGGGGGSGGGSGG SGGGSSGGGGSGGGGSGGGGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFMFKVYGYRGKHLGGSRKPAGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGEMIKAGTLTLEEVRRKFNNGEINF

[0140] Nb.BsaI (C, N441D / R442G);protein sequence;SEQ ID NO:5 CRSHADRGRWEKNLRSYTTDRAFEYWVDGDWVAADKLMGLIRTNEQIKKETCLNDNHPGPCSADHIGPISLGFVHRPEFQLLCNSCNSAKNNRMTFSDVQHLINAENNGEEVASWYCKHIWDLRKHDVKNNENALRLSKILRDNRHTAMFILSELLKDNHYLFL STFLGLQYAERSVSFSNIKIENHIITGQISEQPRDTKYTEEQKARRMRIGFEALKSYIEKEDGNALLVINDKIIDKINEIKNILQDIPDEYKLLNEKISEQFNSEEVSDELLRDLVTHLPTKESEPANFKLARKYLQEIMEIVGDELSKMWEDERYVRQTFADLD

[0141] Nt.BsaI (C, R236D);protein sequence;SEQ ID NO:6 CRSHADRGRWEKNLRSYTTDRDAFEYWVDGDWVAADKLMGLIRTNEQIKKETCLNDNHPGPCSADHIGPISLGFVHRPEFQLLCNSCNSAKNNRMTFSDVQHLINAENNGEEVASWYCKHIWDLRKHDVKNNENALRLSKILRDNRHTAMFILSELLKDNHYLFL STFLGLQYAERSVSFSNIKIENHIITGQISEQPRDTKYTEEQKARRMRIGFEALKSYIEKENNRNALLVINDKIIDKINEIKNILQDIPDEYKLLNEKISEQFNSEEVSDELLRDLVTHLPTKESEPANFKLARKYLQEIMEIVGDELSKMWEDERYVRQTFADLD

[0142] Nb.BsmBI (C, R438D);protein sequence;SEQ ID NO:7 KDPGRHDDNMRLYNHDRRAFMWWSEGDWALADALYNKAGAGKCADPDCQKEVEKISPDHVGPISCGFKQIPFFKPLCASCNSAKNRRFSYQDVKELLKYENYTGDSVASWQVRALWDNCKHLVKNDDDSKLLSNLMRSLQDYYLRSLYKLFSNGFAH LLSYFLTPEYAHYKITFEGLNTSTLEYERYKTFKKTKSTSSLAARIVRIAFEELEIYNSKDINDEKLIKFDTSSWEKDFENIISYATKNLSLDEEASKWNKVLTDKNLSSTEKDKKISSLEDKNYEVYKKQFYILKDLLVEHFNKIGEQIAKDYMK

[0143] Nt.BsmAI (C, R221D);protein sequence;SEQ ID NO:8 CRASQDKGRSKENLKSYTKDRDAYEYWSDGNIHAANQFMGSPFFNNISADHIGPISLGFVHDPRYLQPMSGGDNSSKRDRLQLDDIEKIIETEKRTNVYPMSWYSKLIWEYIKKNYSTHKSLISGVYRDALKQ NMSNFMYILWYILEHCNQDGEHFLEEALLKPNYDYFQYSYTFNELGEIVSINPRHFTDRNQYETERYKRIAFESVYDYNEKENRNIKANLIDNEQRMLNKLCQEISSGVPVEQCKKLLIELMEVIQKRIISTL

[0144] Nb.BsrDI(C); Protein sequence; SEQ ID NO:9 IPEELFNWPRTDKVNFKSPQGLIKYDELCYQLEKAVGSKKAYCLSNNAGAKPQKLESLKEWINSQKKLFDKAPKLTPPAEFNMKLDAFPVTSNNNYYVTTSKNILYLFDYWKD LRIAIETAFPRLKGKLPTDIDEKPALIYICNSVKPGRLFGDPFTGQLSAFSTIFGKKNIDMPRIVVAYYPHQIYSQALPKNNKSNKGITLKKELTDFLIFHGGVVVKLNEGKAY

[0145] Nt.CviPII; Protein sequence; SEQ ID NO:10 MYIYMSTPQAKTKYYEQRFVNDFYKELERNKVSLPVTIVLKDNLGIKQVIQNGSGVRVLRDKANAKSPSKIKSEELGRHVTSKADIALFTEEKNGTKVDVAWISPQSHKDFLGKKITPAQYFDASSDVMFKTIKQPKEIKELKNKMISLSVPLTATKYCWPKYKSGTSLRIWDDVQSTILNMMAIFGVEFGKAYCRNNANILMVGDPLIEVKDDKTIILTTKENGFSLANGFAEYIPSKDKPIFFTKPTSGKKTVVDGKTIEGVSVWIIYRSYAGSKNRKIDDVLKNKIELISSSCSVKKKDNFVSIMQSKKITSPPKSKKITSPKSKKITNFFMKK

[0146] BspQI(C);protein sequence;SEQ ID NO:11 NNSFNPVRTKDQLHESAVITREKKILLKEPEILQKIKNRNNGEGLKSIIWKKFDKKCFNCEKELTIEEVRLDHTRPLAYLWPIDEHATCLCKEKCNNTKHDMFPIDFYQGDEDKLRRLARITGLDYESLVKRDVNEVELARIINNIEDFATNVEARTFRSIRNKVKEVRPDTDLFEILKSKNINLYNELQYELLTRKD

[0147] N.AlwI(C);protein sequence;SEQ ID NO:12 YHLEELLFENNEKKFAENQKNEWDEILAYMDLLISPKPISIEIADKEISIPSGERPAYFEWVLWRAFLANLNHLIIEPQQCRRFKVDQDFKPIHNAPGGGADVIFEYENFKILGEVTSKAGATQFKMESEPVTRHYLNKKKELEKQGVEKELYCLFIAPEINKNTFEFMKYNIVQNTRIIPLSLKQFNMLLMVQKKLIEKGRRLSSYDIKNLMVSLYRTTIECERKYTQIKAGLEETLNNWVVDKEVRF

[0148] Nt.BsrDI; protein sequence; SEQ ID NO:13 MTEYDLHLYADSFHEGHWCCENLAKIAQSDGGKHQIDYLQGFIPRHSLIFSDLIINITVFGSYKSWKHLPKQIKDLLFWGKPDFIAYDPKNDKILFAVEETGAVPTGNQALQRCERIYGSAR KQIPFWYLLSEFGQHKDGGTRRDSIWPTIMGLKLTQLVKTPSIILHYSDINNPEDYNSGNGLFLFKSLLQIIINYCTLKNPLKGMLELLSIQYENMLEFIKSQWKEQIDFLPGEEILNTKT KELARMYASLAIGQTVKIPEELFNWPRTDKVNFKSPQGLIKYDELCYQLEKAVGSKKAYCLSNNAGAKPQKLESLKEWINSQKKLFDKAPKLTPPAEFNMKLDAFPVTSNNNYYVTTSKNILYLFDYWKDLRIAIETAFPRLKGKLPTDIDEKPALIYICNSVKPGRLFGDPFTGQLSAFSTIFGKKNIDMPRIVVAYYPHQIYSQALPKNNKSNKGITLKKELDTFLIFHGGVVVKLNEGKAY

[0149] Nt.BtsI; protein sequence; SEQ ID NO:14 MQIEQLMKSLTIYFDDIQEGLWFKNLHPLLESASLEAITGSLKRNPNLADVLKYDRPDIILTLNQTPILVIERTIEVPSGHNVGQRYGRLAAASEAGVPLVYFGPYAARKHGGATEGPRYMNLRLFYALDVMQKVNGSAITTINWPVDQNFEILQDPSKDKRMK EYLEMFFDNLLKYGIAGINLAIRNSSFQAEQLAEREKFVETMITNPEQYDVPPDSVQILNAERFFNELGISENKRIICDEVVLYQVGMTYVRSDPYTGMALLYKYLYILGSERNRCLILKFPNITTDMWKKVAFGSRERKDVRIYRSVSDGILFADGYLSKEEL

[0150] I-TevI; protein sequence; SEQ ID NO:15 KSGIYQIKNTLNNKVYVGSAKDFEKRWKRHFKDLEKGCHSSIKLQRSFNKHGNVFECSILEEIPYEKDLIIERENFWIKELNSKINGYNIADATFGDTCSTHPLKEEIIKKRSETVKAKMLKLGPDGRKALYSKPGSKNGRWNPETHKFCKCGVRIQTSAYTCSKCRNRSGENNSFFNHKHSQGPSAD

[0151] ss.BspD6I; protein sequence; SEQ ID NO: 16 MQDILDFYEEVEKTINPPNYFEWNTYRVFKKLGSYKNLVPNFKLDDSGHPIGNAIPGVEDILVEYEHFSILIECSLTIGEKQLDYEGDSVVRHLQEYKKKGIEAYTLFLGKSIDLSFARHIGFNKESEPVIPLTVDQFKKLVTQLKGDGEHFNPNKLKEILIKLLRSDLGYDQAEEWLTFIEYNLK

[0152] ss.BsrDI; protein sequence; SEQ ID NO: 17 MTDYRYSFELSEEIARWAFEIKTKNTDWFVAFSNPTAGPWKRVMAIDKASNREGEVHRFGREDERPDIILVNDNISLILILEAKEKLNQLISKSQVDKSVDVFLTLSS ILKEKSDNNYWGDRTKYINVLGILWGSEQETSQKDIDNAFRVYRDSLVKNLKEINPTTPTNICTDILVGVESIKNKKEEISIKIHVSNIYAEIYPKFTGKHLLEKLAVLN

[0153] ss.BtsI; protein sequence; SEQ ID NO:18 MKITEGIVHVAMRHFLKSNGWKLIAGQYPGGSDDELTALNIVDPVVARDNSPDPRRHSLGKIVPDLIAYKNDDLLVIEAKPKYSQDDRDKLLYLLSERKHDFYAALEKFATERNHPELLPVSKLNIIPGLAFSASENKFKKDPGFVYIRVSGIFEAFMEGYDWG

[0154] TadA8e (V106W); protein sequence; SEQ ID NO: 19 SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGWRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN

[0155] rAPOBEC1-2×UGI; Protein sequence; SEQ ID NO:20 SSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEF LSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTF FTIALQSCHYQRLPPHILWATGLKGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSGGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML

[0156] MutH-2AA linker-TadA8e(V106W); protein sequence; SEQ ID NO:21 MSQPRPLLSPPETEEQLLAQAQQLSGYTLGELAALVGLVTPENLKRDKGWIGVLLEIWLGASAGSKPEQDFAALGVELKTIPVDSLGRPLETTFVCVAPLTGNSGVTWETSHVRHKLKRVLWIPVEGERSIPLAQRRVGSPLLWSPNEEDRQLREDWEELMDMIVLGQVERITARHGEYLQIRPKAANAKALTEAIGA RGERILTLPRGFYLKKNFTSALLARHFLIQG*S*SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGWRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN *The amino acid (AA) of the linker is indicated with a trailing asterisk.

[0157] TadA8e(V106W) -2AA linker-MutH; protein sequence; SEQ ID NO:22 SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGWRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSING*S*MSQPRPLLSPPETEEQLLAQAQQLSGYTL GELAALVGLVTPENLKRDKGWIGVLLEIWLGASAGSKPEQDFAALGVELKTIPVDSLGRPLETTFVCVAPLTGNSGVTWETSHVRHKLKRVLWIPVEGERSIPLAQRRVGSPLLWSPNEEDRQLREDWEELMDMIVLGQVERITARHGEYLQIRPKAANAKALTEAIGARGERILTLPRGFYLKKNFTSALLARHFLIQ *Linker amino acids are indicated with a trailing asterisk.

[0158] Nt.BspD6I(C)-2AA linker-TadA8e(V106W); Protein sequence; SEQ ID NO:23 RQLEEVIDLLEVYHEKKNVIEEKIKARFIANKNTVFEWLTWNGFIILGNALEYKNNFVIDEELQPVTHAAGNQPDMEIIYEDFIVLGEVTTSKGATQFKMESEPVTRHYLNKKKELEKQGVEKELYCLFIAPEINKNTFEEFMKYNIVQNTRIIPLSLKQFNMLLMVQKKLIEKGRRLSSYDIKNLMVSLYRTTIE CERKYTQIKAGLEETLNNWVVDKEVRFG*S*SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGWRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN *Linker amino acids are indicated with a trailing asterisk.

[0159] TadA8e(V106W)-2AA linker-Nt.BspD6I(C); protein sequence; SEQ ID NO:24 SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGGWNRAIGLHDPTAPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGWRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSING*S*RQLEEVIDLLEVYHEKKNVIEEKIKA RFIANKNTVFEWLTWNGFIILGNALEYKNNFVIDEELQPVTHAAGNQPDMEIIYEDFIVLGEVTTSKGATQFKMESEPVTRHYLNKKKELEKQGVEKE LYCLFIAPEINKNTFEEFMKYNIVQNTRIIPLSLKQFNMLLMVQKKLIEKGRRLSSYDIKNLMVSLYRTTIECERKYTQIKAGLEETLNNWVVDKEVRF *Linker amino acids are indicated with a trailing asterisk.

[0160] 3*EAAAK linker; protein sequence; SEQ ID NO:25 AEAAAKEAAAKEAAAKEAAAKA

[0161] 32AA linker; protein sequence; SEQ ID NO:26 GSGSSGGSSGSETPGTSESATPESSGGSSGGS

[0162] Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of the present disclosure. The present application is further illustrated by the following examples, which should not be construed as limiting the scope or spirit of the present disclosure to the specific operational flows described therein. Example

[0163] Example 1: Editing mitochondrial DNA using the mitoABE system This example demonstrates efficient editing of mitochondrial DNA by the mitoABE base editor system. Additionally, this example outlines the mechanisms required for efficient A to G mitochondrial base editing.

[0164] In this example, we fused the engineered deoxyadenosine deaminase TadA8e(V106W) with an appropriate TALE array and mitochondrial targeting sequence (MTS) to target transcripts to the mitochondrial matrix and achieve mitochondrial A-to-G base editing. In transfected HEK593T cells, a high mitochondrial DNA editing efficiency of 0.39% was detected at all three target sites, MT-ND1, MT-ND4, and MT-RNR2 (Figures 1A-1C). These low editing efficiencies are only slightly higher than the reported error rates (>0.10%) observed by high-throughput sequencing. TadA is an essential tRNA-specific adenosine deaminase from Escherichia coli that acts on single-stranded DNA. Because TALE arrays can only bind to dsDNA and cannot unwind the DNA double helix, a mechanism for inducing single-stranded DNA structures at the target site is required to achieve TadA8e(V106W)-mediated mitochondrial DNA editing.

[0165] To induce single-stranded DNA structures at target sites, the sequence-specific (5'-GATC-3') nickase MutH was fused to the appropriate TALE array. Introduction of the TALE-MutH and TALE-TadA8e(V106W) constructs into HEK293T cells resulted in targeted A-to-G editing of mitochondrial DNA at all three tested sites (MT-ND1, MT-ND4, and MT-RNR2), with a maximum editing efficiency of 77% (Figure 1D-1F). As shown in Figure 1G-1I, expression of the catalytically inactive TALE-MutH(D70A) together with TALE-TadA8e(V106W) failed to efficiently edit mitochondrial DNA in transfected HEK293T cells. These results indicate that the TALE-nickase activity nicks the single strand, exposing single-stranded DNA and generating a substrate for the deoxyadenosine deaminase activity of TadA8e(V106W). This novel mitochondrial A-to-G base editing system is mitoABE. MutH Of the three sites tested, editing purity exceeding 95% was observed at the MT-ND1 site, and near 100% at the MT-ND4 and MT-RNR2 sites (Figure 1J-1L). In HEK293T cells, durability of mitochondrial DNA editing was confirmed at the three target sites (MT-ND1, MT-ND4, and MT-RNR2) on days 3, 9, and 15, and the percentage of mitochondrial DNA editing was consistent across each time point tested (Figure 1M-O).

[0166] Based on these results, a working model was constructed in which TALE-nickases bind to mitochondrial DNA and generate single-strand breaks at the target site. The resulting DNA nick induces the formation of single-stranded DNA, and TALE-TadA8e(V106W) deaminates the base in the single-stranded DNA. After repair and mitochondrial DNA replication, this edit is retained in only one strand, resulting in a strand-specific A-to-G conversion (Figure 1P).

[0167] Example 2: Strand-specific editing by the mitoABE system This example demonstrates strand specificity in A to G editing using the mitoABE system. Strand specificity is determined by the strand nicked by the TALE-nickase, and adenine deamination is retained only on the unnicked strand.

[0168] As shown in Figures 1D–1F, mitoABE MutH Adenine editing by mitoABE occurred preferentially in the top strand of the target site. Although the edited A within the editing window had multiple adjacent Ts, no editing was detected (Figure 1E-1F) or only minimal editing was detected in the A in its complementary strand (Figure 1D). These results are consistent with those of mitoABE. MutH This suggests that editing by TALE-MutH may be strand-specific. To verify this, we swapped the TALE arrays of each pair, TALE-MutH and TALE-TadA(V106W), and demonstrated that strand-specific editing switched accordingly (Figure 2A-2B). At MT-RNR2 site 1, MT-ND1 site 1, and MT-ND4 site 1, the MutH nicking sequence (5'-GATC-3') is located in the center of the editing window, 3 bp away from each TALE (Figure 2A). In this case, when the left TALE-MutH and the right TALE-TadA8e(V106W) were used, editing occurred primarily on the top DNA strand, whereas when the positions of TALE-MutH and TALE-TadA8e(V106W) were swapped, editing occurred primarily on the bottom DNA strand (Figure 2A). At MT-RNR2 site 2 and MT-ND4 site 1, the MutH nicking sequences were 5 bp and 6 bp away from both ends of the editing window, respectively (Figure 2B). In this case, when the left TALE-MutH and the right TALE-TadA8e(V106W) were used, editing occurred primarily on the bottom DNA strand. After TALE exchange between MutH and TadA8e(V106W), the top DNA strand became the adenine (A)-edited strand (Figure 2B). Therefore, strand-specific editing is related to strand selection at the nick, which in turn is related to the number of bases between the TALE binding site and the MutH recognition motif (5'-GATC-3').

[0169] Next, we fixed the position of the left-TALE-TadA8e(V106W) and moved the position of the right-TALE-MutH from the nicking sequence (5'-GATC-3') in 1-bp increments within a range of 0-10 bp (Figure 2C). When the distance between the TALE binding site and the MutH nicking motif was 0-4 bp or less, editing of MT-ND4 and MT-RNR2 site 1 occurred primarily on the bottom DNA strand. On the other hand, when the distance between the TALE binding site and the MutH nicking motif was 5-9 bp, editing primarily occurred on the top DNA strand (Figure 2C-2D). On the other hand, when the position of TALE-MutH was fixed and the position of TALE-TadA8e(V106W) was varied, the adenine (A)-edited strand did not change, but the observed editing window gradually widened (Figure 2C).

[0170] Because the MutH nicking motif (5'-GATC-3') is a palindromic sequence, we speculated that fusing MutH with either a left-TALE or a right-TALE would produce the same effect. By gradually expanding the editing window of the MT-ND4 site (Figure 2E), we demonstrated that the edited strand switches regardless of whether the TALE-MutH is located on the left or right side of the target site. At this site, efficient editing was achieved with both 6-bp and 24-bp editing windows (Figure 2E). These results support the conclusion that mitoABE MutHThis study highlights the basic operating principle of the TALE-MutH nicking technique, where when the distance between the TALE-binding site and the MutH nicking motif is 0-4 bp, the TALE-MutH nicks the strand opposite the binding strand, whereas when the distance between the TALE-binding site and the MutH nicking motif is 5-9 bp, the nicking occurs on the same strand. This nicking generates two single-stranded DNA strands, and all adenines on both strands are deaminated by TadA8e(V106W) within the window. After repair and DNA replication, only the deaminated A on the unnicked strand is retained (Figure 2F). In summary, the editing outcome of TadA8e(V106W) depends on which strand is nicked by the TALE-nickase, and adenine deamination is retained only on the unnicked strand. Furthermore, the linker sequence between the TALE and MutH is a key factor in determining whether the mitoABE is involved in the editing process. MutH This did not affect strand-specific editing by α-glucan (Figure 2G-J).

[0171] Example 3: mitoABE by site-directed mutagenesis MutH Expanding the system's targeting range This example demonstrates that targeted mutagenesis can broaden the sequence specificity of nickases and further demonstrates that this method can improve editing efficiency in the mitoABE system.

[0172] The combination of TALE-MutH and TALE-TadA(V106W) enabled strand-specific editing of mitochondrial DNA. However, MutH requires a specific nicking sequence (5'-↓GATC-3'), which is essential for the nicking of mitochondrial DNA. MutH Based on the structural information, the editing range of mitoABE is restricted by introducing point mutations into MutH. MutH As a result, the K48A, R184A, and Y212S mutations were found to be involved in the editing of mitoABE at the MT-ND4 site. MutHIn MutH, F94 contributes to the sequence-specific interaction of loop 67 (amino acid residues 184-190) with 5'-GATC-3', whereas F91 interacts with the cytosine of 5'-GATC-3' (Figure 3A). The E91A or F94A variants were shown to eliminate the editing activity of mitoABE. MutH The MutH enzyme containing the E91A and F94A mutations maintained its editing activity, and the combination of these two mutations improved the editing efficiency of the ND4 site (Figure 3B). This MutH enzyme containing the E91A and F94A mutations is designated MutH*. As shown in Figure 3B, MutH* can nick the 5'-GATD-3' (D represents A, T, or G) site, which allows the creation of a new mitochondrial DNA editing tool, mitoABE. MutH The three genes MT-ND5, MT-CO2, and MT-MTTR were targeted (these three gene sites have 5'-GATA-3', 5'-GATG-3', and 5'-GATT-3' sequences in the top strand, respectively). As a result, mitoABE MutH We confirmed that mitoABE functions as an efficient editing tool, generating bottom-strand edits at all three sites (Figures 3C-3E). Importantly, due to the lack of a MutH motif, these sites are mitoABEs. MutH The difference is that the DNA fragments were not edited by the nucleotide sequence (Figures 3F-3H).

[0173] Because the MutH motif 5'-GATC-3' is a palindromic sequence, by design, TALE-MutH*(5'-GATD-3') can only nick the top strand with the 5' guanine and preserve only the adenine edit on the bottom strand (Figure 3C-3E). On the other hand, TALE-MutH cannot nick 5'-GATD-3', resulting in no editing (Figure 3F-3H). Furthermore, MutH* showed the highest editing efficiency when located 3 bp right of 5'-GATD-3'. This is likely due to its high nicking efficiency (Figure 3C-3E). These results are consistent with those of mitoABE. MutHThis supports the presumed operating principle and suggests that TALE-MutH* actually expands the editing range. Human mtDNA has 23 MutH (5'-GATC-3') recognition sites, whereas there are 485 MutH* (5'-GATN-3') recognition sites. Therefore, TALE-TadA8e(V106W) can function within a 20-bp range upstream and downstream of the nicking site. Based on this, we estimated the proportion of designable mitoABEs in the human mitochondrial genome. While the designable target range of TALE-MutH is only approximately 6% of the mitochondrial genome, the range of TALE-MutH* reaches approximately 71%, with an average of two 5'-GATN-3' sites every 40 bp in the mitochondrial genome (Figure 3I-J).

[0174] Example 4: Additional nickases and their use in the mitoABE system This example demonstrates additional nickases that can confer functionality to the mitoABE system. Furthermore, multiple nickases can be used to expand the targeting range of the mitoABE system, allowing editing of more sites in the mitochondrial genome.

[0175] To further expand the editing scope of mitoABE, several enzymes with potential nickase activity were evaluated. Some nucleases have separate active sites for cleaving dsDNA, so mutations that inactivate one active site can convert the nuclease into a nickase. Specifically, the cleavage domain and recognition domain of type IIS restriction endonucleases are independent of each other, making it possible to convert such endonucleases into nickases by semi-inactivating the cleavage domain. For enzymes without crystal structures, the cleavage domain has been predicted for engineering purposes (Figure 4A). mitoABE MutHWe replaced the MutH component of this construct with the native nickase Nt.BspD6I (C) and engineered nickases, such as FokI-FokI (D450A), Nb.BsaI (C, N441D / R442G), Nt.BsaI (C, R236D), Nb.BsmBI (C, R438D), Nt.BsmAI (C, R221D), Nb.BsrDI (C), Nt.CviPII (5'-1CCD-3'), BspQI (C), N.AlwI (C), and I-TEV-I (5'-CNNN1G-3'), to test whether any of these enzymes could nick DNA when fused to the appropriate TALE array. The recognition domains of all the above enzymes were removed, except for Nt.CviPII (5′-1CCD-3′) and I-TEV-I (5′-CNNN1G-3′), and nickases without recognition motifs and relying solely on the TALE array for recognition were identified.

[0176] We fused the above nickases to the left TALE and verified their potential editing activity when combined with the right TALE-TadA8e(V106W) (Figure 4B). Three editing sites, MT-ND1, MT-ND5 site 2, and MT-ND4, were selected for testing. Among all candidate nickases fused to the TALE array, TALE-Nt.BspD6I(C) achieved base editing activity at all three target sites when combined with TALE-TadA8e(V106W) (Figure 4B). Nt.BspD6I is a nickase that forms a heterodimer with BspD6I (small subunit, 20 kDa) and functions as a restriction endonuclease called R.BspD6. The Nt.BspD6I(C) domain fused to the TALE array is only the C-terminal cleavage domain (382-604 aa). Compared to TALE-MutH, TALE-Nt.BspD6I(C) showed some degree of non-strand-specific editing at the ND4 site (Figure 4B-4D). This is likely due to imprecise nicking in dsDNA. This editing tool was developed using mitoABE Nt.BspD6I(C) It is called.

[0177] mitoABENt.BspD6I(C) To further characterize the editing patterns of mitoABEs, Nt.BspD6I(C) targeted more diverse mitochondrial DNA sequences. Among these sites, mitoABE Nt.BspD6I(C) The editing efficiency was as high as 40%, and was strand-specific (Figure 4C). Furthermore, when the TALEs of TALE-Nt.BspD6I(C) and TALE-TadA8e(V106W) were swapped, the edited strand was swapped accordingly (Figure 4D). The linker sequence between the TALE and Nt.BspD6I(C) was similar to that of mitoABE. Nt.BspD6I(C) The editing properties of TALE-Nt.BspD6I(C) were not affected by the TALE-Nt.BspD6I(C) (Figure 4E-H). At all sites tested, TALE-Nt.BspD6I(C) was found to nick the same DNA strand that it recognized and edit an adenine in the strand recognized by TALE-TadA8e(V106W).

[0178] Example 5: Mitochondrial C to T editing deaminase-based mitoCBE system This example demonstrates that programmed C-to-T mitochondrial DNA editing can be achieved by fusing the cytidine deaminase APOBEC1 to a TALE array and inducing single-stranded DNA formation. This system utilizes the same strand-specific mechanism as mitoABE and is called mitoCBE.

[0179] The mitoABE results suggest that a similar strategy can be easily applied to other types of deaminases, such as APOBEC1, which is responsible for C-to-T editing of single-stranded DNA. After substituting TadA8e(V106W) into rAPOBEC1 fused with a uracil glycosylase inhibitor (UGI), we achieved C-to-T editing of mitochondria by using a combination of TALE-rAPOBEC1-2×UGI and TALE-MutH, with a maximum editing efficiency of approximately 30% (Figure 5A-5C). MutH Similarly, mitoABE MutHEditing by DbCBE was also strand-specific. At MT-ND4 and MT-RNR2 site 3, the top strand was edited (Figure 5A, 5B), whereas at MT-RNR2 site 1, the bottom strand was edited (Figure 5C). In contrast, conventional DddA-based mitochondrial C-to-T base editors were not strand-specific. Editing by DbCBE, on the other hand, did not favor any particular strand at these three sites (Figure 5D-F).

[0180] Example 6: A to G conversion by the monomeric mitoABE system In this example, we demonstrate that a single TALE array can be used to encode monomeric mitoABE and mitoCBE systems to achieve efficient A-to-G and C-to-T editing, respectively.

[0181] Although placing the nickase and deaminase domains on two separate TALE arrays was beneficial, we wanted to test whether they would also be effective when fused to the same TALE array. Four versions of this mitoABE were designed: TALE-MutH-TadA8e(V106W), TALE-TadA8e(V106W)-MutH, TALE-Nt.BspD6I(C)-TadA8e(V106W), and TALE-TadA8e(V106W)-Nt.BspD6I(C). Monomeric mitoABE, mitoABE MutH , and mitoABE Nt.BspD6I(C) Both mitoABEs achieved efficient A-to-G editing (Figures 6A and 6B). MutH Compared with the monomeric version of mitoABE MutH showed higher editing efficiency at MT-ND1 target sites, and the dimeric version of mitoABE Nt.BspD6I(C)showed even higher editing efficiency. Furthermore, monomeric mitoABEs had a wider editing window than dimeric mitoABEs, and consistent strand bias was observed for both types within the editing window (Figures 6A and 6B). Due to their relatively small size, monomeric mitoBEs are easy to deliver, especially when AAV is used as a vector. Furthermore, monomeric mitoCBEs (mitoCBEs) MutH and mitoCBE Nt.BspD6I(C) ) was successfully constructed, and efficient C to T editing was achieved at the target site (Figures 6C and 6D).

[0182] Example 7: Editing specificity of the mitoBE system This example demonstrates the editing specificity and safety of the mitoBE system. Using mitochondrial DNA sequencing, we demonstrate that the mitoABE system does not induce off-target A-to-G mitochondrial editing or alter mitochondrial copy number.

[0183] To evaluate the editing specificity of mitoBE, we performed mitochondrial DNA sequencing analysis. MutH or mitoABE Nt.BspD6I(C) Mitochondrial DNA sequencing analysis was performed using HEK293T cells transfected with a plasmid expressing mitoABE (Figure 7A), an untreated group, and a mitoABE (Figure 7B) containing no associated TALE array. MutH and mitoABE Nt.BspD6I(C)The non-targeted group (Figures 7B and 7C) containing TALE-deaminase was used as a control group. The average sequencing coverage of the mitochondrial genome was approximately 1193-fold (Figure 7L). Compared with the control groups (untreated and untargeted), mitochondrial DNA sequencing analysis detected only targeted editing, with no non-specific editing detected in any of the experimental groups (Figures 7A-7I). There was no difference between the non-targeted group (Figures 7B, 7C) and the untreated group (Figure 7A), indicating that the free forms of TALE-deaminase or TALE-nickase did not cause undesired off-target effects. Furthermore, the monomeric mitoABE (monomer mitoABE) MutH and mitoABE Nt.BspD6I(C) We also evaluated the editing specificity of mitoABE and found that its specificity was comparable to that of dimeric mitoABE (Figures 7P-7W). This indicates that both monomeric and dimeric mitoABE exhibit high specificity in mitochondrial genome editing. Furthermore, we compared the off-target editing of mitoCBE and DdCBE, which has the same TALE array, and found that mitoCBE reduced off-target editing in the mitochondrial genome, particularly at the target site of MT-ND4 (Figures 7J, 7K, 7X, and 7Y). These results demonstrate that mitoBE is a reliable mitochondrial editing tool that minimizes off-target editing of mitochondrial DNA.

[0184] Mitochondrial gene editing tools such as DdCBE are known to cause off-target effects in the nuclear genome. To investigate whether mitoBE also has off-target effects in the cell nucleus, we performed genome-wide sequencing (average coverage ~58.4x) and identified a target group (mitoABE) MutH and mitoABE Nt.BspD6I(C)The overall off-target editing effects in the target and control groups (including the TALE array) were compared with EGFP and non-targeted controls. No significant differences were observed between the target and control groups (Figures 6M and 6N). Furthermore, genome-wide sequencing data was used to analyze the presence or absence of TALE-dependent off-target effects. No off-target editing was observed within ±200 bp of the TALE array-bound sequence in the nuclear genome (including 0 or 1 mismatch). These results suggest that mitoBE exhibits low off-target effects in the nuclear genome.

[0185] To further evaluate the effects of mitoABE on mitochondria, we measured the copy number and integrity of mtDNA. Using mitochondrial DNA sequencing data, we searched for the presence of insertion or deletion alterations (indels) within mitochondrial DNA. No differences were observed between the target group (Figures 7CC-7HH) and the control group (Figures 7Z-7BB). Real-time quantitative PCR and long-range PCR analyses showed that the copy number and integrity of mitochondrial DNA in the target group were comparable to those in the control group (Figures 7O, 7II). Overall, mitoABE demonstrated high specificity in human cells.

[0186] Example 8: The circular RNA-encoded mitoABE system can achieve strand-specific editing in multiple cell lines. This example demonstrates that a circular RNA delivery system can effectively induce mitochondrial DNA base editing in various human cell lines.

[0187] Direct delivery of RNA has shown promising potential for the treatment of diseases. Because mitoABEs, unlike CRISPR systems, do not require an RNA component to function, mitochondrial editing was tested using circular RNA to encode mitoABEs. The circular RNA-encoded mitoABEs achieved strand-specific editing in multiple human cell types (including H1299, MCF7, Huh7, and RPE1), demonstrating that mitoABEs are a versatile tool compatible with multiple delivery routes for efficient and precise mitochondrial DNA base editing (Figure 8A-C).

[0188] Example 9: Interference with respiratory chain function by editing the start codon of a mitochondrial gene This example demonstrates that the mitoABE system can efficiently edit mitochondrial DNA to create a cell-based model of mitochondrial dysfunction.

[0189] Mitochondrial diseases are genetic disorders caused by mutations in nuclear DNA or mitochondrial DNA and are characterized by abnormalities in oxidative phosphorylation. Approximately 90% of mitochondrial genetic diseases caused by mutations in mitochondrial DNA result from single-base mutations in mitochondrial coding genes. The primary cause of these genetic diseases is defective assembly of the mitochondrial respiratory complex, resulting in reduced ATP production. The mitoABEs encoded by circRNAs targeted the start codons of three genes in HEK293T cells, and these novel editing tools successfully mimicked the phenotypes of actual mitochondrial diseases (Figure 7D). The three target sites selected were MT-ND4, MT-CYB, and MT-CO1. These encode proteins that are components of mitochondrial complex I, mitochondrial complex III, and mitochondrial complex IV, respectively. mitoABE effectively edited all ATG start codons at these three gene sites by changing them from T (actually edited A on the non-coding strand) to C, with editing efficiencies of 34%, 18%, and 36%, respectively (Figures 7E and 7F). Measurement of intracellular ATP levels revealed that editing at all three gene sites resulted in a decrease in intracellular ATP levels (Figures 7G and 7H). Furthermore, respiratory oxygen consumption rates were reduced in cells in which the MT-ND4 start codon was edited (Figure 7I). Altogether, these results demonstrate that mitoABE can efficiently edit DNA to create mitochondrial disease models with defects in oxidative respiration.

[0190] Example 10: Correction of mitochondrial pathogenic DNA mutations using the mitoABE system This example demonstrates that the mitoABE system can correct inherited mitochondrial mutations that cause mitochondrial disease. Importantly, once mutations within mitochondrial oxidative respiratory chain proteins are corrected, physiological efficacy can be demonstrated by measuring ATP content and respiratory oxygen consumption rates in target cells.

[0191] Leber's hereditary optic neuropathy (LHON) is the most common inherited mitochondrial disease, affecting young and middle-aged adults and ultimately leading to acute or subacute blindness. LHON is usually caused by one of three pathogenic mitochondrial DNA (mtDNA) point mutations. These mutations are located at nucleotide positions 11778 G to A, 3460 G to A, and 14484 T to C in the genes for the MT-ND4, MT-ND1, and MT-ND6 subunits of complex I of the mitochondrial oxidative respiratory chain, respectively. The 11778 G to A mutation in MT-ND4 changes a highly conserved arginine to histidine (R340H) and causes 50% of LHON cases in Caucasians and over 90% of LHON cases in Asians. Using circRNA-encoded mitoABE, we investigated the pathogenesis of LHON in GM10742 cells derived from LHON patients. 37 When we targeted the pathogenic mutation (G11778A), we detected a 20% repair efficiency (Figure 7J, K). Importantly, this correction by mitoABE significantly increased the ATP content and respiratory oxygen consumption rate in GM10742 cells (Figure 7L, M). This result demonstrates the strong therapeutic potential of mitoABE in treating LHON disease and many other mitochondrial genetic diseases caused by SNPs. Currently, 97 mtDNA mutations have been associated with human diseases, most of which are point mutations (MITOMAP, Table 1). Of these, 46% are due to A·T to G·C mutations and 41% are due to C·G to T·A mutations. Theoretically, mitoBE may mimic or correct these disease-associated mutations (Figure 8N).

[0192] [Table 1A]

[0193] [Table 1B]

[0194] [Table 1C]

[0195]

Table 1D

Claims

1. 1. A base editor system comprising: a first unit comprising a first double-stranded (ds) DNA-binding polypeptide bound to a single-stranded (ss-) nickase; a second unit comprising a second dsDNA-binding polypeptide bound to a deaminase; The base editor system is configured so that when the first dsDNA-binding polypeptide of the first unit and the second dsDNA-binding polypeptide of the second unit are simultaneously bound to dsDNA, the ss-nickase and the deaminase can be precisely localized to their respective action sites within the same editing region of the dsDNA.

2. The base editor system of claim 1, wherein the ss-nickases of the first unit recognize a recognition sequence present within the editing region of the dsDNA.

3. The base editor system of claim 2, wherein the recognition sequence of the ss-nickase is a palindrome recognition sequence.

4. The base editor system of claim 2 or 3, wherein the recognition sequence of the ss-nickase is 5'-GATC-3'.

5. The base editor system of claim 2, wherein the recognition sequence of the ss-nickase is a non-palindromic recognition sequence.

6. 6. The base editor system of claim 5, wherein the recognition sequence of the ss-nickase is 5'-GATD-3', where D is a base selected from G, A, and T.

7. The base editor system of claim 5, wherein the recognition sequence of the ss-nickase is 5'-GAGTC-3'.

8. The base editor system of any one of claims 2 to 7, wherein the recognition sequence of the ss-nickase is hemimethylated.

9. The base editor system of any one of claims 1 to 8, wherein the first dsDNA-binding polypeptide of the first unit comprises a transcription activator-like effector (TALE) domain.

10. The base editor system of any one of claims 1 to 8, wherein the first dsDNA-binding polypeptide of the first unit comprises a zinc finger (ZF) domain.

11. The base editor system of any one of claims 1 to 10, wherein the first dsDNA-binding polypeptide of the first unit specifically binds to a portion of the dsDNA upstream of the edited region in the dsDNA sequence.

12. The base editor system of any one of claims 1 to 10, wherein the first dsDNA-binding polypeptide of the first unit specifically binds to a portion of the dsDNA downstream of the edited region in the dsDNA sequence.

13. The base editor system of any one of claims 2 to 12, wherein the ss-nickase action site is located on the same strand of dsDNA to which the first dsDNA-binding polypeptide of the first unit is bound.

14. The base editor system of claim 13, wherein the first dsDNA-binding polypeptide of the first unit is bound to a dsDNA position 5 to 9 bases away from the recognition sequence of the ss-nickase.

15. The base editor system of any one of claims 2 to 12, wherein the ss-nickase action site is located on the complementary strand of the dsDNA bound to the first dsDNA-binding polypeptide of the first unit.

16. The base editor system of claim 15, wherein the first dsDNA-binding polypeptide of the first unit is bound to a dsDNA position 0 to 4 bases away from the recognition sequence of the ss-nickase.

17. The base editor system of any one of claims 1 to 16, wherein the ss-nickase is heterologous.

18. The base editor system of any one of claims 1 to 17, wherein the ss-nickase of the first unit is a type I nickase, a type II nickase, a type III nickase, or a type IV nickase.

19. The base editor system according to claims 1 to 18, wherein the ss-nickase is MutH or Nt.BspD6I, or a nickase derived therefrom.

20. The base editor system of any one of claims 1 to 19, wherein the second dsDNA-binding polypeptide of the second unit comprises a transcription activator-like effector (TALE) domain.

21. The base editor system of any one of claims 1 to 19, wherein the second dsDNA-binding polypeptide of the second unit comprises a zinc finger (ZF) domain.

22. The base editor system of any one of claims 1 to 21, wherein the second dsDNA-binding polypeptide is bound to a dsDNA strand to which the first dsDNA-binding polypeptide of the first unit is not bound.

23. The base editor system of claim 11, wherein the second dsDNA-binding polypeptide of the second unit specifically binds to a dsDNA downstream of the edited region in the dsDNA sequence.

24. The base editor system of claim 12, wherein the second dsDNA-binding polypeptide of the second unit specifically binds to a dsDNA upstream of the edited region in the dsDNA sequence.

25. The base editor system of any one of claims 1 to 24, wherein the site of action of the deaminase is part of the unnicked strand of the dsDNA.

26. 26. The base editor system of any one of claims 1 to 25, wherein the deaminase is heterologous.

27. 27. The base editor system of any one of claims 1 to 26, wherein the deaminase is a single-stranded deaminase (ss-deaminase).

28. 28. The base editor system of any one of claims 1 to 27, wherein the deaminase of the second unit is a cytosine to uracil deaminase, a 5-methylcytosine to thymine deaminase, a guanine to xanthine deaminase, an adenine to hypoxanthine deaminase, or an adenine to inosine deaminase.

29. The base editor system of claims 1 to 28, wherein the deaminase is TadA8e, APOBEC, or AID.

30. The base editor system of any one of claims 1 to 29, wherein the edited region in the dsDNA is 1 to 24 base pairs in length.

31. 31. The base editor system of any one of claims 1 to 30, wherein the site of action of the ss-nickase is separated from the site of action of the deaminase by 10 base pairs or less.

32. 32. The base editor system of any one of claims 1 to 31, wherein the first unit is a fusion polypeptide, and a first dsDNA-binding polypeptide of the first unit is fused to an ss-nickase of the first unit.

33. 33. The base editor system of claim 32, wherein the first dsDNA-binding polypeptide of the first unit is fused to the C-terminus of the ss-nickase of the first unit.

34. 33. The base editor system of claim 32, wherein the first dsDNA-binding polypeptide of the first unit is fused to the N-terminus of the ss-nickase of the first unit.

35. The base editor system of any one of claims 32 to 34, wherein the first unit further comprises a linker connecting the first dsDNA-binding polypeptide and the ss-nickase.

36. 36. The base editor system of any one of claims 1 to 35, wherein the second unit is a fusion polypeptide, and a second dsDNA-binding polypeptide of the second unit is fused to a deaminase of the second unit.

37. 37. The base editor system of claim 36, wherein the second dsDNA-binding polypeptide of the second unit is fused to the C-terminus of the deaminase of the second unit.

38. 37. The base editor system of claim 36, wherein the second dsDNA-binding polypeptide of the second unit is fused to the N-terminus of the deaminase of the second unit.

39. The base editor system of any one of claims 36 to 38, wherein the second unit further comprises a linker connecting the second dsDNA-binding polypeptide and the deaminase.

40. 39. The base editor system of claim 35 or 38, wherein the linker of the first unit and / or the linker of the second unit comprises a polypeptide linker.

41. 41. The base editor system of claim 40, wherein the length of the polypeptide linker is 2 to 100 amino acid residues.

42. 32. The base editor system of any one of claims 1 to 31, wherein the first dsDNA-binding polypeptide is non-covalently bound to the ss-nickase of the first unit.

43. 32. The base editor system of any one of claims 1 to 31, wherein the second dsDNA-binding polypeptide is non-covalently bound to the deaminase of the second unit.

44. The base editor system of any one of claims 1 to 43, wherein the first unit further comprises a mitochondrial localization signal (MLS).

45. 45. The base editor system of claim 44, wherein the MLS is located at the N-terminus of the first unit.

46. The base editor system of any one of claims 1 to 45, wherein the second unit further comprises a mitochondrial localization signal (MLS).

47. 47. The base editor system of claim 46, wherein the MLS is located at the N-terminus of the second unit.

48. The base editor system of any one of claims 1 to 47, wherein the dsDNA is a circularized dsDNA.

49. The base editor system of any one of claims 1 to 48, wherein the dsDNA is mitochondrial DNA (mtDNA).

50. The base editor system of any one of claims 1 to 49, wherein the dsDNA has a B-DNA structure.

51. 1. A base editor system comprising: single-stranded (ss-) nickase; Deaminase and a double-stranded (ds) DNA binding polypeptide, The ss-nickase, the deaminase, and the dsDNA-binding polypeptide form a complex, and the complex is configured so that, when the dsDNA-binding polypeptide is bound to dsDNA, the ss-nickase and the deaminase can be precisely localized to their respective sites of action within the same editing region of the dsDNA.

52. A non-naturally occurring polynucleotide, a first unit comprising a first double-stranded (ds) DNA-binding polypeptide bound to a single-stranded (ss-) nickase; and / or A non-naturally occurring polynucleotide encoding a second unit comprising a second dsDNA-binding polypeptide that is bound to the deaminase.

53. A non-naturally occurring polynucleotide, a first unit comprising a first double-stranded (ds) DNA-binding polypeptide bound to a single-stranded (ss-) nickase; a second unit comprising a second dsDNA-binding polypeptide that is bound to the deaminase; The first unit and the second unit are configured such that, when expressed, the first dsDNA-binding polypeptide of the first unit and the second dsDNA-binding polypeptide of the second unit are simultaneously bound to dsDNA, allowing the ss-nickase and the deaminase to be precisely localized to their respective sites of action within the same editing region of the dsDNA.

54. A non-naturally occurring polynucleotide, single-stranded (ss-) nickase; Deaminase and a double-stranded (ds) DNA-binding polypeptide; The non-naturally occurring polynucleotide, wherein the ss-nickase, the deaminase, and the dsDNA-binding polypeptide, when expressed, form a complex, and the complex is configured such that, when the dsDNA-binding polypeptide is bound to the dsDNA, the ss-nickase and the deaminase can be precisely localized to their respective sites of action within the same editing region of the dsDNA.

55. 55. A method of editing a target nucleotide in an edited region of a cell, the method comprising delivering to a cell a base editor system of any one of claims 1-51 or a polynucleotide of any one of claims 52-54.

56. 56. The method of claim 55, wherein the edited region is located in mitochondrial DNA.

57. 1. A method of treating an individual suffering from a disease associated with a DNA mutation, the method comprising: a first unit comprising a first double-stranded (ds) DNA-binding polypeptide that is bound to a single-stranded (ss-) nickase; a second unit comprising a second dsDNA-binding polypeptide bound to a deaminase; the first unit and the second unit are configured such that, when expressed in the individual and bound to dsDNA, the first dsDNA-binding polypeptide of the first unit and the second dsDNA-binding polypeptide of the second unit can precisely localize the ss-nickase and the deaminase to their respective sites of action within the same editing region of the dsDNA.

58. 58. The method of claim 57, wherein the mutant DNA is mitochondrial DNA.

59. 1. A kit for a base editor system, the kit comprising: a first unit comprising a first double-stranded (ds) DNA-binding polypeptide bound to a single-stranded (ss-) nickase, and a second unit comprising a second ds DNA-binding polypeptide bound to a deaminase; The base editor system is configured so that when the first dsDNA-binding polypeptide of the first unit and the second dsDNA-binding polypeptide of the second unit are simultaneously bound to dsDNA, the ss-nickase and the deaminase can be precisely localized to their respective action sites within the same editing region of the dsDNA.

60. 1. A kit for a base editor system, the kit comprising: a first unit comprising a first double-stranded (ds) DNA-binding polypeptide bound to a single-stranded (ss-) nickase; and a second unit comprising a second dsDNA-binding polypeptide that is bound to a deaminase; The base editor system is configured, when expressed, so that when the first dsDNA-binding polypeptide of the first unit and the second dsDNA-binding polypeptide of the second unit are simultaneously bound to dsDNA, the ss-nickase and the deaminase can be precisely localized to their respective action sites within the same editing region of the dsDNA.

61. A non-naturally occurring polypeptide having nickase activity, said non-naturally occurring polypeptide comprising a variant of the amino acid sequence of SEQ ID NO: 1 with E91A and F94A mutations (SEQ ID NO: 2).

62. 62. The non-naturally occurring polypeptide of claim 61, wherein the polypeptide is isolated.

63. 62. A polynucleotide encoding the non-naturally occurring polypeptide of claim 61.

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