Base editor and application thereof

Abstract

The invention discloses a base editor and application thereof, provides a nucleic acid base editor, and particularly relates to a base editor not based on a CRISPR (clustered regularly interspaced short palindromic repeats) technology. The base editor comprises a sequence specific DNA binding protein, a nicking enzyme, an exonuclease and a base specific deaminase. The base editor has single-chain specificity, and compared with a traditional base editor, the base editor has wide applicability in cells and can play a role in cell nuclei and mitochondrial DNA and / or chloroplast DNA. While realizing efficient base editing, the base editor also has the characteristics of high purity of base editing products and few byproducts with insertion and deletion, and is beneficial to being used as an efficient and safe gene editing tool.

Description

[0001] This application is a divisional application of the Chinese Patent Application No. 202311622479.8, filed on November 30, 2023, with the title of “A Base Editor and Its Application”, which claims priority to the Chinese Patent Application No. 202211613160.4, filed on December 15, 2022, with the title of “A Base Editor and Its Application”, and the Chinese Patent Application No. 202311017698.3, filed on August 14, 2023, with the title of “A Base Editor and Its Application”, the contents of which are incorporated herein by reference in their entirety.

[0002] Priority and Related Applications

[0003] This application claims priority to the Chinese Patent Application No. 202211613160.4, filed on December 15, 2022, with the title of “A Base Editor and Its Application”, and the Chinese Patent Application No. 202311017698.3, filed on August 14, 2023, with the title of “A Base Editor and Its Application”, the contents of which are incorporated herein by reference in their entirety. TECHNICAL FIELD

[0004] The present application relates to the field of gene editing, in particular to a nucleic acid base editor, especially to a base editor comprising a sequence-specific DNA binding protein, a nickase, an exonuclease and a base-specific deaminase and its application. BACKGROUND

[0005] It is known that mutations in the genome and mitochondrial DNA can cause various genetic diseases (Newby et al., 2021, Nature 595:295-302), and correcting these mutations is expected to effectively treat or improve some serious diseases. In plants, some important agronomic traits are related to single nucleotide variations (SNVs) occurring in the plant genome, plant mitochondrial genome or plant chloroplast genome; introducing these SNVs into plants can promote plant performance, molecular breeding, restore gene function to alleviate disease states, etc.

[0006] Genome editing has shown great potential for genome modification; among genome editing tools, targeted base substitution without introducing DNA double-strand breaks (DSBs) can be achieved through base editing, thus achieving more precise and accurate editing (Gaudelli et al., 2017, Nature 551:464-471; Komor et al., 2016, Nature 533:420-424), which has broad prospects in disease treatment and crop improvement.

[0007] The cytosine base editor (CBE) (Komor et al., 2016, Nature 533:420-424) and the adenine base editor (ABE) (Gaudelli et al., 2017, Nature 551:464-471) are the most widely used base editors. In the CBE system, a CRISPR-Cas9 nickase (nCas9) with single-stranded DNA cleavage activity is guided by sgRNA to the target dsDNA, nCas9 cleaves the sgRNA-targeted strand, forming an R-loop. Subsequently, a single-stranded specific cytosine deaminase converts cytosine (C) to uracil (U) within a window of about five nucleotides in the single-stranded DNA bubble structure generated by nCas9, and U is replaced by T after DNA repair, resulting in a C:G base pair to T:A base pair conversion. In addition, the addition of a uracil glycosylase inhibitor (UGI) that hinders the uracil excision and its downstream process can improve the base editing efficiency and product purity. Cytosine deaminases suitable for Cas-mediated CBE systems include but are not limited to APOBEC1, hAID, and hAPOBEC3A. Some new deaminase systems have recently been found to be suitable for the deaminase described in the present invention (Huang, J. et al. Discovery of new deaminase functions by structure-based protein clustering. bioRxiv (2023).).

[0008] Fusion of nCas9 with an artificially evolved single-stranded DNA adenine deaminase TadA generates the ABE system (Gaudelli et al., 2017, Nature 551:464-471). The working principle of ABE is similar to CBE, nCas9 will cut a gap on the target strand of DNA under the guidance of sgRNA, and adenine deaminase TadA will convert adenine (A) to inosine (I), which will be replaced by G after DNA repair, resulting in a A:T base pair to G:C base pair conversion. However, the ABE system does not need UGI to improve its editing efficiency or product concentration, because no uracil intermediate is involved in the process.

[0009] The ABE and CBE mentioned above can work effectively in the nucleus, but they cannot work in chloroplasts or mitochondria, because the sgRNA in the CRISPR system cannot be effectively transferred to these organelles.

[0010] In 2020, researchers developed a non-CRISPR base editor system consisting of only protein components. This new base editor system was named DdCBE (Mok et al., 2020, Nature 583:631-637). The core components of DdCBE include a double-stranded DNA cytosine deaminase, DddA, which can convert C to U on double-stranded DNA without the need for CRISPR-Cas9 to create single-stranded DNA. However, the complete DddA is cytotoxic, so it is split into two halves, DddA-N and DddA-C, each fused to a pair of TALE proteins. DddA-N and DddA-C are guided to the target DNA sequence by the TALE pair, and the two recombine to restore their cytosine deaminase activity; similar to the CRISPR-based CBE system, this system can also convert C:G base pairs to T:A base pairs; the addition of UGI can improve the base editing efficiency and product purity of DdCBE. Due to the all-protein component nature of the DdCBE system, it can function both in the nucleus and be transported to chloroplasts and mitochondria, enabling targeted cytosine base editing of chloroplast DNA and mitochondrial DNA.

[0011] However, because DddA toxin is a cytosine deaminase, it can only act on cytosine bases in the CBE system, and cannot act on adenine bases required by the ABE system, severely limiting its range of application. In 2022, researchers fused an artificially directed evolution adenine deaminase TadA-8e with DdCBE to generate a TALED system that can achieve base editing of A to G (Cho et al., 2022, Cell 185:1764-1776). In the TALED system, adenine deaminase TadA-8e is fused to one of the split DddA, and this combination successfully induces simultaneous base conversion of C to T and A to G in mitochondrial DNA. In addition, when the deaminase activity of DddA is inactivated, TadA-8e-mediated base editing of A to G is still effective.

[0012] Although the DdCBE and TALED systems expand the scope of base editing to mitochondrial DNA and / or chloroplast DNA, there are still some limitations. First, due to the intrinsic double-stranded DNA cytosine deaminase activity of DddA, cytosine in the deamination window on both strands will undergo deamination reaction, which means that deamination reaction cannot occur only on the selected single strand, and therefore is not safe and accurate enough for safe use. Second, compared with CBE and ABE-mediated base editing in the nucleus, the base editing product of DddA contains a relatively high indel frequency, resulting in a lower product purity. Third, it has been reported that the DddA-based mitochondrial base editor induces extensive off-target mutations in the nucleus when performing mitochondrial base editing (Lei et al., 2022, Nature 606: 804-811), and it is worth noting that most of the off-target mutations are not TALE-dependent but caused by DddA. A large number of nuclear off-target mutations can have a significant adverse impact on the safety of using these base editors.

[0013] Therefore, there is an urgent need in the art to develop a new type of base editor with single-strand specificity and high product purity that can function on the nucleus, as well as mitochondrial DNA and / or chloroplast DNA. SUMMARY

[0014] To solve the above technical problems, the present application provides a new type of base editor independent of CRISPR technology, which has single-strand specificity, can function on the nucleus, mitochondrial DNA or chloroplast DNA, and can obtain high-purity editing products.

[0015] Specifically, the present application provides a new nucleic acid base editor protein composition, a recombinant expression construct encoding a new synthetic nucleic acid base editor protein, a genetically engineered cell containing one or more recombinant expression constructs encoding a new synthetic nucleic acid base editor protein, and a method of using the above new nucleic acid base editor protein, recombinant expression construct and genetically engineered cell.

[0016] The nucleic acid base editor of the present application comprises: a sequence-specific DNA binding protein; a nickase; an exonuclease; and a base-specific deaminase. In certain embodiments, the nucleic acid base editor further comprises a uracil glycosylase inhibitor. In particular embodiments, the sequence-specific DNA binding protein, the nickase, the exonuclease, and the base-specific deaminase form one or more fusion proteins. In advantageous embodiments of the nucleic acid base editor provided by the present application, the sequence-specific DNA binding protein is selected from a TALE protein, a ZFA protein, a Cas protein, or a meganuclease. And in certain particular embodiments, the sequence-specific DNA binding protein is preferably a TALE protein. In specific embodiments of the nucleic acid base editor of the present application, the nickase is a Fokl nickase. In the nucleic acid base editor of the present application, the deaminase is selected from a cytosine-specific deaminase or an adenine-specific deaminase. In advantageous embodiments of the nucleic acid base editor of the present application comprising a cytosine-specific deaminase, the cytosine deaminase is selected from hAPOBEC3A, rAPOBEC1, hAID, pmCDA1, or a Sdd deaminase. In advantageous embodiments of the nucleic acid base editor of the present application comprising an adenine-specific deaminase, the adenine deaminase is TadA-8e.

[0017] In another preferred embodiment, the compositions provided by the present application comprise one or more recombinant expression constructs encoding a sequence-specific DNA binding protein, a nickase, an exonuclease, a base-specific deaminase, wherein each of the sequence-specific DNA binding protein, the nickase, the exonuclease, and the base-specific deaminase is capable of being expressed in a cell. In certain embodiments, these nucleic acid compositions further comprise a recombinant expression construct encoding a uracil glycosylase inhibitor. In particular embodiments, the compositions comprise one or more recombinant expression constructs encoding a sequence-specific DNA binding protein, a nickase, an exonuclease, a base-specific deaminase as a fusion protein, wherein the fusion protein contained is capable of being expressed in a cell. In advantageous embodiments of the nucleic acid base editors provided herein, the sequence-specific DNA binding protein is selected from a TALE protein, a ZFA protein, a Cas protein, or a meganuclease, and in certain particular embodiments, the sequence-specific DNA binding protein is a TALE protein. In particular embodiments of the nucleic acid base editors of the present application, the nickase is a Fokl nickase. The deaminase in the nucleic acid base editors of the present application is selected from a cytosine-specific deaminase or an adenine-specific deaminase, preferably from a deaminase as set forth in SEQ ID NO. 36-59, 80-86. In advantageous embodiments of the nucleic acid base editors described above comprising a cytosine-specific deaminase, the cytosine deaminase is selected from hAPOBEC3A, rAPOBEC1, hAID, pmCDA1, or a Sdd deaminase. In embodiments of the nucleic acid base editors of the present application comprising an adenine-specific deaminase, the adenine deaminase is TadA-8e.

[0018] In another preferred embodiment, the present application also provides a recombinant cell comprising one or more recombinant expression constructs comprising a coding sequence for a sequence-specific DNA binding protein, a nickase, an exonuclease, and a base- specific deaminase; wherein each of the sequence-specific DNA binding protein, the nickase, the exonuclease, and the base-specific deaminase is capable of being expressed in the cell. In certain embodiments, the recombinant cells contain a nucleic acid composition further comprising a recombinant expression construct encoding a uracil glycosylase inhibitor. In specific embodiments, the recombinant cell comprises one or more recombinant expression constructs encoding a sequence-specific DNA binding protein, a nickase, an exonuclease, a base-specific deaminase as a fusion protein, wherein the contained fusion protein is capable of being expressed in the cell. In advantageous embodiments of the recombinant cells provided herein, the sequence-specific DNA binding protein is selected from a TALE protein, a ZFA protein, a Cas protein, or a meganuclease, and in certain specific embodiments, the sequence-specific DNA binding protein is a TALE protein. In specific embodiments of the recombinant cells provided herein, the nickase is selected as Fokl. Further provided are recombinant cells of the present application comprising one or more recombinant expression constructs encoding a deaminase, wherein the deaminase is a cytosine-specific deaminase or an adenine-specific deaminase, preferably selected from the deaminases as shown in SEQ ID NOs. 36-59, 80-86. Advantageous embodiments of the recombinant cells provided herein comprise one or more recombinant expression constructs encoding a cytosine-specific deaminase, wherein in advantageous embodiments the cytosine deaminase is selected from hAPOBEC3A, rAPOBEC1, hAID, pmCDA1, or a Sdd deaminase. In further advantageous embodiments, the recombinant cell comprises one or more recombinant expression constructs encoding an adenine-specific deaminase, wherein in non-limiting examples the adenine deaminase is selected as TadA-8e.

[0019] In another preferred embodiment, the present application also provides a method of performing base editing in a cell, comprising the step of introducing into the cell a nucleic acid base editor, or a recombinant expression construct encoding a nucleic acid base editor of the present application, or a fusion protein encoding a nucleic acid base editor of the present application. In the practice of the method described herein, the base editing is performed on a target nucleic acid recognized by the specific binding protein and results in a change of a cytosine residue or an adenine residue.

[0020] In another preferred embodiment, the present application provides a nucleic acid base editor with specificity for base editing activity in the nucleus or organelle. Further, a nuclear localization signal (NLS) can be included in the nucleic acid base editor targeting the nucleus. Further, a base editor targeting mitochondria or chloroplast can comprise a mitochondrial targeting sequence (MTS) or a chloroplast transit peptide (CTP), respectively. In these embodiments, the NLS, MTS or CTP can be substituted for each other depending on the specific target organelle or base editor, which will be further elaborated herein.

[0021] The following are exemplary technical solutions of the present application:

[0022] A first object of the present application is to provide a nucleic acid base editor comprising the following components: a) a sequence-specific DNA binding protein; b) a nickase; c) an exonuclease; and d) a base-specific deaminase.

[0023] Preferably, each component of the nucleic acid base editor exists separately or constitutes one or more fusion proteins.

[0024] Preferably, the sequence-specific DNA binding protein is one or more of a TALE protein, a ZFA protein, a Cas protein, and a meganuclease.

[0025] Preferably, the sequence-specific DNA binding protein is a TALE protein.

[0026] Preferably, the nickase is a dimer of a cleavage functional domain monomer of FokI or a mutant thereof, the dimer of a cleavage functional domain monomer of FokI or a mutant thereof is composed of a pair of interacting cleavage functional domain monomers of FokI, and only one of the FokI monomers in the dimer of a cleavage functional domain monomer of FokI or a mutant thereof has DNA endonuclease activity.

[0027] Preferably, the cleavage functional domain monomer of FokI is isolated from a mutant of wild-type FokI protein that is mutated at position 450 and / or 467, or has an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a cleavage functional domain monomer of FokI.

[0028] Further preferably, the mutation renders the FokI monomer DNA endonuclease-inactive.

[0029] Preferably, the cleavage functional domain monomer of FokI, FokICD, is isolated from a mutant of wild-type FokI protein, the mutation makes the FokICD monomer unable to form a dimer with a FokICD monomer containing the same site mutation by self-aggregation.

[0030] Further preferably, the FokICD monomer sequence is selected from SEQ ID No. 87-88.

[0031] Preferably, the amino acid sequence of the cleavage functional domain monomer of FokI, FokICD, is selected from SEQ No. 60-63.

[0032] Preferably, the base-specific deaminase is selected from cytosine-specific deaminase, or adenine-specific deaminase.

[0033] Further preferably, the base deaminase is selected from SEQ ID NO. 36-59, 80-86.

[0034] Further preferably, the base-specific deaminase is cytosine-specific deaminase.

[0035] Further preferably, the cytosine-specific deaminase is one or more of hAPOBEC3A, rAPOBEC1, hAID, pmCDA1, or Sdd deaminase.

[0036] Further, the nucleic acid base editor further contains:

[0037] e) uracil glycosylase inhibitor (UGI);

[0038] and the uracil glycosylase inhibitor exists alone, or forms at least one fusion protein with other nucleic acid base editor components.

[0039] Preferably, the base-specific deaminase is adenine-specific deaminase.

[0040] Preferably, the adenine-specific deaminase is TadA-8e.

[0041] Further, the nucleic acid base editor further contains:

[0042] f) γb;

[0043] The γb forms at least one fusion protein with other nucleic acid base editor components.

[0044] The second object of the present application is to provide a fusion protein as a nucleic acid base editor, the fusion protein containing the protein domain of the base editor as described in the first object.

[0045] Another object of the present application is to provide a fusion protein as a nucleic acid base editor, which is arranged in a linear order from the amino terminal end of the protein, comprising: an exonuclease, an XTEN spacer peptide, a base-specific deaminase, an XTEN spacer peptide, a uracil glycosylase inhibitor (UGI), and a nuclear localization signal.

[0046] Another object of the present application is to provide a fusion protein as a nucleic acid base editor, which is arranged in a linear order from the amino terminal end of the protein, comprising: an exonuclease, a 48-amino acid spacer peptide, a base-specific deaminase, an XTEN spacer peptide, a uracil glycosylase inhibitor (UGI), and a nuclear localization signal.

[0047] Another object of the present application is to provide a composition of fusion proteins having nucleic acid base editor activity, the composition comprising:

[0048] a first fusion protein comprising: a nuclear localization signal (NLS), a sequence-specific DNA binding protein, and a base-specific deaminase;

[0049] a second fusion protein comprising: an exonuclease, and a nuclear localization signal (NLS); and,

[0050] a third fusion protein comprising: a uracil glycosylase inhibitor (UGI), and a nuclear localization signal (NLS).

[0051] Another object of the present application is to provide a composition of fusion proteins having nucleic acid base editor activity, the composition comprising:

[0052] a first fusion protein, arranged in a linear order from the amino terminal end of the protein, comprising: a nuclear localization signal (NLS), a base-specific deaminase, a TALE-L protein, a FokI-L D450A protein, a T2A sequence, an NLS, a TALE-R protein, and a FokI-R protein;

[0053] a second fusion protein comprising: an exonuclease, and a nuclear localization signal (NLS); and,

[0054] a third fusion protein comprising: a uracil glycosylase inhibitor (UGI), and a nuclear localization signal (NLS).

[0055] Another object of the present application is to provide a composition of fusion proteins having nucleic acid base editor activity, the composition comprising:

[0056] a first fusion protein, arranged in a linear order from the amino terminal end of the protein, comprising: a nuclear localization signal (NLS), a TALE-L protein, a FokI-L D450Aa protein, a T2A sequence, an NLS, a base-specific deaminase, a 48-amino acid spacer peptide, a TALE-R protein, and a FokI-R protein;

[0057] a second fusion protein comprising: an exonuclease and a nuclear localization signal (NLS); and,

[0058] a third fusion protein comprising: a uracil glycosylase inhibitor (UGI) and a nuclear localization signal (NLS).

[0059] Another object of the present application is to provide a composition of fusion proteins having nucleic acid base editor activity, the composition comprising:

[0060] a first fusion protein comprising: a nuclear localization signal (NLS), a sequence-specific DNA binding protein, a base-specific deaminase, and a uracil glycosylase inhibitor (UGI); and,

[0061] a second fusion protein comprising: an exonuclease and a nuclear localization signal (NLS).

[0062] Another object of the present application is to provide a composition of fusion proteins having nucleic acid base editor activity, the composition comprising:

[0063] a first fusion protein, arranged in a linear order from the amino-terminal end of the protein, comprising: a nuclear localization signal (NLS), a base-specific deaminase, a 48-amino acid spacer peptide, a TALE-L protein, a FokI-L D450A a protein, a T2A sequence, an NLS, a TALE-R protein, a FokI-R protein, a 4-amino acid spacer peptide, and a uracil glycosylase inhibitor (UGI); and,

[0064] a second fusion protein comprising: an exonuclease and a nuclear localization signal (NLS).

[0065] Another object of the present application is to provide a composition of fusion proteins having nucleic acid base editor activity, the composition comprising:

[0066] a first fusion protein, arranged in a linear order from the amino-terminal end of the protein, comprising: a nuclear localization signal (NLS), a uracil glycosylase inhibitor (UGI), a 4-amino acid spacer peptide,

[0067] a base-specific deaminase, a 48-amino acid spacer peptide, a TALE-L protein, a FokI-L D450A a protein, a T2A sequence, an NLS, a TALE-R protein, a FokI-R protein; and,

[0068] a second fusion protein comprising: an exonuclease and a nuclear localization signal (NLS).

[0069] Another object of the present application is to provide a composition of fusion proteins having nucleic acid base editing activity and capable of base editing on mitochondria, the composition comprising:

[0070] a first fusion protein, arranged in a linear order from the amino terminus of the protein, comprising: a mitochondrial targeting sequence (MTS), a TALE-L protein, and a FokI-L D450A protein;

[0071] a second fusion protein, arranged in a linear order from the amino terminus of the protein, comprising: a mitochondrial targeting sequence (MTS), a TALE-R protein, and a FokI-R protein;

[0072] a third fusion protein, arranged in a linear order from the amino terminus of the protein, comprising: a mitochondrial targeting sequence (MTS), and an exonuclease;

[0073] a fourth fusion protein, arranged in a linear order from the amino terminus of the protein, comprising: a mitochondrial targeting sequence (MTS), and a base-specific deaminase; and,

[0074] a fifth fusion protein, arranged in a linear order from the amino terminus of the protein, comprising: a mitochondrial targeting sequence (MTS), and a uracil glycosylase inhibitor (UGI).

[0075] Another object of the present application is to provide a composition of fusion proteins having nucleic acid base editing activity and capable of base editing on mitochondria, the composition comprising:

[0076] a first fusion protein, arranged in a linear order from the amino terminus of the protein, comprising: a mitochondrial targeting sequence (MTS), a TALE-L protein, and a FokI-L D450A protein;

[0077] a second fusion protein, arranged in a linear order from the amino terminus of the protein, comprising: a mitochondrial targeting sequence (MTS), a TALE-R protein, and a FokI-R protein;

[0078] a third fusion protein, arranged in a linear order from the amino terminus of the protein, comprising: a mitochondrial targeting sequence (MTS), a γb, and an exonuclease;

[0079] a fourth fusion protein, arranged in a linear order from the amino terminus of the protein, comprising: a mitochondrial targeting sequence (MTS), and a base-specific deaminase; and,

[0080] A fifth fusion protein, arranged in a linear order starting from the amino terminus of the protein, comprising: a mitochondrial targeting sequence (MTS), a gammadelta and a uracil glycosylase inhibitor (UGI).

[0081] Another object of the present application is to provide a composition of a fusion protein having nucleic acid base editor activity, the composition comprising:

[0082] A first fusion protein, comprising: a nuclear localization signal (NLS) / chloroplast transit peptide (CTP) / mitochondrial targeting sequence (MTS), a sequence-specific DNA binding protein, and a nickase;

[0083] A second fusion protein, comprising: an exonuclease and a nuclear localization signal (NLS) / chloroplast transit peptide (CTP) / mitochondrial targeting sequence (MTS); and,

[0084] A third fusion protein, comprising: a base-specific deaminase, a uracil glycosylase inhibitor (UGI), and a nuclear localization signal (NLS) / chloroplast transit peptide (CTP) / mitochondrial targeting sequence (MTS).

[0085] Another object of the present application is to provide a composition of a fusion protein having nucleic acid base editor activity, the composition comprising:

[0086] A first fusion protein, arranged in a linear order starting from the amino terminus of the protein, comprising: a nuclear localization signal (NLS) / chloroplast transit peptide (CTP) / mitochondrial targeting sequence (MTS), a TALE-L protein, a FokI-L D450A protein, a T2A sequence, an NLS, a TALE-R protein, a FokI-R protein, or a TALE-L protein, a FokI-L protein, a T2A sequence, an NLS, a TALE-R protein, a FokI-R D450A protein, a TALE-L protein, a FokI-L protein, a T2A sequence, an NLS, a TALE-R protein, a FokI-R

[0087] A second fusion protein, arranged in a linear order starting from the amino terminus of the protein, comprising: a nuclear localization signal (NLS) / chloroplast transit peptide (CTP) / mitochondrial targeting sequence (MTS) and an exonuclease; and,

[0088] A third fusion protein, arranged in a linear order starting from the amino terminus of the protein, comprising: a nuclear localization signal (NLS) / chloroplast transit peptide (CTP) / mitochondrial targeting sequence (MTS), a base-specific deaminase, an XTEN spacer peptide, and a uracil glycosylase inhibitor (UGI).

[0089] Another object of the present application is to provide a composition of a fusion protein having nucleic acid base editing activity and capable of performing base editing on mitochondria, characterized in that the composition comprises:

[0090] a first fusion protein arranged in a linear order from the amino terminus of the protein, comprising: a mitochondrial targeting sequence (MTS), a base-specific deaminase, a 48-amino acid spacer peptide, a TALE-L protein, a FokI-L D450A protein, an 11-amino acid spacer peptide, and a uracil glycosylase inhibitor (UGI); and,

[0091] a second fusion protein arranged in a linear order from the amino terminus of the protein, comprising: a mitochondrial targeting sequence (MTS), a 48-amino acid spacer peptide, a TALE-R protein, a uracil glycosylase inhibitor (UGI), a 14-amino acid spacer peptide, and a FokI-R protein.

[0092] Another object of the present application is to provide a recombinant expression construct for nucleic acid base editing, the recombinant expression construct being used to express the nucleic acid base editor of the above first object or the fusion protein or composition of the above other objects.

[0093] Another object of the present application is to provide a genetically engineered cell for transforming the recombinant expression construct of the above object.

[0094] Another object of the present application is to provide a method for performing nucleic acid base editing in a cell, introducing the nucleic acid base editor or the recombinant expression construct of the above object into the cell, and editing a target gene.

[0095] Preferably, the target gene is selected from nuclear genomic DNA, mitochondrial genomic DNA, or chloroplast genomic DNA.

[0096] Further preferably, the target gene is nuclear genomic DNA, and the nucleic acid base editor further comprises a nuclear localization signal (NLS).

[0097] Further preferably, the target gene is mitochondrial genomic DNA, and the nucleic acid base editor further comprises a mitochondrial targeting sequence (MTS).

[0098] Further preferably, the target gene is chloroplast genomic DNA, and the nucleic acid base editor further comprises a chloroplast transit peptide (CTP).

[0099] Another object of the present application is to fuse γb to the end of each component.

[0100] Further preferably, the γb is fused to UGI and Trex2, respectively.

[0101] Another object of the present application is to provide an application of the base editor in base editing, using the base editor, fusion protein, composition, recombinant expression construct, genetically engineered cell or method as described in the above objects for base editing of DNA in a cell, which is a mammalian cell, a bacterium, a protist, a fungus, an insect cell, a yeast, an unconventional yeast or a plant cell.

[0102] Preferably, the plant cell is derived from a whole plant, seedling, meristem, ground tissue, vascular tissue, epidermal tissue, seed, leaf, root, shoot, stem, flower, fruit, stolon, bulb, tuber, corm, vegetative offshoot, bud, sprout, or tumor tissue of a monocotyledonous or dicotyledonous plant.

[0103] Preferably, the mammalian cell is selected from a human reproductive cell, neuron, muscle cell, endocrine / exocrine cell, epithelial cell, muscle cell, tumor cell, embryonic cell, hematopoietic cell, bone cell, germ line cell, somatic cell, stem cell, pluripotent stem cell, induced pluripotent stem cell, progenitor cell, meiotic cell, and mitotic cell.

[0104] Preferably, the base editing is performed on a nuclear genome or a organelle genome using the editor.

[0105] Preferably, the organelle is a mitochondrion or a chloroplast.

[0106] Another object of the present application is to provide a use of the base editor, fusion protein, composition, recombinant expression construct, genetically engineered cell as described in the above objects in the preparation of a pharmaceutical composition for treating a disease in a subject in need thereof.

[0107] Another object of the present application is to provide a pharmaceutical composition for treating a disease in a subject in need thereof, which comprises the base editor, fusion protein, composition, recombinant expression construct, genetically engineered cell as described in the above objects, and, optionally, a pharmaceutically acceptable carrier.

[0108] Another object of the present application is to provide a method for producing a genetically modified plant, which is characterized in that the method comprises introducing the base editor, fusion protein, composition, recombinant expression construct, genetically engineered cell as described in the above objects into at least one of the plants.

[0109] The base editor and its application provided by the present application have the following beneficial effects:

[0110] (1) The base editor of the present application only occurs base editing on selected single strands, has good safety and accuracy.

[0111] (2) The base editor of the present application has high editing product purity and low insertion-deletion byproduct rate, and has excellent editing efficiency.

[0112] (3) The base editor of the present application has low off-target rate, effectively improving its therapeutic effect and safety.

[0113] (4) The base editor of the present application is not based on CRISPR technology, has a wider application range and scene, and its components can function in cell nucleus or mitochondria, chloroplast and other organelles. BRIEF DESCRIPTION OF DRAWINGS

[0114] In order to better understand the technical solutions disclosed in the present application, the following drawings will be described.

[0115] Figure 1 The figure is a functional diagram of the nucleic acid base editor of the present application, wherein in the first step, the sequence-specific DNA binding protein (SSDBP) is positioned and combined to the target DNA sequence; in the second step, the nicking enzyme preferentially cuts one DNA strand at the target site, and then the exonuclease digests the cut DNA strand to the SSDBP binding site. This will expose a ssDNA fragment in the complementary strand, which will then become the substrate of the deaminase to realize deamination, and then cause the corresponding base to change after DNA repair (C:G pair to T:A pair or A:T pair to G:C pair, the conversion type depends on the deaminase used).

[0116] Figure 2A And Figure 2B The figure is the application effect of the nucleic acid base editor of the present application in high-purity base editing in the nucleus of rice cells. Among them Figure 2A The figure is the C to T base editing efficiency of the OsBADH2 target in different treatment methods in rice protoplast. Figure 2B The figure is the C to T base editing efficiency and the frequency of generating insertion-deletion (indel) byproducts of the OsBADH2 target in different treatment methods in rice protoplast.

[0117] Figure 3A And Figure 3B The figure is the analysis of the base editing window of the base editor of the present application. After the nucleic acid base editor of the present application is transformed into rice protoplast, DNA is extracted, and high-throughput sequencing is performed on the target site to obtain the editing efficiency of different bases on the target sequence. Figure 3A The figure is a schematic diagram of the OsBADH2 target sequence. The gray sequences on both sides are TALE binding sites, and the black region in the middle is a spacer sequence. Figure 3BTo analyze the base editing window of the base editor according to the high-throughput sequencing result. Wherein, CK is a blank control without transforming any plasmid, TALEN WT and TALEN WT +ExoI is a combination of only transforming wild-type TALEN or TALEN+ exonuclease ExoI, and the two treatments are used as negative controls.

[0118] Figure 4A and Figure 4B It is shown that after the base editor of the application is transformed into rice protoplast to target OsDEP1, the editing efficiency of cytosine nucleotide at the target point ( Figure 4A ) and the frequency of indel byproduct ( Figure 4B ) are analyzed by high-throughput sequencing. Wherein, CK is a blank control without transforming any plasmid, TALEN WT and TALEN WT +ExoI is a combination of only transforming wild-type TALEN or TALEN+ exonuclease ExoI, and the two treatments are used as negative controls.

[0119] Figure 5A and Figure 5B It is shown that the implementation effect of the base editor using combinations containing different FokI nicking enzymes, different exonucleases, and cytosine deaminases. Different editing windows are produced when using different digestion directions of exonucleases; different DNA single strands at the target are specifically edited using different nicking enzymes ( Figure 5A ). And the analysis of the editing product purity and byproduct frequency of the base editor of the application under different combinations ( Figure 5B ).

[0120] Figure 6A and Figure 6B It is shown that the base editing efficiency and indel byproduct frequency detected by high-throughput sequencing of the target sequence (OsBADH2 in rice protoplast) introduced by the base editor of the application including the combination of cytosine deaminase and exonuclease. Wherein the exonuclease is 5' exonuclease or 3' exonuclease.

[0121] Figure 7A and Figure 7B It is shown that the base editing efficiency and editing window detected by high-throughput sequencing of the target sequence (OsBADH2 in rice protoplast) introduced by the base editor of the application including the combination of different cytosine deaminases and exonucleases.

[0122] Figure 8The base editing efficiency detected by high-throughput sequencing is shown by introducing the target sequence (OsCKX2 in rice protoplasts) using the base editor containing adenine deaminase of the present invention.

[0123] Figure 9 This is a schematic diagram of the base editor of the present invention, which comprises a fusion protein of an exonuclease, a deaminase, a uracil DNA glycosylase inhibitor, and a nuclear localization signal (NLS) separated by an XTEN spacer peptide or a 48-amino acid spacer peptide.

[0124] Figure 10A and Figure 10B The base editors of different constructs of this invention introduce the target sequence (OsDEP1 in rice protoplasts), and the base editing efficiency is detected by high-throughput sequencing. Figure 10A ) and different base editor editing windows ( Figure 10B ).

[0125] Figure 11A and Figure 11B This is a schematic diagram of a base editor for a vector containing the deaminase-TALE fusion protein of the present invention. In each embodiment, the NLS-exonuclease fusion protein and the NLS-uracil glycosylation inhibitor (UGI) are provided in separate vectors.

[0126] Figure 12A and Figure 12B This demonstrates the introduction of the target sequence (OsDEP1 in rice protoplasts) by the base editor of the fusion protein of this invention. Figure 12A OsCKX2 in rice protoplasts Figure 12B A bar chart showing the base editing rate and insertion / deletion rate of deaminase-TALE-FokI-R. nickase The results of the protein fusion protein are shown in Figure 12A Deaminase-TALE-FokI-L nickase The results of the protein fusion protein are shown in Figure 12B As shown.

[0127] Figure 13A and Figure 13B This is a schematic diagram of the base editor containing the deaminase-TALE fusion protein of the present invention. In each embodiment, the fusion protein of NLS and exonuclease is provided in a separate vector.

[0128] Figure 14 Using the base editor of this invention, such as Figure 13A and Figure 13B The fusion protein and the individual expression of each component show the base editing efficiency of the target sequence (OsDEP1 in rice protoplasts).

[0129] Figure 15A A schematic diagram of a vector for use of the base editor of the present application in mitochondrial editing, comprising a construct expressing MTS-Deaminase, MTS-UGI, MTS-TALE-R-Fokl-R (or MTS-TALE-R-Fokl-R D450A , MTS-TALE-L-Fokl-L D450A (or MTS-TALE-L-Fokl-L) nickase and MTS-Exonuclease.

[0130] Figure 15B A schematic diagram of a target sequence for use of the base editor of the present application Figure 15A , showing the TALE-R and TALE-L binding sites and cytosine residues targeted by certain nucleic acid base editors of the present application, i.e. a schematic diagram of the mitochondrial ND6 target sequence and TALE binding sites.

[0131] Figure 15C A schematic diagram of the base editor of the present application Figure 15A for use of the construct shown in

[0132] Figure 16A - Figure 16E A representative diagram of a recombinant expression construct encoding a base editor used in the examples described herein in rice, in Figure 16A - Figure 16E , FokK-L-nickase corresponds to FOKI-L; Fokl-R corresponds to FOKI-R (D450A / D467A).

[0133] Figure 16A represents a recombinant expression construct encoding a wild-type TALEN used in Example 2 and other examples (NLS-TALEN WTFigure 1 is a schematic diagram of a vector (i.e., a vector schematic diagram, using TALEs targeting OsBADH2 as an example) that can cause double-strand breaks in the target DNA and randomly induce indel mutations, which are used as a control in various embodiments. In this construct, a stable expression T-DNA vector with a UBI promoter from maize and a Nos terminator is used to drive the expression of wild-type TALENs (including TALE-L-Fokl-L and TALE-R-Fokl-R fusion proteins, where Fokl does not contain D450A or D467A mutations), where the N- and C-terminal regions of the TALEs contain the corresponding truncations (AN152 / C63) flanking the DNA-binding domain of the TALE. The TALE-L-Fokl-L and TALE-R-Fokl-R fusion proteins are linked by a T2A self-cleaving peptide. Other components shown in the figure include a CaMV 35S promoter (a cauliflower mosaic virus-derived promoter), a hygromycin resistance selection gene Hyg, a nopaline synthase terminator Nos of Agrobacterium tumefaciens, etc.

[0134] Figure 16B Figure 2 is a schematic diagram of a recombinant expression construct containing sequence-specific DNA binding proteins TALE-L, TALE-R, and nicking enzyme Fokl nickase (i.e., a vector schematic diagram, using TALEs targeting OsBADH2 as an example; corresponding TALE coding sequences can be designed according to the target sequence), as well as two additional constructs: NLS-deaminase-UGI and exonuclease-NLS. These constructs contain a UBI promoter from maize and a Nos terminator, which drive the expression of the deaminase-UGI fusion protein and the exonuclease, respectively. UGI, a uracil-DNA glycosylase inhibitor from a Bacillus subtilis phage, protects uracils in DNA by irreversibly inhibiting the key DNA repair enzyme uracil-DNA glycosylase. Other components shown in the figure include a CaMV 35S promoter (a cauliflower mosaic virus-derived promoter), a hygromycin resistance selection gene Hyg, a nopaline synthase terminator Nos of Agrobacterium tumefaciens, a CaMV polyadenylation signal terminator.

[0135] Figure 16Cis a schematic of a recombinant expression construct comprising a fusion protein of sequence-specific DNA binding protein TALE-L, TALE-R, nickase Fokl nickase, and deaminase (i.e., a vector schematic comprising a nickase, exonuclease, deaminase, and uracil glycosylase inhibitor, with TALEs targeting OsBADH2 as an example; corresponding TALE coding sequences can be designed according to the target sequence), and two additional constructs: UGI-NLS and exonuclease-NLS. The recombinant expression constructs UGI-NLS and exonuclease-NLS each have a UBI promoter and a CaMV terminator to drive expression of UGI and exonuclease, respectively. UGI, a uracil-DNA glycosylase inhibitor from a bacteriophage of Bacillus subtilis, protects uracil in DNA by irreversibly inhibiting the key DNA repair enzyme uracil-DNA glycosylase. Other components shown in the figure include a CaMV 35S promoter (a cauliflower mosaic virus-derived promoter), a hygromycin resistance selection gene Hyg, a nopaline synthase terminator Nos of Agrobacterium tumefaciens, and a CaMV polyadenylation signal terminator.

[0136] Figure 16D is a schematic of a recombinant expression construct comprising a fusion protein of sequence-specific DNA binding protein TALE-L, TALE-R, nickase Fokl nickase, deaminase, and UGI (i.e., a vector schematic comprising NLS-deaminase-TALE-L-Fokl- nickase -TALEN-R-UGI, exonuclease-NLS, with TALEs targeting OsBADH2 as an example; corresponding TALE coding sequences can be designed according to the target sequence), and one additional construct: exonuclease-NLS. The recombinant expression construct exonuclease-NLS has a UBI promoter and a CaMV terminator to drive expression of exonuclease. UGI, a uracil-DNA glycosylase inhibitor from a bacteriophage of Bacillus subtilis, protects uracil in DNA by irreversibly inhibiting the key DNA repair enzyme uracil-DNA glycosylase. Other components shown in the figure include a CaMV 35S promoter (a cauliflower mosaic virus-derived promoter), a hygromycin resistance selection gene Hyg, a nopaline synthase terminator Nos of Agrobacterium tumefaciens, and a CaMV polyadenylation signal terminator.

[0137] Figure 16E is a schematic of a recombinant expression construct comprising a fusion protein of sequence-specific DNA binding protein TALE-L, TALE-R, nickase Fokl nickase, deaminase, exonuclease, and UGI (i.e., a vector schematic comprising NLS-deaminase-TALE-L-Fokl- nickaseTALEN-R-UGI-Exonuclease vector schematic, with TALE targeting OsBADH2 as an example, the TALE coding sequence can be designed according to the target sequence, and the additional features of UGI and exonuclease are encoded in the construct rather than introduced into the cell in a separate construct.

[0138] Figure 17A to Figure 17H is a representative illustration of a recombinant expression construct encoding a base editor for use in the embodiments described herein for mitochondrial editing in human cells.

[0139] Figure 17A is a representative of the mitochondrial recombinant expression construct MTS-TALE-L-FokI-L (schematic of MTS-TALE-L-FokI-L vector targeting mitochondrial ND6). The TALE sequence in this construct can be replaced according to the target. The expression vector MTS-TALE-L-FokI-L has a CMV promoter and a bGH poly(A) signal terminator to drive the expression of the MTS-TALE-L-FokI-L fusion protein, in which the N and C terminal regions of TALE contain the corresponding truncations (ΔN152 / C63) and are located on the flanks of the DNA binding domain of TALE (see Mok et al., 2020, Nature 583:631-637). MTS is a mitochondrial targeting sequence from human superoxide dismutase 2, which helps to transport proteins into mitochondria. The CMV promoter is a human herpesvirus 5-derived promoter that has been shown to be highly active in animal cells. The CMV enhancer is a fragment containing the region of the cytomegalovirus promoter that enhances the transcription efficiency of the CMV promoter. The bGH poly(A) signal is a growth hormone polyadenylation signal-derived terminator.

[0140] Figure 17Bis a representative of the mitochondrial recombinant expression construct MTS-TALE-R-Fokl-R (schematic diagram of MTS-TALE-R-Fokl-R vector targeting mitochondria ND6). The TALE sequence therein can be replaced according to the target point. The expression vector MTS-TALE-R-Fokl-R has a CMV promoter and a bGH poly(A) signal terminator to drive the expression of the MTS-TALE-R-Fokl-R fusion protein, wherein the N and C terminal regions of the TALE contain the corresponding truncation (AN152 / C63) and are located on the flanks of the DNA binding domain of the TALE (see Mok et al., 2020, Nature 583:631-637). The MTS in the vector is the mitochondrial targeting sequence of cytochrome c oxidase subunit 8, which helps to transport the protein into the mitochondria. The CMV promoter is a human herpes virus 5 derived promoter, which has been proven to be highly active in animal cells. The CMV enhancer is a fragment containing the CMV promoter region, which can enhance the transcription efficiency of the CMV promoter. The bGH poly(A) signal is a growth hormone polyadenylation signal derived terminator.

[0141] Figure 17C is a schematic diagram of the mitochondrial recombinant expression construct MTS- deaminase (schematic diagram of MTS-deaminase vector). The recombinant expression construct has a CMV promoter and a bGH poly(A) signal terminator, which can drive the expression of MTS-deaminase in human mitochondria. The MTS, CMV promoter, CMV enhancer and bGH poly(A) signal terminator are as described in Figure 17A .

[0142] Figure 17D is a representative of the mitochondrial recombinant expression construct MTS- deaminase (schematic diagram of MTS-deaminase vector). The recombinant expression construct has a CMV promoter and a bGH poly(A) signal terminator, which can drive the expression of MTS-deaminase in human mitochondria. The MTS, CMV promoter, CMV enhancer and bGH poly(A) signal terminator are as described in Figure 17A .

[0143] Figure 17E is a representative of the mitochondrial recombinant expression construct MTS- deaminase (schematic diagram of MTS-deaminase vector). The recombinant expression construct has a CMV promoter and a bGH poly(A) signal terminator, which can drive the expression of MTS-deaminase in human mitochondria. The MTS, CMV promoter, CMV enhancer and bGH poly(A) signal terminator are as described in Figure 17A .

[0144] Figure 17F is a schematic of the mitochondrial recombinant expression construct MTS-UGI-exonuclease-TALE-R-FokI-R (MTS-UGI-exonuclease-TALE-R-FokI-R vector schematic.). The recombinant expression construct MTS-UGI-exonuclease-TALE-R-FokI-R has a CMV promoter and a bGH poly(A) signal terminator to drive expression of the MTS-UGI-exonuclease-TALE-R fusion protein. Components such as the MTS, CMV promoter, CMV enhancer, and bGH poly(A) signal terminator are as described in Figure 17A .

[0145] Figure 17G is a schematic of the mitochondrial recombinant expression construct MTS-UGI-exonuclease-TALE-R-FokI-R (MTS-UGI-exonuclease-TALE-R-FokI-R vector schematic.). The recombinant expression construct MTS-UGI-exonuclease-TALE-R-FokI-R has a CMV promoter and a bGH poly(A) signal terminator to drive expression of the MTS-UGI-exonuclease-TALE-R fusion protein. Components such as the MTS, CMV promoter, CMV enhancer, and bGH poly(A) signal terminator are as described in Figure 17B .

[0146] Figure 17H is a schematic of the mitochondrial recombinant expression construct MTS-UGI-exonuclease-TALE-R-FokI-R (MTS-UGI-exonuclease-TALE-R-FokI-R vector schematic.). The recombinant expression construct MTS-UGI-exonuclease-TALE-R-FokI-R has a CMV promoter and a bGH poly(A) signal terminator to drive expression of the MTS-UGI-exonuclease-TALE-R fusion protein. Components such as the MTS, CMV promoter, CMV enhancer, and bGH poly(A) signal terminator are as described in Figure 17B .

[0147] Figure 18 is a schematic of the CyDENT structure for nuclear genome editing.

[0148] Figure 19A is the C-to-T conversion frequency and indel frequency of nuCyDENT-R and TALEN at the OsDEP1, OsSD1, OsCKX2, and OsBADH2 loci in rice protoplasts.

[0149] Figure 19Bis the base editing window of CyDENT at OsDEP1, OsSD1, OsCKX2 and OsBADH2 loci in rice protoplast. The gray area represents the TALE binding site, and the middle area is the spacer.

[0150] Figure 20 is the base editing of CyDENT at OsCKX2 and OsSD1 loci in rice protoplast. The gray area is the TALE binding site.

[0151] Figure 21 is the base editing of CyDENT at the SIRT6 locus in humans. The gray area is the TALE binding site.

[0152] Figure 22A is a schematic diagram of the modular CyDENT construct used in chloroplast genome editing, taking cpCyDENT-R as an example.

[0153] Figure 22B is the base editing window of CyDENT at the OsrbcL locus in rice protoplast. The gray area is the TALE binding site.

[0154] Figure 23A is a schematic diagram of the modular CyDENT structure used in mitochondria. Take mtCyDENT-R as an example.

[0155] Figure 23B is the base editing of mtCyDENT-L or mtCyDENT-R with various γb fusion states at the mitochondrial ND6 locus in HEK293T cells.

[0156] Figure 24 is the editing frequency of DdCBE, mtCyDENT-R, mtCyDENT1b-R, mtCyDENT-L and mtCyDENT1b-L at the mitochondrial ND1.2, ND1.3, ND3 and ND6.2 loci in HEK293T cells.

[0157] Figure 25 is the indel frequency of DdCBE, mtCyDENT1b-R and mtCyDENT1b-L at different loci in the mitochondria of HEK293T cells.

[0158] Figure 26 is the base editing site of mtCyDENT at different loci in the mitochondria of HEK293T cells. The gray area is the TALE binding site.

[0159] Figure 27Figure 1 is the editing frequency of mtCyDENTl b-L and mtCyDENTl b-R at HEK293T cell ND5.1, ND6 and ND1.3 sites using Sdd7 deaminase.

[0160] Figure 28A Figure 2 is a schematic of mtCyDENT2 construct in mitochondrial genome.

[0161] Figure 28B Figure 3 is the efficiency of base editing and the proportion of various editing events of DdCBE and mtCyDENT2-L, mtCyDENT2-R containing different deaminases at ND6 site in HEK293T cells.

[0162] Figure 29 Figure 4 is the editing frequency and editing strand bias of DdCBE and mtCyDENT2-L containing Sdd3 deaminase at HEK293T cell ND1.2 and ND6.2 sites. The gray color indicates TALE binding site.

[0163] Figure 30 Figure 5 is the editing strand bias of mtCyDENT2-L (Sdd3 deaminase + TALE-L1 + TALE-R1) designed for the pathogenic mutation at Leigh's syndrome ND6.2 site at HEK293T cell ND6.2 site.

[0164] Figure 31A Figure 6 is WGS and NGS analysis of target editing frequency at ND3 and ND6.2

[0165] Figure 31B Figure 7 is the Logo plot of off-target C:G to T:A and G:C to A:T base conversion for each editor.

[0166] Figure 31C Figure 8 is the SNV and indel frequency distribution in potential TALE-dependent off-target sites. DETAILED DESCRIPTION

[0167] Terminology:

[0168] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0169] Numerical ranges include the numbers defining the range and expressly include each integer and non-integer fraction of the range. All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art unless otherwise defined.

[0170] The term "structure," "recombinant expression structure," or "recombinant expression construct" as used herein refers to an artificially designed piece of DNA that can be used to introduce genetic material into a target cell (e.g., a recombinant expression structure is used to produce a base editor or component thereof). The term "expression" refers to the transcription and translation of a nucleic acid coding sequence, resulting in the production of a polypeptide.

[0171] The term "genetically engineered" as used herein refers to the alteration of the genetic makeup of a cell through biotechnology, including gene transfer within and across species, to produce a modified or non-naturally occurring cell. In a specific use of this term, the construct encodes a base editor or component thereof and the genetically engineered cell produces the base editor. A cell that contains a foreign, recombinant, synthetic, and / or otherwise modified polynucleotide is considered a genetically engineered cell and, therefore, is non-naturally occurring relative to any naturally occurring counterpart. In some cases, a genetically engineered cell comprises one or more recombinant nucleic acids. In other cases, a genetically engineered cell comprises one or more synthetic or genetically engineered nucleic acids (e.g., a nucleic acid comprises at least one human- made insertion, deletion, inversion, or substitution relative to the sequence of its naturally occurring counterpart). Methods for producing genetically engineered cells are known in the art, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual (Fourth Edition), Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2012).

[0172] The term "genetically engineered cell" or "genetically engineered host cell" or "recombinant expression host cell" as used herein can be a cell that has been modified with gene editing technology. Gene editing refers to a form of genetic engineering in which DNA is inserted, deleted, modified, or replaced in the genome of a living cell. In contrast to other genetic engineering techniques that can randomly insert genetic material into a host genome, gene editing can position the insertion to a specific location (e.g., the AAVS1 allele). Examples of gene editing technologies include, but are not limited to, restriction enzymes, zinc finger nucleases, TALENs, and CRISPR-Cas9. The base editors disclosed herein are a specific example of gene editing that allow for one or more single nucleotide changes, particularly resulting in a change in cell phenotype.

[0173] The term "deaminase," "base-specific deaminase," or "deaminase domain" as used herein refers to a protein or enzyme that catalyzes a deamination reaction. "Deaminase" and "base-specific deaminase" are used interchangeably in the present application. In some embodiments, the deaminase or deaminase domain is a cytosine deaminase that catalyzes the hydrolytic deamination of cytosine or deoxycytosine to uracil or deoxyuracil, respectively, which is ultimately converted to thymine (T) during cellular modification and DNA replication. In some embodiments, the deaminase or deaminase domain is an adenine deaminase domain that catalyzes the hydrolytic deamination of adenine or deoxyadenine to inosine or deoxyinosine (I), which is ultimately converted to guanine or deoxyguanine nucleotides (G) during cellular modification and DNA replication. In some embodiments, the deaminase or deaminase domain is a naturally occurring deaminase from an organism, such as a microorganism, a plant, an animal, for example, a human, a chimpanzee, a gorilla, a monkey, a cow, a dog, a rat, or a mouse. In some embodiments, the deaminase or deaminase domain is a variant of a naturally occurring deaminase from an organism that does not exist in nature. For example, in some embodiments, the deaminase or deaminase domain is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally occurring deaminase from an organism.

[0174] The term "spacer peptide" or "Linker" as used herein refers to an element that connects two molecules or moieties, such as two domains of a fusion protein. In some embodiments, the spacer peptide is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the spacer peptide is 5-100 amino acids in length, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids. Longer or shorter spacer peptides are also contemplated.

[0175] The term "mutation" as used herein refers to the substitution of a residue within a sequence (e.g., a nucleic acid or amino acid sequence) with another residue or the deletion or insertion of one or more residues within a sequence. Mutations are generally described herein by identifying the original residue, followed by the position of the residue within the sequence and the identity of the new substituted residue. Various methods for generating the amino acid substitutions (mutations) provided herein are well known in the art and provided by, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)).

[0176] The term "uracil glycosylase inhibitor" or "UGI" as used herein refers to a protein that is capable of inhibiting uracil-DNA glycosylase as a base excision repair enzyme.

[0177] The terms "top strand" or "A strand" and "bottom strand" or "B strand" as used herein are merely for distinguishing the relative positions of the two strands of a DNA target site in a certain embodiment, and are not intended to have any special limitation on the specific DNA double-stranded structure. The "top strand" can be interchangeable with "A strand", and the "bottom strand" can be interchangeable with "B strand". Unless otherwise specified, the "top strand" or "A strand" in accordance with the schematic diagram of the present application Figure 1 ) is the DNA single strand interacting with TALE-L, and correspondingly the "bottom strand" or "B strand" is the DNA single strand interacting with TALE-R.

[0178] Various embodiments of the compositions and methods according to the present application are now described in the following non-limiting examples. The examples are provided for illustrative purposes only and are not intended to limit the scope of the present application in any way.

[0179] Nucleic acid base editor

[0180] The base editing function of the nucleic acid base editors of the present application is shown in Figure 1 . The components include a sequence-specific DNA binding protein (SSDBP), a nickase, an exonuclease (with 5' or 3' exonuclease activity), a cytosine or adenine deaminase, and optionally a uracil glycosylase inhibitor (UGI), and optionally a localization sequence. These components can be expressed in separate constructs or fused in a single or multiple constructs using appropriate spacer peptides.

[0181] Sequence-specific DNA binding protein

[0182] In the base editors disclosed herein, the SSDBP can be a TALE protein, a zinc finger protein (ZFA protein), a CRISPR-Cas endonuclease (Cas protein), or a meganuclease, wherein in some particular embodiments, a TALE protein is selected. A TALE protein (Transcription activator-like effector) is a transcriptional activator effector derived from Xanthomonas spp. that has been engineered to be a sequence-specific DNA binding protein. A TALE protein comprises 1 to 33 repeat units (or repeat units) of 33-35 amino acid residues in length, wherein each repeat unit and the half repeat unit at the end can specifically recognize and bind to a specific nucleotide target site. In each repeat sequence, the type of DNA base that the TALE can recognize and bind to is determined by the two highly variable residues (referred to as repeat variable diresidues (RVDs)) at positions 12 and 13 that target a specific base pair. The RVDs that recognize the type of DNA have been deciphered: RVDs His / Asp (HD), Asn / Gly (NG), Asn / Asn (NN), and Asn / Ile (NI) recognize cytosine (C), thymine (T), guanine (G), and adenine (A), respectively (see, Boch & Bonas, 2010, Annu. Rev. Phytopathol. 48:419-436; Deng et al., 2012, Cell Res. 22: 1502-1504). The TALE repeat units are modular, and the RVDs can be designed to target and bind to DNA. As disclosed herein, a pair of TALE proteins (referred to as TALE-L or TALE-L protein, and TALE-R or TALE-R protein, respectively) are used to bind to DNA at two adjacent sites on the DNA, wherein the DNA sequence between the adjacent sites is the spacer sequence, also referred to as the target sequence; wherein the binding sites of TALE-L and TALE-R are defined as the Left Binding Site and the Right Binding Site. The sequence specificity of the TALE proteins is used to determine the target site in the base editors disclosed herein. In addition, in some cases, only one TALE (rather than a pair) is needed to bind to and target the dsDNA to achieve the base editing function of the present disclosure.

[0183] The following provides exemplary TALE protein structures that can be used as components of the base editors disclosed herein, including but not limited to N-terminal as shown in SEQ ID NO. 1, C-terminal as shown in SEQ ID NO. 2, repeat units as shown in SEQ ID NO. 3-35.

[0184] TALE-NTD (Δ152)

[0185] MVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAV

[0186] TYQHIITALPEATHEDIVGVGKQWSGARALEALLTDAGELRGPPLQLDTGQLVKIAKRGGVTAMEAVHASRNALTGAPLN (SEQ ID NO. 1)

[0187] TALE-CTD (C63)

[0188] SIVAQLSRPDPALAALTNDHLVALACLGGRPAMDAVKKGLPHAPELIRRVNRRIGERTSHRVA (SEQ ID NO. 2)

[0189] OsBADH2-TALE-Left repeat

[0190] LTPDQVVAIASNNGGKQALETVQRLLPVLCQDHG (SEQ ID NO. 3)

[0191] LTPDQVVAIASHDGGKQALETVQRLLPVLCQDHG (SEQ ID NO. 4)

[0192] LTPDQVVAIASNGGGKQALETVQRLLPVLCQDHG (SEQ ID NO. 5)

[0193] LTPDQVVAIASNNGGKQALETVQRLLPVLCQDHG (SEQ ID NO. 6)

[0194] LTPDQVVAIASNNGGKQALETVQRLLPVLCQDHG (SEQ ID NO. 7)

[0195] LTPDQVVAIASNIGGKQALETVQRLLPVLCQDHG (SEQ ID NO. 8)

[0196] LTPDQVVAIASNGGGKQALETVQRLLPVLCQDHG (SEQ ID NO. 9)

[0197] LTPDQVVAIASNNGGKQALETVQRLLPVLCQDHG (SEQ ID NO. 10)

[0198] ​​​LTPDQVVAIASHDGGKQALETVQRLLPVLCQDHG (SEQ ID NO. 11)

[0199] LTPDQVVAIASNGGGKQALETVQRLLPVLCQDHG (SEQ ID NO. 12)

[0200] LTPDQVVAIASNGGGKQALETVQRLLPVLCQDHG (SEQ ID NO. 13)

[0201] LTPDQVVAIASNGGGKQALETVQRLLPVLCQDHG (SEQ ID NO. 14)

[0202] LTPDQVVAIASNNGGKQALETVQRLLPVLCQDHG (SEQ ID NO. 15)

[0203] LTPDQVVAIASNIGGKQALETVQRLLPVLCQDHG (SEQ ID NO. 16)

[0204] LTPDQVVAIASNNGGKQALETVQRLLPVLCQDHG (SEQ ID NO. 17)

[0205] LTPDQVVAIASNGGGKQALETVQRLLPVLCQDHG (SEQ ID NO. 18)

[0206] LTPDQVVAIASNIGGKQALE (SEQ ID NO. 19)

[0207] OsBADH2-TALE-Right repeat :

[0208] LTPDQVVAIASNNGGKQALETVQRLLPVLCQDHG (SEQ ID NO. 20)

[0209] LTPDQVVAIASHDGGKQALETVQRLLPVLCQDHG (SEQ ID NO. 21)

[0210] LTPDQVVAIASHDGGKQALETVQRLLPVLCQDHG (SEQ ID NO. 22)

[0211] LTPDQVVAIASNGGGKQALETVQRLLPVLCQDHG (SEQ ID NO. 23)

[0212] LTPDQVVAIASNGGGKQALETVQRLLPVLCQDHG (SEQ ID NO. 24)

[0213] LTPDQVVAIASNGGGKQALETVQRLLPVLCQDHG (SEQ ID NO. 25)

[0214] LTPDQVVAIASNGGGKQALETVQRLLPVLCQDHG (SEQ ID NO. 26)

[0215] LTPDQVVAIASNNGGKQALETVQRLLPVLCQDHG (SEQ ID NO. 27)

[0216] LTPDQVVAIASNGGGKQALETVQRLLPVLCQDHG (SEQ ID NO. 28)

[0217] LTPDQVVAIASHDGGKQALETVQRLLPVLCQDHG (SEQ ID NO. 29)

[0218] LTPDQVVAIASHDGGKQALETVQRLLPVLCQDHG (SEQ ID NO. 30)

[0219] LTPDQVVAIASNIGGKQALETVQRLLPVLCQDHG (SEQ ID NO. 31)

[0220] LTPDQVVAIASNIGGKQALETVQRLLPVLCQDHG (SEQ ID NO. 32)

[0221] LTPDQVVAIASNNGGKQALETVQRLLPVLCQDHG (SEQ ID NO. 33)

[0222] LTPDQVVAIASNNGGKQALETVQRLLPVLCQDHG (SEQ ID NO. 34)

[0223] LTPDQVVAIASNIGGKQALETVQRLLPVLCQDHG (SEQ ID NO. 35)

[0224] Nicking enzyme

[0225] Nucleases used as components of the base editors disclosed herein are capable of cleaving one of the two strands of a target DNA. In the base editors disclosed herein, an exemplary nuclease is Fokl from Flavobacterium okeanokoites, or Fokl protein, particularly an amino acid sequence variant thereof that transforms the dsDNA cleavage activity of Fokl into a nick generated on only one strand of the target DNA, including but not limited to the D450A / D467A mutant. In addition, alternative nucleases comprising bacterial type IIS restriction enzymes can also be used as components of the base editors disclosed herein.

[0226] Wild-type Fokl is composed of two functional domains, a recognition domain and a cleavage domain. The recognition domain is artificially removed to obtain Fokl CD, which only retains the cleavage domain. When two molecules of Fokl CD monomers interact to form a dimer, the cleavage activity of Fokl CD will be activated, and both strands of double-stranded DNA can be cleaved. The following provides exemplary Fokl CD monomers that can be used as components of the base editors disclosed herein, including but not limited to SEQ ID NO. 87-88:

[0227] Fokl-L:

[0228] QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGK

[0229] HLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVKENQTRNKHI

[0230] NPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHKTNCNGAVLSVEELLIGGE

[0231] MIKAGTLTLEEVRRKFNNGEINF (SEQ ID NO. 87)

[0232] Fokl-R:

[0233] QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGK

[0234] HLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMERYVEENQTRNKHL

[0235] NPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGE

[0236] MIKAGTLTLEEVRRKFNNGEINF (SEQ ID NO. 88)

[0237] When the aspartic acid at position 450 (1st amino acid when counting from the first amino acid of wild type FokI when the recognition domain is included; 67th if counting from the first amino acid of the cleavage domain only FokICD) and / or 467 (1st amino acid when counting from the first amino acid of wild type FokI when the recognition domain is included; 84th if counting from the first amino acid of the cleavage domain only FokICD) is mutated to alanine (D450A or D467A) in one FokICD monomer of a dimer, the FokICD monomer will lose cleavage activity, while the other FokICD monomer in the dimer which has not been mutated will retain cleavage activity, and the resulting FokICD dimer can and only can cleave one strand of double stranded DNA, and cannot cleave the other strand. The FokICD dimer is referred to as FokI nickase. For the convenience of description, the inventors refer to the FokICD monomer fused to TALE-L as FokI-L (e.g. SEQ ID NO. 87), and the FokICD monomer fused to TALE-R as FokI-R (e.g. SEQ ID NO. 88). Further, the FokICD mutant monomer which contains the D450A and / or D467A mutation and thus loses cleavage activity is referred to as FokI-L nickase and FokI-R D450A / D467A respectively. In the present invention, the FokI-L D450A / D467A and FokI-R D450A / D467A interact to form a FokICD dimer which retains only the cleavage activity of FokI-L, and this dimer is referred to as FokI-L nickase (or FokI-L nickase); correspondingly, the FokI-L D450A / D467A and FokI-R nickase interact to form a FokICD dimer which retains only the cleavage activity of FokI-R, and this dimer is referred to as FokI-R

[0238] It should be noted that FokI-L nickase and FokI-R nickase tend to cleave different single strands of double stranded DNA, i.e. FokI-L nickase and FokI-Rnickase has single strand specificity or preference when cutting DNA. As shown in Figure 1 , at this target site, FokI-R nickase tends to cut the B strand, correspondingly, if FokI-L nickase is used, it tends to cut the A strand (as shown in Figure 1 ). FokI-L nickase and FokI-R nickase exhibit strand specificity that is advantageous for selecting the desired DNA single strand for the subsequent deamination step. With the sequence-specific binding of TALE-L and TALE-R to the left and right binding sites, FokI-L nickase or FokI-R nickase cuts the target sequence, leaving a nick in the A strand or B strand, respectively. The strand specificity of the nicking enzyme determines the DNA single strand that is further deaminated by the base editor of the present application.

[0239] The following provides nicking enzyme protein monomers that can be used as exemplary nucleic acid base editor components of the present application including but not limited to SEQ ID NO. 60-63:

[0240] FokI-L D450A :

[0241] QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGK

[0242] HLGGSRKPAGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVKENQTRNKHI

[0243] NPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHKTNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF (SEQ ID NO. 60)

[0244] FokI-L D467A :

[0245] QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGK

[0246] HLGGSRKPDGAIYTVGSPIDYGVIVATKAYSGGYNLPIGQADEMQRYVKENQTRNKHI

[0247] NPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF (SEQ ID NO. 62)

[0248] FokI-R D450A :

[0249] QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGK

[0250] HLGGSRKPDGAIYTVGSPIDYGVIVATKAYSGGYNLPIGQADEMERYVEENQTRNKHL

[0251] NPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF (SEQ ID NO. 62)

[0252] FokI-R D467A :

[0253] QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGK

[0254] HLGGSRKPDGAIYTVGSPIDYGVIVATKAYSGGYNLPIGQADEMERYVEENQTRNKHL

[0255] NPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF (SEQ ID NO. 62)

[0256] Exonuclease

[0257] Depending on the type of exonuclease used, the exonuclease component of the nucleic acid base editors of the present application digests the cleaved DNA strand in either the 5' to 3' direction or in the 3' to 5' direction from the nicked location. After exonuclease digestion, a short ssDNA fragment is exposed on the complementary DNA strand. The type of exonuclease determines the region of ssDNA that is deaminated (or the editing window). Exonucleases that can be used as components of the base editors disclosed herein include, but are not limited to, DNA polymerases I and III (E. coli), mammalian p53 protein, exonucleases I-VII (E. coli) (e.g., exonucleases I and V (with 3' to 5' exonuclease activity)), bacteriophage derived polymerases (e.g., T4 DNA polymerase (with 3' to 5' exonuclease activity)), Thermus aquaticus polymerase (with 5' to 3' exonuclease activity), and 3' to 5' exonucleases reported by Shevelev and Hubscher (Shevelev & Hubscher, 2002, Nat. Rev. Molec. Cell Biol. 3:364-376).

[0258] The following provides exonuclease proteins that can be used as exemplary base editor components of the present application include, but are not limited to, proteins as set forth in SEQ ID NOS. 64-67, 153.

[0259] Exonuclease V (ExoV):

[0260] MAETGEEETASAEASGFSDLSDSELVEFLDLEEAKESAVSLSKPGPSAELPGKDDKPVS

[0261] LQNWKGGLDVLSPMERFHLKYLYVTDLCTQNWCELQMVYGKELPGSLTPEKAAVLD

[0262] TGASIHLAKELELHDLVTVPIATKEDAWAVKFLNILAMIPALQSEGRVREFPVFGEVEG

[0263] IFLVGVIDELHYTSKGELELAELKTRRRPVLPLPAQKKKDYFQVSLYKYIFDAMVQGK

[0264] VTPASLIHHTKLCLDKPLGPSVLRHARQGGVSVKSLGDLMELVFLSLTLSDLPAIDTLK

[0265] LEYIHQETATILGTEIVAFEEKEVKSKVQHYVAYWMGHRDPQGVDVEEAWKCRTCDYVDICEWRRGSGVLSSSWEPKAKKFK (SEQ ID NO. 153)

[0266] mExoI:

[0267] MGIQGLLQFIQEASEPVNVKKYKGQAVAVDTYCWLHKGAIACAEKLAKGEPTDRYV

[0268] GFCMKFVNMLLSYGVKPILIFDGCTLPSKKEVERSRRERRQSNLLKGKQLLREGKVSE

[0269] ARDCFARSINITHAMAHKVIKAARALGVDCLVAPYEADAQLAYLNKAGIVQAVITED

[0270] SDLLAFGCKKVILKMDQFGNGLEVDQARLGMCKQLGDVFTEEKFRYMCILSGCDYLA

[0271] SLRGIGLAKACKVLRLANNPDIVKVIKKIGHYLRMNITVPEDYITGFIRANNTFLYQLV

[0272] FDPIQRKLVPLNAYGDDVNPETLTYAGQYVGDSVALQIALGNRDVNTFEQIDDYSPDT

[0273] MPAHSRSHSWNEKAGQKPPGTNSIWHKNYCPRLEVNSVSHAPQLKEKPSTLGLKQVIS

[0274] TKGLNLPRKSCVLKRPRNEALAEDDLLSQYSSVSKKIKENGCGDGTSPNSSKMSKSCP

[0275] DSGTAHKTDAHTPSKMRNKFATFLQRRNEESGAVVVPGTRSRFFCSSQDFDNFIPKKE

[0276] SGQPLNETVATGKATTSLLGALDCPDTEGHKPVDANGTHNLSSQIPGNAAVSPEDEAQ

[0277] SSETSKLLGAMSPPSLGTLRSCFSWSGTLREFSRTPSPSASTTLQQFRRKSDPPACLPEA

[0278] SAVVTDRCDSKSEMLGETSQPLHELGCSSRSQESMDSSCGLNTSSLSQPSSRDSGSEES

[0279] DCNNKSLDNQGEQNSKQHLPHFSKKDGLRRNKVPGLCRSSSMDSFSTTKIKPLVPARV

[0280] SGLSKKSGSMQTRKHHDVENKPGLQTKISELWKNFGFKKDSEKLPSCKKPLSPVKDNIQLTPETEDEIFNKPECVRAQRAIFH (SEQ ID NO. 64)

[0281] mTrex2:

[0282] MSEPPRAETFVFLDLEATGLPNMDPEIAEISLFAVHRSSLENPERDDSGSLVLPRVLDKL

[0283] TLCMCPERPFTAKASEITGLSSESLMHCGKAGFNGAVVRTLQGFLSRQEGPICLVAHN

[0284] GFDYDFPLLCTELQRLGAHLPQDTVCLDTLPALRGLDRAHSHGTRAQGRKSYSLASLF

[0285] HRYFQAEPSAAHSAEGDVHTLLLIFLHRAPELLAWADEQARSWAHIEPMYVPPDGPSLEA (SEQ ID NO. 65)

[0286] mArtimes:

[0287] MSSGMAYTSDRDRNKARAYSHCHKDHMKGRASKRRCSKVYCSVTKTSKYRWNRTT

[0288] TSVDASGKVVVTAGHCGSVMGSNGTVYTGDRAKGASRMHSGGRVKDSVYDTTCDR

[0289] YSRCRGVRSWVTRSHHVVWNCKAAYGYYTNSGVVHVDKDMKNMDHHTTDRNTHA

[0290] CRHKACWNKCGTSNKTAHTSKSTMWGRTRKTNVVRTGSSYRACSHSSSKDSYCVNV

[0291] YNVVGTVDKVMDVKCRSSVKYKGKKRARTHDSDDDDDTRHKVYTSMKADRSGGC

[0292] KASVWSSANDCSNSDSGTSGGGSTVNADDVDWVKRRDTGCHSSTGGSSKCSDSKCS

[0293] DSKCSDSDGDSTHSSNSSSTHTDGSGWDSCDTVSSKSGGDSTSNKGAYKKKSSASDAC

[0294] DTHCDKSRAVNGACVDTSGRKSKTSSTRADSSSSDSTATHCYRKATGSVVKRKCSDS (SEQ ID NO. 66)

[0295] T5exo:

[0296] MSKSWGKFIEEEEAEMASRRNLMIVDGTNLGFRFKHNNSKKPFASSYVSTIQSLAKSY

[0297] SARTTIVLGDKGKSVFRLEHLPEYKGNRDEKYAQRTEEEKALDEQFFEYLKDAFELCK

[0298] TTFPTFTIRGVEADDMAAYIVKLIGHLYDHVWLISTDGDWDTLLTDKVSRFSFTTRRE

[0299] YHLRDMYEHHNVDDVEQFISLKAIMGDLGDNIRGVEGIGAKRGYNIIREFGNVLDIIDQ

[0300] LPLPGKQKYIQNLNASEELLFRNLILVDLPTYCVDAIAAVGQDVLDKFTKDILEIAEQ (SEQ ID NO. 67)

[0301] Deaminase

[0302] Deaminases useful as base editor components of the present application include cytosine deaminases and adenine deaminases. Cytosine deaminases include, but are not limited to, hAPOBEC3A (Zong et al., 2018, Nat. Biotechnol. Oct 1. doi: 10.1038 / nbt.4261), rAPOBEC1, C57, Sdd (Huang J et al., 2023, Cell, doi: 10.1101 / 2023.05.21.541555), which cause a conversion of a base site from C to T. Alternative adenine deaminases include TadA-8e (Richter et al., 2020, Nat. Biotechnol. 38:883-891), which produces a conversion of a base site from A to G.

[0303] The following provides deaminases useful as exemplary base editor components of the present application, including but not limited to the deaminases listed in Table 1 (e.g., proteins shown in SEQ ID NO. 36-59, 80-86):

[0304] Table 1 Deaminase species

[0305]

[0306] Deaminase rAPOBEC1:

[0307] MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTN

[0308] KHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLY

[0309] HHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK (SEQ ID NO. 36)

[0310] Deaminase hAPOBEC3A:

[0311] MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGFLHN

[0312] QAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGEVRAFLQ

[0313] ENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIMTYDEFKHCWDTFVDHQGCPFQPWDGLDEHSQALSGRLRAILQNQGN (SEQ ID NO. 37)

[0314] hAPOBEC3G-CTD:

[0315] MDPPTFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHGF

[0316] LEGRHAELCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCIFT

[0317] ARIYDDQGRCQEGLRTLAEAGAKISIMTYSEFKHCWDTFVDHQGCPFQPWDGLDEHSQDLSGRLRAILQNQEN (SEQ ID NO. 38)

[0318] PmCDA1:

[0319] MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFWGYAVNKP

[0320] QSGTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKILEWYNQELRGNG

[0321] HTLKIWACKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIFIQSSHNQLNENRWLEKTLKRAEKRRSELSIMIQVKILHTTKSPAVSRGSG (SEQ ID NO. 39) tCDA1EQ:

[0322] SHRCYVLFELKRRGERRACFWGYAVNKPQSGTERGIHAEIFSIRKVEEYLRDNPGQFTI

[0323] NWYSSWSPCADCAEKILEWYNQELRGNGHTLKIEACKLYYEKNARNQIGLQNLRDNGVGLNV (SEQ ID NO. 40)

[0324] hAID:

[0325] MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLRNKNGCH

[0326] VELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTARLY

[0327] FCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENHERTFKAWEGLHENSVRLSRQLRRILLPLYEVDDLRDAFRTLGL (SEQ ID NO. 41)

[0328] PpAPOBEC1:

[0329] MTSEKGPSTGDPTLRRRIESWEFDVFYDPRELRKETCLLYEIKWGMSRKIWRSSGKNT

[0330] TNHVEVNFIKKFTSERRFHSSISCSITWFLSWSPCWECSQAIREFLSQHPGVTLVIYVAR

[0331] LFWHMDQRNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPL

[0332] WMMLYALELHCIILSLPPCLKISRRWQNHLAFFRLHLQNCHYQTIPPHILLATGLIHPSVTWR (SEQ ID NO. 42)

[0333] RrA3F:

[0334] MKPQIRDHRPNPMEAMYPHIFYFHFENLEKAYGRNETWLCFTVEIIKQYLPVPWKKG

[0335] VFRNQVDPETHCHAEKCFLSWFCNNTLSPKKNYQVTWYTSWSPCPECAGEVAEFLAE

[0336] HSNVKLTIYTARLYYFWDTDYQEGLRSLSEEGASVEIMDYEDFQYCWENFVYDDGEPFKRWKGLKYNFQSLTRRLREILQ (SEQ ID NO. 43)

[0337] AmAPOBEC1:

[0338] MADSSEKMRGQYISRDTFEKNYKPIDGTKEAHLLCEIKWGKYGKPWLHWCQNQRMN

[0339] IHAEDYFMNNIFKAKKHPVHCYVTWYLSWSPCADCASKIVKFLEERPYLKLTIYVAQL

[0340] YYHTEEENRKGLRLLRSKKVIIRVMDISDYNYCWKVFVSNQNGNEDYWPLQFDPWVKENYSRLLDIFWESKCRSPNPW (SEQ ID NO. 44)

[0341] SsAPOBEC3B:

[0342] MDPQRLRQWPGPGPASRGGYGQRPRIRNPEEWFHELSPRTFSFHFRNLRFASGRNRSYI

[0343] CCQVEGKNCFFQGIFQNQVPPDPPCHAELCFLSWFQSWGLSPDEHYYVTWFISWSPCC

[0344] ECAAKVAQFLEENRNVSLSLSAARLYYFWKSESREGLRRLSDLGAQVGIMSFQDFQH

[0345] CWNNFVHNLGMPFQPWKKLHKNYQRLVTELKQILREEPATYGSPQAQGKVRIGSTAAGLRHSHSHTRSEAHLRPNHSSRQHRILNPPREARARTCVLVDASWICYR (SEQ ID NO. 45)

[0346] hA3B:

[0347] MNPQIRNPMERMYRDTFYDNFENEPILYGRSYTWLCYEVKIKRGRSNLLWDTGVFRG

[0348] QVYFKPQYHAEMCFLSWFCGNQLPAYKCFQITWFVSWTPCPDCVAKLAEFLSEHPNV

[0349] TLTISAARLYYYWERDYRRALCRLSQAGARVKIMDYEEFAYCWENFVYNEGQQFMP

[0350] WYKFDENYAFLHRTLKEILRYLMDPDTFTFNFNNDPLVLRRRQTYLCYEVERLDNGT

[0351] WVLMDQHMGFLCNEAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPC

[0352] FSWGCAGEVRAFLQENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIMTYDEFEYCWDTFVYRQGCPFQPWDGLEEHSQALSGRLRAILQNQGN (SEQ ID NO. 46) hA3C:

[0353] MNPQIRNPMKAMYPGTFYFQFKNLWEANDRNETWLCFTVEGIKRRSVVSWKTGVFR

[0354] NQVDSETHCHAERCFLSWFCDDILSPNTKYQVTWYTSWSPCPDCAGEVAEFLARHSN

[0355] VNLTIFTARLYYFQYPCYQEGLRSLSQEGVAVEIMDYEDFKYCWENFVYNDNEPFKPWKGLKTNFRLLKRRLRESLQ (SEQ ID NO. 47)

[0356] hA3D:

[0357] MNPQIRNPMERMYRDTFYDNFENEPILYGRSYTWLCYEVKIKRGRSNLLWDTGVFRG

[0358] PVLPKRQSNHRQEVYFRFENHAEMCFLSWFCGNRLPANRRFQITWFVSWNPCLPCVV

[0359] KVTKFLAEHPNVTLTISAARLYYYRDRDWRWVLLRLHKAGARVKIMDYEDFAYCWE

[0360] NFVCNEGQPFMPWYKFDDNYASLHRTLKEILRNPMEAMYPHIFYFHFKNLLKACGRN

[0361] ESWLCFTMEVTKHHSAVFRKRGVFRNQVDPETHCHAERCFLSWFCDDILSPNTNYEV

[0362] TWYTSWSPCPECAGEVAEFLARHSNVNLTIFTARLCYFWDTDYQEGLCSLSQEGASVKIMGYKDFVSCWKNFVYSDDEPFKPWKGLQTNFRLLKRRLREILQ (SEQ ID NO. 48) hA3F:

[0363] MKPHFRNTVERMYRDTFSYNFYNRPILSRRNTVWLCYEVKTKGPSRPRLDAKIFRGQ

[0364] VYSQPEHHAEMCFLSWFCGNQLPAYKCFQITWFVSWTPCPDCVAKLAEFLAEHPNVT

[0365] LTISAARLYYYWERDYRRALCRLSQAGARVKIMDDEEFAYCWENFVYSEGQPFMPW

[0366] YKFDDNYAFLHRTLKEILRNPMEAMYPHIFYFHFKNLRKAYGRNESWLCFTMEVVKH

[0367] HSPVSWKRGVFRNQVDPETHCHAERCFLSWFCDDILSPNTNYEVTWYTSWSPCPECA

[0368] GEVAEFLARHSNVNLTIFTARLYYFWDTDYQEGLRSLSQEGASVEIMGYKDFKYCWENFVYNDDEPFKPWKGLKYNFLFLDSKLQEILE (SEQ ID NO. 49) hA3G:

[0369] MKPHFRNTVERMYRDTFSYNFYNRPILSRRNTVWLCYEVKTKGPSRPPLDAKIFRGQV

[0370] YSELKYHPEMRFFHWFSKWRKLHRDQEYEVTWYISWSPCTKCTRDMATFLAEDPKV

[0371] TLTIFVARLYYFWDPDYQEALRSLCQKRDGPRATMKIMNYDEFQHCWSKFVYSQREL

[0372] FEPWNNLPKYYILLHIMLGEILRHSMDPPTFTFNFNNEPWVRGRHETYLCYEVERMHN

[0373] DTWVLLNQRRGFLCNQAPHKHGFLEGRHAELCFLDVIPFWKLDLDQDYRVTCFTSWS

[0374] PCFSCAQEMAKFISKNKHVSLCIFTARIYDDQGRCQEGLRTLAEAGAKISIMTYSEFKHCWDTFVDHQGCPFQPWDGLDEHSQDLSGRLRAILQNQEN (SEQ ID NO. 50)

[0375] hA3H:

[0376] MALLTAETFRLQFNNKRRLRRPYYPRKALLCYQLTPQNGSTPTRGYFENKKKCHAEIC

[0377] FINEIKSMGLDETQCYQVTCYLTWSPCSSCAWELVDFIKAHDHLNLRIFASRLYYHWC

[0378] KPQQDGLRLLCGSQVPVEVMGFPEFADCWENFVDHEKPLSFNPYKMLEELDKNSRAIKRRLDRIKS (SEQ ID NO. 51)

[0379] hA3Bctd:

[0380] MEILRYLMDPDTFTFNFNNDPLVLRRRQTYLCYEVERLDNGTWVLMDQHMGFLCNE

[0381] AKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGEVRAFLQE

[0382] NTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIMTYDEFEYCWDTFVYRQGCPFQPWDGLEEHSQALSGRLRAILQNQGN (SEQ ID NO. 52)

[0383] FERNY:

[0384] FERNY DPRELRKETYLLYEIKWGKSGKLWRHWCQNNRTQHAEVYFLENIFNARRFNP

[0385] STHCSITWYLSWSPCAECSQKIVDFLKEHPNVNLEIYVARLYYHEDERNRQGLRDLVNSGVTIRIMDLPDYNYCWKTFVSDQGGDEDYWPGHFAPWIKQYSLKL (SEQ ID NO. 53) ecTadA:

[0386] MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTA

[0387] HAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTG

[0388] AAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD (SEQ ID NO. 54)

[0389] mADA:

[0390] MAQTPAFNKPKVELHVHLDGAIKPETILYFGKKRGIALPADTVEELRNIIGMDKPLSLP

[0391] GFLAKFDYYMPVIAGCREAIKRIAYEFVEMKAKEGVVYVEVRYSPHLLANSKVDPMP

[0392] WNQTEGDVTPDDVVDLVNQGLQEGEQAFGIKVRSILCCMRHQPSWSLEVLELCKKY

[0393] NQKTVVAMDLAGDETIEGSSLFPGHVEAYEGAVKNGIHRTVHAGEVGSPEVVREAVD

[0394] ILKTERVGHGYHTIEDEALYNRLLKENMHFEVCPWSSYLTGAWDPKTTHAVVRFKND

[0395] KANYSLNTDDPLIFKSTLDTDYQMTKKDMGFTEEEFKRLNINAAKSSFLPEEEKKELLERLYREYQ (SEQ ID NO. 55)

[0396] hADAR2:

[0397] MHLDQTPSRQPIPSEGLQLHLPQVLADAVSRLVLGKFGDLTDNFSSPHARRKVLAGVV

[0398] MTTGTDVKDAKVISVSTGTKCINGEYMSDRGLALNDCHAEIISRRSLLRFLYTQLELYL

[0399] NNKDDQKRSIFQKSERGGFRLKENVQFHLYISTSPCGDARIFSPHEPILEEPADRHPNRK

[0400] ARGQLRTKIESGEGTIPVRSNASIQTWDGVLQGERLLTMSCSDKIARWNVVGIQGSLLS

[0401] IFVEPIYFSSIILGSLYHGDHLSRAMYQRISNIEDLPPLYTLNKPLLSGISNAEARQPGKAP

[0402] NFSVNWTVGDSAIEVINATTGKDELGRASRLCKHALYCRWMRVHGKVPSHLLRSKITKPNVYHESKLAAKEYQAAKARLFTAFIKAGLGAWVEKPTEQDQFSLTP (SEQ ID NO. 56)

[0403] hADAT2:

[0404] MEAKAAPKPAASGACSVSAEETEKWMEEAMHMAKEALENTEVPVGCLMVYNNEVV

[0405] GKGRNEVNQTKNATRHAEMVAIDQVLDWCRQSGKSPSEVFEHTVLYVTVEPCIMCA

[0406] AALRLMKIPLVVYGCQNERFGGCGSVLNIASADLPNTGRPFQCIPGYRAEEAVEMLKTFYKQENPNAPKSKVRKKECQKS (SEQ ID NO. 57)

[0407] ecTadA*(7.10) :

[0408] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTA

[0409] HAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGA

[0410] AGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO. 58)

[0411] TadA*ABE8e (TadA-8e) :

[0412] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTA

[0413] HAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGA

[0414] AGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN (SEQ ID NO. 59)

[0415] Sdd2

[0416] MAPDSLVWFDPLGLIVLQQVPYNDHPLFGAVSEFIQGKSRSDLRGRNVAAVLLDDGT

[0417] VIVRASEGGGNHAERVLMGLSEVDPAKVVAVYTERSPCTGRINCHDLLDSSLGADVPVYYTHEMIRGQEGKTAQQIEADRNQFCRGG (SEQ ID NO. 80) Sdd3

[0418] MSASAQLNTYLAAIGNSTTTVEAQPEAAPPPAAAESLDSTPRLPDGGIDFHALAKRLGL

[0419] LEARPTEQPPFDPRRFNPACWQGLKPYDQAGTAEGNLFIAPGKRWNTRPMQASKLEV

[0420] GPQSDLHPQWRSRKAPWHIEGKIAAYMRQKGFTDGCVYLNARPCSGPDGCARNLPDLLPVGSTLHVHARYIDRTGETRFYYREYRGTGKALT (SEQ ID NO. 81) Sdd4

[0421] MLDAMDAYLSEIAGGNAPARAGPKAPEPKQPGGSSSPRARDGRIDFRALLERLKAQG

[0422] VVGLEGRSDDPIPDFDPKKQNPACYQGLAPRQKGKPVRGNLFFPDGRRWNDVALESS

[0423] RGEPAFDLNIIKPEYRSLSPARGHLEGNVAAWMRSTFHQEMVLYINESPCRKHGKGCLYTLEHFLPRGYVLHVWSRNDRGEWRGNTFRGSGEAFTEGA (SEQ ID NO. 82) Sdd6

[0424] MVETRDKIIAAKSRSDAGLLAFQQATNGSIDSRPAEAIANLQRAKTHLDEAQRLVANS

[0425] DAAVDNYINAILGGASAATAQPSAVIPASKPSRFKPMRTDPAKADEIRPHVGKDRAVA

[0426] TLWDADGNRVLGLHSADDDGPAATAAWKPPWRDYVRLRRHVEAHAAARMHQDGH

[0427] KTMVMYINLPPCKYFDGCKLNLEDILPKGSTLWMHRVFQNGGTKIYQFNGTGRAYV (SEQ ID NO. 83)

[0428] Sdd7 (also denoted as C57 in this specification)

[0429] MLEAVRARLIGEGGGPGAVPEGGDGPPAVPAEEVERLRGELPPPVVPGTGQKTHGRW

[0430] IGPDGRVRAIVSGRDEDAALVHAQLAAKGIPDEPTRNSDVEQKLAAHMVANGIRHVT LVINHRPCRGFDDSCDTLVPIILPEGCTLTVHGQTDKGMRVRVRYTGGARPWWS (SEQ ID NO. 84)

[0431] Sdd10

[0432] MLDAALGAVRRIIAALGTSGAERASPGANGSERVDELAERLPPTVVPNTSAKTHGWW

[0433] FTGQGAAQELISGEGPDARAAYEALREEGYPRPGMPFVAMHVEIKLAAHMRRNDIEH ATVVINNIPCPLVWGCENLIGVVLPEGSSLTVHGSNGYERTFTGGRKPPWPR (SEQ ID NO. 85)

[0434] Sdd59

[0435] MLLTPPPRPAAPPTTRPKPLVARTGDAYPPGTEWALPLIVQPHPPVGGTVPVEGHVRA

[0436] LRPESQISHVFHPGGGHWTEQARARLRVLPGFGWAVNLGHHVELQIAAWMTACGIH HAELVLNRPPCGERYGLGCHQALPVLLPRGYRLTVSSTRGGPQPYQHHYEGKA (SEQ ID NO. 86)

[0437] Uracil glycosylase inhibitor (UGI)

[0438] In some embodiments, when cytosine deaminase is used, a uracil glycosylase inhibitor (UGI) is fused to the N-terminus of the deaminase, while no UGI is needed when adenine deaminase is used.

[0439] The following discloses exemplary UGI proteins useful in the base editors of the present application include, but are not limited to, the protein as set forth in SEQ ID NO. 68:

[0440] TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML (SEQ ID NO. 68)

[0441] nuclear localization sequence (NLS)

[0442] In some embodiments of the application, the NLS of the fusion protein of the application can be located at the N-terminus and / or the C-terminus. In some embodiments of the application, the NLS of the fusion protein of the application can be located between the adenine deamination domain, the cytosine deamination domain, the nucleic acid targeting domain, and / or the UGI. In some embodiments, the fusion protein comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLSs. In some embodiments, the fusion protein comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLSs at or near the N-terminus. In some embodiments, the fusion protein comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLSs at or near the C-terminus. In some embodiments, the polypeptide comprises a combination of these, such as one or more NLSs at the N-terminus and one or more NLSs at the C-terminus. When there is more than one NLS, each can be selected independently of the others.

[0443] Generally, an NLS consists of one or more short sequences of positively charged lysines or arginines exposed on the surface of a protein, although other types of NLSs are known. Non-limiting examples of NLSs include: KKRKV (SEQ ID NO. 150), PKKKRKV (SEQ ID NO. 151), or KRPAATKKAGQAKKKK (SEQ ID NO. 152).

[0444] Recombinant expression construct

[0445] The components of the base editors of the application can be expressed individually, or as one or more fusion proteins, or using recombinant expression constructs used in recombinant genetic engineering techniques to express the above elements or components individually or collectively. Exemplary recombinant expression constructs of the application are set forth in, for example, Figure 16A to Figure 16E and Figure 17A to Figure 17H .

[0446] The types, functions, and references of the genes and regulatory elements in the above exemplary recombinant expression constructs Figure 16A to Figure 16E and Figure 17A to 17H are explained and exemplified as shown in Table 2 below:

[0447] Table 2: Examples of genes and regulatory elements in constructs

[0448]

[0449]

[0450]

[0451]

[0452]

[0453] Specifically, the genes and regulatory elements in the exemplary recombinant constructs used in the present application include, but are not limited to, the following sequences: promoter sequences as shown in SEQ ID NO. 69-72; terminator sequences as shown in SEQ ID NO. 73-76; mitochondrial targeting sequences (MTS) as shown in SEQ ID NO. 77-78; chloroplast transit peptide (CTP) sequences as shown in SEQ ID NO. 79.

[0454] UBI promoter:

[0455] TGACCCGGTCGTGCCCCTCTCTAGAGATAATGAGCATTGCATGTCTAAGTTATAAA

[0456] AAATTACCACATATTTTTTTTGTCACACTTGTTTGAAGTGCAGTTTATCTATCTTTA

[0457] TACATATATTTAAACTTTACTCTACGAATAATATAATCTATAGTACTACAATAATA

[0458] TCAGTGTTTTAGAGAATCATATAAATGAACAGTTAGACATGGTCTAAAGGACAATT

[0459] GAGTATTTTGACAACAGGACTCTACAGTTTTATCTTTTTAGTGTGCATGTGTTCTCC

[0460] TTTTTTTTTGCAAATAGCTTCACCTATATAATACTTCATCCATTTTATTAGTACATCC

[0461] ATTTAGGGTTTAGGGTTAATGGTTTTTATAGACTAATTTTTTTAGTACATCTATTTT

[0462] ATTCTATTTTAGCCTCTAAATTAAGAAAACTAAAACTCTATTTTAGTTTTTTTATTT

[0463] AATAATTTAGATATAAAATAGAATAAAATAAAGTGACTAAAAATTAAACAAATAC

[0464] CCTTTAAGAAATTAAAAAAACTAAGGAAACATTTTTCTTGTTTCGAGTAGATAATG

[0465] CCAGCCTGTTAAACGCCGTCGACGAGTCTAACGGACACCAACCAGCGAACCAGCA

[0466] GCGTCGCGTCGGGCCAAGCGAAGCAGACGGCACGGCATCTCTGTCGCTGCCTCTG

[0467] GACCCCTCTCGATCGAGAGTTCCGCTCCACCGTTGGACTTGCTCCGCTGTCGGCAT

[0468] CCAGAAATTGCGTGGCGGAGCGGCAGACGTGAGCCGGCACGGCAGGCGGCCTCCT

[0469] CCTCCTCTCACGGCACCGGCAGCTACGGGGGATTCCTTTCCCACCGCTCCTTCGCTT

[0470] TCCCTTCCTCGCCCGCCGTAATAAATAGACACCCCCTCCACACCCTCTTTCCCCAAC

[0471] CTCGTGTTGTTCGGAGCGCACACACACACAACCAGATCTCCCCCAAATCCACCCGT

[0472] CGGCACCTCCGCTTCAAGGTACGCCGCTCGTCCTCCCCCCCCCCCCCTCTCTACCTT

[0473] CTCTAGATCGGCGTTCCGGTCCATGGTTAGGGCCCGGTAGTTCTACTTCTGTTCATG

[0474] TTTGTGTTAGATCCGTGTTTGTGTTAGATCCGTGCTGCTAGCGTTCGTACACGGATG

[0475] CGACCTGTACGTCAGACACGTTCTGATTGCTAACTTGCCAGTGTTTCTCTTTGGGG

[0476] AATCCTGGGATGGCTCTAGCCGTTCCGCAGACGGGATCGATTTCATGATTTTTTTT

[0477] GTTTCGTTGCATAGGGTTTGGTTTGCCCTTTTCCTTTATTTCAATATATGCCGTGCA

[0478] CTTGTTTGTCGGGTCATCTTTTCATGCTTTTTTTTGTCTTGGTTGTGATGATGTGGTC

[0479] TGGTTGGGCGGTCGTTCTAGATCGGAGTAGAATTAATTCTGTTTCAAACTACCTGG

[0480] TGGATTTATTAATTTTGGATCTGTATGTGTGTGCCATACATATTCATAGTTACGAAT

[0481] TGAAGATGATGGATGGAAATATCGATCTAGGATAGGTATACATGTTGATGCGGGT

[0482] TTTACTGATGCATATACAGAGATGCTTTTTGTTCGCTTGGTTGTGATGATGTGGTGT

[0483] GGTTGGGCGGTCGTTCATTCGTTCTAGATCGGAGTAGAATACTGTTTCAAACTACC

[0484] TGGTGTATTTATTAATTTTGGAACTGTATGTGTGTGTCATACATCTTCATAGTTACG

[0485] AGTTTAAGATGGATGGAAATATCGATCTAGGATAGGTATACATGTTGATGTGGGTT

[0486] TTACTGATGCATATACATGATGGCATATGCAGCATCTATTCATATGCTCTAACCTT

[0487] GAGTACCTATCTATTATAATAAACAAGTATGTTTTATAATTATTTTGATCTTGATAT

[0488] ACTTGGATGATGGCATATGCAGCAGCTATATGTGGATTTTTTTAGCCCTGCCTTCAT

[0489] ACGCTATTTATTTGCTTGGTACTGTTTCTTTTGTCGATGCTCACCCTGTTGTTTGGTGTTACTTCTGCA (SEQ ID NO. 69)

[0490] CaMV 35S promoter (enhanced):

[0491] TGAGACTTTTCAACAAAGGGTAATATCGGGAAACCTCCTCGGATTCCATTGCCCAG

[0492] CTATCTGTCACTTCATCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTACAAATG

[0493] CCATCATTGCGATAAAGGAAAGGCTATCGTTCAAGATGCCTCTGCCGACAGTGGTC

[0494] CCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAAC

[0495] CACGTCTTCAAAGCAAGTGGATTGATGTGATAACATGGTGGAGCACGACACTCTC

[0496] GTCTACTCCAAGAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCTATTGAGA

[0497] GATACAGTCTCAGAAGACCAAAGGGCAATTGAGACTTTTCAACAAAGGGTAATATCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATC

[0498] TGTCACTTCATCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTACAAATGCCATC

[0499] ATTGCGATAAAGGAAAGGCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAA

[0500] AGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACG

[0501] TCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCAC

[0502] AATCCCACTATCCTTCGCAAGACCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACACGCTGA(SEQ ID NO.70)

[0503] CaMV 2 x 35S promoter

[0504] CCTGCAGGTCAACATGGTGGAGCACGACACACTTGTCTACTCCAAAAATATCAAA

[0505] GATACAGTCTCAGAAGACCAAAGGGCAATTGAGACTTTTCAACAAAGGGTAATAT

[0506] CCGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTTATTGTGAAGATA

[0507] GTGGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCCA

[0508] TCGTTGAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAG

[0509] GAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGA

[0510] TGTGATAACATGGTGGAGCACGACACACTTGTCTACTCCAAAAATATCAAAGATA

[0511] CAGTCTCAGAAGACCAAAGGGCAATTGAGACTTTTCAACAAAGGGTAATATCCGG

[0512] AAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTTATTGTGAAGATAGTGG

[0513] AAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCCATCGT

[0514] TGAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGC

[0515] ATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTG

[0516] ATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCCT

[0517] TCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACCTCGACCTCAACACAACA

[0518] TATACAAAACAAACGAATCTCAAGCAATCAAGCATTCTACTTCTATTGCAGCAATT

[0519] TAAATCATTTCTTTTAAAGCAAAAGCAATTTTCTGAAAATTTTCACCATTTACGAACGATA(SEQ IDNO.71)

[0520] CMV promoter:

[0521] GTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGG

[0522] GATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAAT

[0523] CAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCT(SEQ ID NO.72)Nos terminator:

[0524] GAATTTCCCCGATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTG

[0525] TTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAA

[0526] TAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTC

[0527] CCGCAATTATACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACT(SEQ ID NO.73)E9terminator:

[0528] AGAGCTTTCGTTCGTATCATCGGTTTCGACAACGTTCGTCAAGTTCAATGCATCAG

[0529] TTTCATTGCGCACACACCAGAATCCTACTGAGTTTGAGTATTATGGCATTGGGAAA

[0530] ACTGTTTTTCTTGTACCATTTGTTGTGCTTGTAATTTACTGTGTTTTTTATTCGGTTT

[0531] TCGCTATCGAACTGTGAAATGGAAATGGATGGAGAAGAGTTAATGAATGATATGG

[0532] TCCTTTTGTTCATTCTCAAATTAATATTATTTGTTTTTTCTCTTATTTGTTGTGTGTTG

[0533] AATTTGAAATTATAAGAGATATGCAAACATTTTGTTTTGAGTAAAAATGTGTCAAA

[0534] TCGTGGCCTCTAATGACCGAAGTTAATATGAGGAGTAAAACACTTGTAGTTGTACC

[0535] ATTATGCTTATTCACTAGGCAACAAATATATTTTCAGACCTAGAAAAGCTGCAAAT

[0536] GTTACTGAATACAAGTATGTCCTCTTGTGTTTTAGACATTTATGAACTTTCCTTTAT

[0537] GTAATTTTCCAGAATCCTTGTCAGATTCTAATCATTGCTTTATAATTATAGTTATAC

[0538] TCATGGATTTGTAGTTGAGTATGAAAATATTTTTTAATGCATTTTATGACTTGCCAATTGATTGACAAC(SEQ ID NO.74)

[0539] CaMV poly(A) signal:

[0540] TTTCTCCATAATAATGTGTGAGTAGTTCCCAGATAAGGGAATTAGGGTTCCTATAG

[0541] GGTTTCGCTCATGTGTTGAGCATATAAGAAACCCTTAGTATGTATTTGTATTTGTAA

[0542] AATACTTCTATCAATAAAATTTCTAATTCCTAAAACCAAAATCCAGTACTAAAATCCAGATC(SEQ IDNO.75)

[0543] bGH poly(A) signal:

[0544] CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGA

[0545] CCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCG

[0546] CATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCA

[0547] AGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGG(SEQ ID NO.76).

[0548] SOD2 MTS:

[0549] MLSRAVCGTSRQLAPVLGYLGSRQKHSLPD (SEQ ID NO. 77)

[0550] COX8 MTS:

[0551] MSVLTPLLLRGLTGSARRLPVPRAK (SEQ ID NO. 78)

[0552] CTP:

[0553] MAPTVMMASSATAVAPFQGLKSAASLPVARRSTRSLGNVSNGGRIRCMQ (SEQ ID NO. 79)

[0554] Target cell of interest

[0555] The recombinant expression construct provided by the present application can be produced according to the gene engineering method known in the art. In some embodiments, the base editor or the recombinant expression construct thereof is introduced into a cell to edit and express the target gene, forming an edited genetically engineered cell.

[0556] Any cell from any organism can be used with the nucleic acids, polypeptides, compositions, and methods described herein. Cells include, but are not limited to, human, non-human, animal, mammalian, bacterial, protist, fungal, insect, yeast, non-conventional yeast, and plant cells, including monocot and dicot plants and plant elements, as well as plants and seeds produced by the methods described herein, and in some aspects, the cells of the organism are germ cells, somatic cells, meiotic cells, mitotic cells, stem cells, or pluripotent stem cells.

[0557] In some embodiments, animal cells can include, but are not limited to, organisms of the phylum that includes Chordata, Arthropoda, Mollusca, Annelida, Eumetazoa, or Echinodermata; organisms of the class that includes Mammalia, Insecta, Aves, Amphibia, Reptilia, or Pisces. In some aspects, the animal is a human, a mouse, a Caenorhabditis elegans, a rat, a fruit fly, a zebrafish, a chicken, a dog, a cow, a sheep, a pig, a guinea pig, a hamster, a chicken, a Japanese rice fish, a sea lamprey, a puffer fish, a tree frog, a monkey, or a chimpanzee.

[0558] Particular animal cell types include haploid cells, diploid cells, germ cells, neurons, muscle cells, endocrine or exocrine cells, epithelial cells, muscle cells, tumor cells, embryonic cells, hematopoietic cells, bone cells, germ line cells, somatic cells, stem cells, pluripotent stem cells, induced pluripotent stem cells, progenitor cells, meiotic cells, and mitotic cells. In some aspects, multiple cells from an organism can be used.

[0559] In some embodiments, the plant cells are derived from monocotyledonous and dicotyledonous plants. Examples of monocotyledonous plants that can be used include, but are not limited to, corn (Zea mays), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet, Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), fonio (Eleusine coracana), wheat (Triticum species, e.g., Triticum aestivum, Triticum monococcum), sugarcane (Saccharum spp.), oats (Avena), barley (Hordeum), switchgrass (Panicum virgatum), pineapple (Ananas comosus), banana (Musa spp.), palm, ornamental plants, turf grasses, and other grasses. Examples of dicotyledonous plants that can be used include, but are not limited to, soybean (Glycine max), Brassica species (e.g., but not limited to: oilseed rape or canola) (Brassica napus, B. campestris, Brassica rapa, Brassica juncea), alfalfa (Medicago sativa), tobacco (Nicotiana tabacum), Arabidopsis (Arabidopsis thaliana), sunflower (Helianthus annuus), cotton (Gossypium arboreum, Gossypium barbadense), peanut (Arachis hypogaea), tomato (Solanum lycopersicum), potato (Solanum tuberosum).Additional plants that can be used include safflower (Carthamus tinctorius), sweet potato (Ipomoea batatas), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), vegetables, ornamentals, and conifers. Vegetables that can be used include tomato (Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima bean (Phaseolus limensis), peas (Lathyrus spp.), and members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo).Ornamental plants include azaleas (Rhododendron spp.), bigleaf hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnations (Dianthus caryophyllus), poinsettias (Euphorbia pulcherrima), and chrysanthemums. Coniferous trees that can be used include pines such as loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata); Douglas fir (Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); true firs such as silver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such as Western red cedar (Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis).

[0560] Particular plant cell types include, but are not limited to, cells obtained from whole plants, seedlings, meristematic tissues, ground tissues, vascular tissues, epidermal tissues, seeds, leaves, roots, shoots, stems, flowers, fruits, stolons, bulbs, tubers, corms, rhizomes, vegetative offshoots, buds, young shoots, tumor tissues, and various forms of cells and cultures (e.g., single cells, protoplasts, embryos, callus tissues). Cells can be present in a plant or in a plant organ, tissue culture, or cell culture

[0561] Therapeutic applications

[0562] The present application also encompasses the use of the base editors of the present application in the treatment of disease.

[0563] Modifying a disease-associated gene with the base editor of the present application can achieve up-regulation, down-regulation, inactivation, activation, mutation correction or introduction of a disease-associated site of the disease-associated gene, thereby achieving prevention and / or treatment of the disease and / or creation of a disease-associated model. For example, the target nucleic acid region described in the present application can be located in the protein coding region of the disease-associated gene, or for example, can be located in the gene expression regulatory region such as the promoter region or enhancer region, so as to achieve modification of the function of the disease-associated gene or modification of the expression of the disease-associated gene. Therefore, the modification of the disease-associated gene described herein includes modification of the disease-associated gene itself (e.g., protein coding region), and also includes modification of the expression regulatory region (such as promoter, enhancer, intron, etc.) thereof.

[0564] A "disease-associated" gene refers to any gene that produces a transcriptional or translational product at an abnormal level or in an abnormal form in a cell derived from a tissue affected by a disease, as compared to a tissue or cell that is not affected by the disease. It can be a gene that is expressed at an abnormally high level in cases where altered expression is associated with the occurrence and / or progression of the disease; it can be a gene that is expressed at an abnormally low level. A disease-associated gene also refers to a gene having one or more mutations or genetic variations that are directly responsible or in linkage disequilibrium with one or more genes responsible for the etiology of the disease. The mutation or genetic variation is, for example, a single nucleotide variation (SNV). The transcriptional or translational product can be known or unknown, and can be at a normal or abnormal level.

[0565] Therefore, the present application also provides a method of treating a disease in a subject in need thereof, comprising delivering to the subject an effective amount of the base editor of the present application to modify a gene associated with the disease (e.g., by fusing the protein or multiple proteins to deaminate mitochondrial DNA). The present application also provides use of a base editor in the manufacture of a pharmaceutical composition for treating a disease in a subject in need thereof, wherein the base editor is used to modify a gene associated with the disease. The present application also provides a pharmaceutical composition for treating a disease in a subject in need thereof, comprising a base editor of the present application, and optionally a pharmaceutically acceptable carrier, wherein the base editor is used to modify a gene associated with the disease.

[0566] In some embodiments, the fusion proteins or base editors described herein are used to introduce point mutations into a nucleic acid by deaminating a target nucleobase (e.g., a C residue). In some embodiments, deamination of the target nucleobase results in correction of a genetic deficiency, e.g., in correcting a point mutation that results in loss of function in a gene product. In some embodiments, the genetic deficiency is associated with a disease or disorder (e.g., a lysosomal storage disease or a metabolic disease such as, e.g., Type I diabetes). In some embodiments, the methods provided herein can be used to introduce an inactivating point mutation into a gene or allele that encodes a gene product associated with a disease or disorder.

[0567] In some embodiments, the aim of the schemes described herein is to restore function to a dysregulated gene via genome editing. The nucleobase editing proteins provided herein are for use in in vitro gene editing of human cells, such as to correct disease-associated mutations in human cell cultures.

[0568] In some embodiments, the aim of the schemes described herein is to treat a disease associated with or caused by a point mutation that can be corrected by the DNA base editing fusion proteins provided herein. In some embodiments, the disease is a proliferative disease. In some embodiments, the disease is a genetic disease. In some embodiments, the disease is a de novo disease. In some embodiments, the disease is a metabolic disease. In some embodiments, the disease is a lysosomal storage disease.

[0569] In some embodiments, the aim of the schemes described herein is to be useful in treating a mitochondrial disease or disorder. As used herein, a “mitochondrial disease” relates to a disease caused by abnormal mitochondria, e.g., mitochondrial gene mutations, gene mutations in enzyme pathways, etc. Examples of diseases include, but are not limited to, neurological disease, loss of motor control, muscle weakness and pain, gastrointestinal disease and difficulty swallowing, poor growth, heart disease, liver disease, diabetes, respiratory complications, seizures, vision / hearing problems, lactic acidosis, developmental delays, and susceptibility to infection.

[0570] Examples of diseases contemplated in the present application include, but are not limited to, genetic diseases, circulatory diseases, muscle diseases, diseases of the brain, central nervous system, and immune system, Alzheimer’s disease, secretase disorders, amyotrophic lateral sclerosis (ALS), autism, trinucleotide repeat expansion disorders, hearing diseases, gene targeting therapy of non-dividing cells (neurons, muscle), liver and kidney diseases, epithelial cell and lung diseases, cancer, Usher syndrome or retinitis pigmentosa-39, cystic fibrosis, HIV and AIDS, beta thalassemia, sickle cell disease, herpes simplex virus, autism, drug addiction, age-related macular degeneration, schizophrenia. Other diseases treated by correcting point mutations or introducing inactivating mutations into disease-associated genes are known to those skilled in the art, and thus the present application is not limited in this respect. In addition to the diseases exemplarily described in the present application, other related diseases can also be treated using the strategies and fusion proteins provided in the present application, which will be apparent to those skilled in the art. Diseases or targets to which the present application can be applied are referred to the related diseases listed in WO2015089465A1 (PCT / US2014 / 070135), WO2016205711A1 (PCT / US2016 / 038181), WO2018141835A1 (PCT / EP2018 / 052491), WO2020191234A1 (PCT / US2020 / 023713), WO2020191233A1 (PCT / US2020 / 023712), WO2019079347A1 (PCT / US2018 / 056146), WO2021155065A1 (PCT / US2021 / 015580) for base editors.

[0571] Applications in plants

[0572] The base editing fusion proteins, base editors, and methods of producing genetically modified cells of the present application are particularly suitable for use in genetically modifying plants. Preferably, the plant is a crop plant, including but not limited to wheat, rice, maize, soybean, sunflower, sorghum, oilseed rape, alfalfa, cotton, barley, millet, sugarcane, tomato, tobacco, cassava, and potato. More preferably, the plant is rice.

[0573] In another aspect, the present application provides a method of producing a genetically modified plant, comprising introducing a base editor of the present application into at least one of said plants, thereby causing one or more nucleotide substitutions within a target nucleic acid region in the genome of said at least one plant.

[0574] In some embodiments, the method further comprises screening the at least one plant for plants having the desired one or more nucleotide substitutions.

[0575] In the methods of the application, the base editing composition can be introduced into the plant by various methods well known to those skilled in the art. Methods that can be used to introduce the base editors of the application into plants include, but are not limited to, biolistics, PEG-mediated protoplast transformation, Agrobacterium-mediated transformation, plant virus-mediated transformation, pollen tube pathway, and ovary injection. Preferably, the base editing composition is introduced into the plant by transient transformation.

[0576] In the methods of the application, the modification of the target sequence is achieved simply by introducing or producing the base editing fusion protein in the plant cell, and the modification can be stably inherited without the need to stably transform the plant with an exogenous polynucleotide encoding the components of the base editor. This avoids potential off-target effects of the stably present (constitutively produced) base editing composition, and also avoids integration of the exogenous nucleotide sequence into the plant genome, thereby providing higher biosafety.

[0577] In some preferred embodiments, the introduction is performed in the absence of selection pressure, thereby avoiding integration of the exogenous nucleotide sequence into the plant genome.

[0578] In some embodiments, the introduction comprises transforming the base editor of the application into an isolated plant cell or tissue, and then regenerating the transformed plant cell or tissue into a whole plant. Preferably, the regeneration is performed in the absence of selection pressure, i.e. without using any selection agent for the selection gene carried on the expression vector during tissue culture. The absence of selection agent can improve the efficiency of plant regeneration, and allow obtaining a modified plant that is free of the exogenous nucleotide sequence.

[0579] In other embodiments, the base editor of the application can be transformed into a specific location on a whole plant, such as a leaf, a shoot tip, a pollen tube, a young ear, or a hypocotyl. This is particularly suitable for transformation of plants that are difficult to regenerate by tissue culture.

[0580] Thus, in some embodiments, the genetic modification and breeding of plants using the methods of the application can result in plants whose genome is free of integration of the exogenous polynucleotide, i.e. transgene-free modified plants.

[0581] In some embodiments of the application, wherein the modified target nucleic acid region is associated with a plant trait, such as an agronomic trait, the one or more nucleotide substitutions result in the plant having an altered, preferably improved, trait, such as an agronomic trait, relative to the wild type plant.

[0582] In some embodiments, the method further comprises the step of screening the plant for the desired one or more nucleotide substitutions and / or the desired trait, such as an agronomic trait.

[0583] In some embodiments of the application, the method further comprises obtaining progeny of the genetically modified plant. Preferably, the genetically modified plant or its progeny has the desired one or more nucleotide substitutions and / or the desired trait, such as an agronomic trait.

[0584] In another aspect, the present application also provides a genetically modified plant or its progeny or a part thereof, wherein the plant is obtained by the above-mentioned method of the present application. In some embodiments, the genetically modified plant or its progeny or a part thereof is non-transgenic. Preferably, the genetically modified plant or its progeny has the desired genetic modification and / or the desired trait, such as an agronomic trait.

[0585] In another aspect, the present application also provides a plant breeding method, comprising crossing a genetically modified first plant comprising one or more nucleotide substitutions in a target nucleic acid region with a second plant not containing the one or more nucleotide substitutions, thereby introducing the one or more nucleotide substitutions into the second plant. Preferably, the genetically modified first plant has the desired trait, such as an agronomic trait.

[0586] Examples

[0587] A further understanding of the application can be obtained by reference to the specific examples given herein, which are meant to be purely exemplary and are not intended to be limiting to the scope of the present application. It will be apparent to those skilled in the art that various modifications and variations can be made to the present application without departing from the spirit or scope of the application. Thus, it is intended that the present application cover modifications and variations of this application provided they come within the scope of the appended claims and their equivalents.

[0588] Some of the sequences of the elements used in the subsequent examples are shown below:

[0589] OsBADH2 Left TALE repeat

[0590] LTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQ

[0591] RLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASN

[0592] NGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGL

[0593] TPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQR

[0594] LLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHD

[0595] GGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLT

[0596] PDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRL

[0597] LPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGG

[0598] KQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPD

[0599] QVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALE(SEQ ID NO.89)

[0600] OsBADH2Right TALE repeat

[0601] LTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQ

[0602] RLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASN

[0603] GGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGL

[0604] TPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQR

[0605] LLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNG

[0606] GGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLT

[0607] PDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRL

[0608] LPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGG

[0609] KQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKALETVQRLPVLCQDHGLTPD

[0610] QVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALE(SEQ ID NO.90)

[0611] OsDEP1Left TALErepeat

[0612] LTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQ

[0613] RLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNI

[0614] GGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGGKQALETVQRLLPVLCQDHGLT

[0615] PDQVVAIASNIGGKQALETVQRLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRL

[0616] LPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGG

[0617] KQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPD

[0618] QVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPV

[0619] LCQDHGLTPDQVVAIASNNGGKQALETVQRLPVLCQDHGLTPDQVVAIASNNGGKQ

[0620] ALETVQRLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLPVLCQDHGLTPDQV

[0621] VIASNNGGKQALETVQRLLPVQDHGLTPDQVVAIASHDGGKQALETVQRLPVL

[0622] CQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQ

[0623] ALE

[0624] (SEQ ID NO.91)

[0625] OsDEP1 Right TALE repeat

[0626] LTPDQVVAIASNNGGKQALETVQRLPVLCQDHGLTPDQVVAIASNNGGKQALETVQ

[0627] RLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLPVLCQDHGLTPDQVVAIASN

[0628] GGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGL

[0629] TPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQR

[0630] LLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNN

[0631] GGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLT

[0632] PDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRL

[0633] LPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGG

[0634] KQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALE

[0635] (SEQ ID NO. 92)

[0636] OsCKX2 Left TALE repeat

[0637] LTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQ

[0638] RLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASN

[0639] NGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGL

[0640] TPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQR

[0641] LLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHD

[0642] GGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLT

[0643] PDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRL

[0644] LPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGG

[0645] KQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTP

[0646] QVVAIASNNGGKQALE

[0647] (SEQ ID NO. 93)

[0648] OsCKX2 Right TALE repeat

[0649] LTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQ

[0650] RLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNI

[0651] GGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLT

[0652] PDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRL

[0653] LPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDG

[0654] GKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTP

[0655] DQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLL

[0656] PVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGG

[0657] KQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPD

[0658] QVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLP

[0659] VLCQDHGLTPDQVVAIASHDGGKQALE

[0660] (SEQ ID NO. 94)

[0661] OsSD1 Left TALE repeat

[0662] LTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQ

[0663] RLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNN

[0664] GGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLT

[0665] PDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRL

[0666] LPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGG

[0667] KQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPD

[0668] QVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLP

[0669] VLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGK

[0670] QALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQ

[0671] VVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPV

[0672] LCQDHGLTPDQVVAIASNNGGKQALE

[0673] (SEQ ID NO. 95)

[0674] OsSD1 Right TALE repeat

[0675] LTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQ

[0676] RLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASN

[0677] NGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGL

[0678] TPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQR

[0679] LLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNN

[0680] GGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLT

[0681] PDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRL

[0682] LPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGG

[0683] GKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTP

[0684] DQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLL

[0685] PVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGG

[0686] KQALE

[0687] (SEQ ID NO.96)

[0688] SIRT6 Left TALE repeat

[0689] LTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQR

[0690] LLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIANNN

[0691] GGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLT

[0692] PEQVVAIANNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIANNNGGKQALETVQR

[0693] LLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIANNN

[0694] GGKQALETVQRLLPVLCQDHGLTPEQVVAIANNNGGKQALETVQRLLPVLCQAHGLT

[0695] PDQVVAIANNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIANNNGGKQALETVQR

[0696] LLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNG

[0697] GGKQALETVQRLLPVLCQAHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQAHGL

[0698] TPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGRPALE (SEQ ID NO. 97)

[0699] SIRT6 Right TALE repeat

[0700] LTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQ

[0701] RLLPVLCQAHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIANN

[0702] NGGKQALETVQRLLPVLCQDHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQDHG

[0703] LTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIANNNGGKQALETVQ

[0704] RLLPVLCQAHGLTPAQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASH

[0705] DGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGL

[0706] TPDQVVAIANNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQR

[0707] LLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDG

[0708] GKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTP

[0709] AQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNNGGRPALE (SEQ ID NO. 98)

[0710] OsRbcL Left TALE repeat

[0711] LTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQ

[0712] RLLPVLCQDHGLTPDQVVAIASNIGGKQAVETVQRLLPVLCQAHGLTPAQVVAIASHD

[0713] GGKQAVETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQAVETVQRLLPVLCQDHGL

[0714] TPDQVVAIASNIGGKQALETLQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRL

[0715] LPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGG

[0716] KQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPD

[0717] QVVAIASNGGGKQAVETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQAVETVQRLLP

[0718] VLCQAHGLTPAQVVAIASNIGGKQAVETVQRLLPVLCQDHGLTPEQVVAIASNGGGK

[0719] QALE

[0720] (SEQ ID NO. 99)

[0721] OsRbcL Right TALE repeat

[0722] LTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQAVETVQ

[0723] RLLPVLCQDHGLTPDQVVAIASNIGGKQAVETVQRLLPVLCQAHGLTPAQVVAIASNI

[0724] GGKQAVETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETLQRLLPVLCQDHGLT

[0725] PDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRL

[0726] LPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGG

[0727] KQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQAVETVQRLLPVLCQDHGLTPD

[0728] QVVAIASNIGGKQAVETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQAVETVQRLLPV

[0729] LCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQ

[0730] ALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQV

[0731] VAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALE (SEQ ID NO. 100)

[0732] ND6 Left TALE repeat

[0733] LLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHD

[0734] LLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHD

[0735] LLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHD

[0736] LLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHD

[0737] LLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHD

[0738] LLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHD

[0739] (SEQ ID NO. 101)

[0740] ND6 Right TALE repeat

[0741] LLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHD

[0742] LLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHD

[0743] LLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHD

[0744] LLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHD

[0745] LLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHD

[0746] KQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPE

[0747] QVVAIASNGGGRPALE

[0748] (SEQ ID NO. 102)

[0749] ND5.1 Left TALE repeat

[0750] LTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQR

[0751] LLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIG

[0752] GKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTP

[0753] EQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLP

[0754] VLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGK

[0755] QALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQV

[0756] VAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVL

[0757] CQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQAL

[0758] ETVQRLLPVLCQAHGLTPEQVVAIASNGGGRPALE

[0759] (SEQ ID NO. 103)

[0760] ND5.1 Right TALE repeat

[0761] LTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQ

[0762] RLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNG

[0763] GGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLT

[0764] PEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLL

[0765] PVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGG

[0766] KQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQ

[0767] VVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVL

[0768] CQAHGLTPEQVVAIASNIGGRPALE

[0769] (SEQ ID NO. 104)

[0770] ND3 Left TALE repeat

[0771] LTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASHDGGKQALETVQR

[0772] LLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIG

[0773] GKQALETVQALLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGLTP

[0774] EQVVAIASNGGGKQALETVQALLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLL

[0775] PVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQVVAIASNNGG

[0776] KQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGLTPE

[0777] QVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLP

[0778] VLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASHDGGK

[0779] QALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGLTPQQ

[0780] VVAIASNIGGRPALE

[0781] (SEQ ID NO. 105)

[0782] ND3 Right TALE repeat

[0783] LTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGGKQALETVQR

[0784] LLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNN

[0785] GGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLT

[0786] PEQVVAIASNNGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLL

[0787] PVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGK

[0788] QALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGLTPEQ

[0789] VVAIASNNGGKQALETVQALLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPV

[0790] LCQAHGLTPEQVVAIASNIGGKQLETVQRLLPVLCQAHGLTPQQVVAIASHDGGKQA

[0791] LETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGLTPQQVV

[0792] AIASHDGGRPALE

[0793] (SEQ ID NO. 106)

[0794] ND1.3 Left TALE repeat

[0795] LTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGGKQALETVQ

[0796] RLLPVLCQAHGLTPQQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASH

[0797] DGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGL

[0798] TPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRL

[0799] LPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGG

[0800] GKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPE

[0801] QVVAIASNNGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLP

[0802] VLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGK

[0803] QALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQV

[0804] VAIASNNGGRPALE

[0805] (SEQ ID NO. 107)

[0806] ND1.3 Right TALE repeat

[0807] LTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPQQVVAIASHDGGKQALETVQ

[0808] RLLPVLCQAHGLTPQQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASH

[0809] DGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGL

[0810] TPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQR

[0811] LLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGLTPEQVVAIASNN

[0812] GGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLT

[0813] PEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALL

[0814] PVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGG

[0815] KQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPE

[0816] QVVAIASNGGGKQALETVQALLPVLCQAHGLTPQQVVAIASHDGGRPALE (SEQ ID NO. 108)

[0817] ND1.2 Left TALE repeat

[0818] LTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPQQVVAIASHDGGKQALETVQ

[0819] RLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNI

[0820] GGKQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLT

[0821] PEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRL

[0822] LPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGGG

[0823] KQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPE

[0824] QVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLP

[0825] VLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNNGGRP

[0826] ALE

[0827] (SEQ ID NO. 109)

[0828] ND1.2 Right TALE repeat

[0829] LTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPQQVVAIASNNGGKQALETVQ

[0830] RLLPVLCQAHGLTPQQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNN

[0831] GGKQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLT

[0832] PEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLL

[0833] PVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGK

[0834] QALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGLTPEQ

[0835] VVAIASNNGGKQALETVQALLPVLCQAHGLTPQQVVAIASHDGGRPALE (SEQ ID NO. 110)

[0836] ND6.2 Left TALE repeat (TALE-L2)

[0837] LTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPQQVVAIASHDGGKQALETVQ

[0838] RLLPVLCQAHGLTPQQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNI

[0839] GGKQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLT

[0840] PEQVVAIASNNGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRL

[0841] LPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGG

[0842] GKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTP

[0843] EQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLP

[0844] VLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNIGGK

[0845] QALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPQQ

[0846] VVAIASNIGGRPALE

[0847] (SEQ ID NO.111)

[0848] ND6.2 Right TALE repeat(TALE-R2)

[0849] LTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGGKQALETVQ

[0850] RLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASN

[0851] NGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGL

[0852] TPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRL

[0853] LPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGG

[0854] KQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPE

[0855] QVVAIASNGGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLP

[0856] VLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNIGGK

[0857] QALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGLTPQQ

[0858] VVAIASHDGGRPALE

[0859] (SEQ ID NO.112)

[0860] ND6.2 Left TALE repeat(TALE-L1)

[0861] LTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQ

[0862] RLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASH

[0863] DGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGL

[0864] TPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQR

[0865] LLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLPVLCQDHGLTPDQVVAIASNGG

[0866] GKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHG

[0867] (SEQ ID NO.185)

[0868] ND6.2Left TALErepeat(TALE-L3)

[0869] LTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQ

[0870] RLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLPVLCQDHGLTPDQVVAIASN

[0871] GGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGL

[0872] TPDQVVAIASNIGGKQALETVQRLPVLCQDHGLTPDQVVAIASNNGGKQALETVQR

[0873] LLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLPVLCQDHGLTPDQVVAIASNIG

[0874] GKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTP

[0875] DQVVAIASNIGGKQALETVQRLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLL

[0876] PVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGG

[0877] KQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPD

[0878] QVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLP

[0879] VLCQDHG

[0880] (SEQ ID NO. 186)

[0881] ND6.2 Right TALE repeat (TALE-R1)

[0882] LTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQ

[0883] RLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNG

[0884] GGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLT

[0885] PDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRL

[0886] LPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGG

[0887] KQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPD

[0888] QVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLP

[0889] VLCQDHG

[0890] (SEQ ID NO. 187)

[0891] XTEN spacer peptide NSGSETPGTSESATPES

[0892] (SEQ ID NO. 113)

[0893] 48 amino acid spacer peptide SGSETPGTSESATPESSGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO. 114)

[0894] 16 amino acid spacer peptide SGSETPGTSESATPES

[0895] (SEQ ID NO. 115)

[0896] 14 amino acid spacer peptide SGGGSGGSGGSGGS

[0897] (SEQ ID NO. 116)

[0898] 11 amino acid spacer peptide SGGSGGSGGSS

[0899] (SEQ ID NO. 117)

[0900] 4 amino acid spacer peptide SGGS

[0901] (SEQ ID NO. 118)

[0902] Yb

[0903] MMATFSCVCCGTLTTSTYCGKRCERKHVYSETRNKRLELYKKYLLEPQKCALNGIVG

[0904] HSCGMPCSIAEEACDQLPIVSRFCGQKHADLYDSLLKRSEQELLLEFLQKKMQELKLS

[0905] HIVKMAKLESEVNAIRKSVASSFEDSVGCDDSSSVSK

[0906] (SEQ ID NO. 119)

[0907] Figure 16A to Figure 16E and Figure 17A to Figure 17H The amino acid sequences of the vectors or elements involved in the above-mentioned embodiments are shown below, and the corresponding fusion proteins can be constructed according to the schematic diagrams of the constructs shown in Figure 16A to Figure 16E and Figure 17A to 17H the present specification, and the sequences disclosed in the present specification. OsBADH2-NLS-TALEN WT ( Figure 16A )

[0908] MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPSRMVDLRTLGYSQQQQE

[0909] KIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVTYQHIITALPEATHEDI

[0910] VGVGKQWSGARALEALLTDAGELRGPPLQLDTGQLVKIAKRGGVTAMEAVHASRNA

[0911] LTGAPLNLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGK

[0912] QALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQ

[0913] VVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPV

[0914] LCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQ

[0915] ALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQV

[0916] VAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVL

[0917] CQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQ

[0918] ALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQV

[0919] VAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLC

[0920] QDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQAL

[0921] ESIVAQLSRPDPALAALTNDHLVALACLGGRPAMDAVKKGLPHAPELIRRVNRRIGER

[0922] TSHRVAGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMK

[0923] VYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVKE

[0924] NQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHKTNCNGAVLS

[0925] VEELLIGGEMIKAGTLTLEEVRRKFNNGEINFEGRGSLLTCGDVEENPGPRMDYKDHD

[0926] GDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPARMVDLRTLGYSQQQQEKIKPKVRS

[0927] TVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVTYQHIITALPEATHEDIVGVGKQ

[0928] WSGARALEALLTDAGELRGPPLQLDTGQLVKIAKRGGVTAMEAVHASRNALTGAPL

[0929] NLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETV

[0930] QRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIAS

[0931] NGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDH

[0932] GLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETV

[0933] QRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIAS

[0934] NGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDH

[0935] GLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETV

[0936] QRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASN

[0937] NGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGL

[0938] TPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALESIVAQ

[0939] LSRPDPALAALTNDHLVALACLGGRPAMDAVKKGLPHAPELIRRVNRRIGERTSHRV

[0940] AGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGY

[0941] RGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMERYVEENQTR

[0942] NKHLNPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLI

[0943] GGEMIKAGTLTLEEVRRKFNNGEINF

[0944] (SEQ ID NO.120)

[0945] OsBADH2-NLS-TALE-L-FokI-L-T2A-TALE-R-FokI-RD450A ( Figure 16B

[0946] (SEQ ID NO. 121)

[0947] OsBADH2-NLS-TALE-L-FokI-L D450A -T2A-TALE-R-FokI-R( Figure 16B

[0948] (SEQ ID NO. 122)

[0949] NLS-A3A-XTEN-UGI( Figure 16B )

[0950] MKRTADGSEFESPKKKRKVMEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVER

[0951] LDNGTSVKMDQHRGFLHNQAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFI

[0952] SWSPCFSWGCAGEVRAFLQENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIM

[0953] TYDEFKHCWDTFVDHQGCPFQPWDGLDEHSQALSGRLRAILQNQGNSGSETPGTSES

[0954] ATPESTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVM

[0955] LLTSDAPEYKPWALVIQDSNGENKIKML

[0956] (SEQ ID NO. 123)

[0957] NLS-UGI( Figure 16B )

[0958] MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTS

[0959] DAPEYKPWALVIQDSNGENKIKMLMKRTADGSEFESPKKKRKV (SEQ ID NO. 163)

[0960] NLS-C57-XTEN-UGI( Figure 16B )

[0961] MKRTADGSEFESPKKKRKVLEAVRARLIGEGGGPGAVPEGGDGPPAVPAEEVERLRG

[0962] ELPPPVVPGTGQKTHGRWIGPDGRVRAIVSGRDEDAALVHAQLAAKGIPDEPTRNSDV

[0963] EQKLAAHMVANGIRHVTLVINHRPCRGFDDSCDTLVPIILPEGCTLTVHGQTDKGMRV

[0964] RVRYTGGARPWWSNSGSETPGTSESATPESTNLSDIIEKETGKQLVIQESILMLPEEVEE

[0965] VIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML (SEQ ID NO. 124)

[0966] NLS-rAPOBEC1-XTEN-UGI( Figure 16B )

[0967] MKRTADGSEFESPKKKRKVSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEI

[0968] NWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAIT

[0969] EFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYS

[0970] PSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPP

[0971] HILWATGLKNSGSETPGTSESATPESTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNK

[0972] PESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML (SEQ ID NO. 164)

[0973] TadA8e-NLS( Figure 16B )

[0974] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTA

[0975] HAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGA

[0976] AGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSINSGG

[0977] SMKRTADGSEFESPKKKRKV

[0978] (SEQ ID NO. 166)

[0979] mExoI-NLS( Figure 16B )

[0980] MGIQGLLQFIQEASEPVNVKKYKGQAVAVDTYCWLHKGAIACAEKLAKGEPTDRYV

[0981] GFCMKFVNMLLSYGVKPILIFDGCTLPSKKEVERSRRERRQSNLLKGKQLLREGKVSE

[0982] ARDCFARSINITHAMAHKVIKAARALGVDCLVAPYEADAQLAYLNKAGIVQAVITED

[0983] SDLLAFGCKKVILKMDQFGNGLEVDQARLGMCKQLGDVFTEEKFRYMCILSGCDYLA

[0984] SLRGIGLAKACKVLRLANNPDIVKVIKKIGHYLRMNITVPEDYITGFIRANNTFLYQLV

[0985] FDPIQRKLVPLNAYGDDVNPETLTYAGQYVGDSVALQIALGNRDVNTFEQIDDYSPDT

[0986] MPAHSRSHSWNEKAGQKPPGTNSIWHKNYCPRLEVNSVSHAPQLKEKPSTLGLKQVIS

[0987] TKGLNLPRKSCVLKRPRNEALAEDDLLSQYSSVSKKIKENGCGDGTSPNSSKMSKSCP

[0988] DSGTAHKTDAHTPSKMRNKFATFLQRRNEESGAVVVPGTRSRFFCSSQDFDNFIPKKE

[0989] SGQPLNETVATGKATTSLLGALDCPDTEGHKPVDANGTHNLSSQIPGNAAVSPEDEAQ

[0990] SSETSKLLGAMSPPSLGTLRSCFSWSGTLREFSRTPSPSASTTLQQFRRKSDPPACLPEA

[0991] SAVVTDRCDSKSEMLGETSQPLHELGCSSRSQESMDSSCGLNTSSLSQPSSRDSGSEES

[0992] DCNNKSLDNQGEQNSKQHLPHFSKKDGLRRNKVPGLCRSSSMDSFSTTKIKPLVPARV

[0993] SGLSKKSGSMQTRKHHDVENKPGLQTKISELWKNFGFKKDSEKLPSCKKPLSPVKDNI

[0994] QLTPETEDEIFNKPECVRAQRAIFHMKRTADGSEFESPKKKRKV (SEQ ID NO. 125)

[0995] Trex2-NLS( Figure 16B )

[0996] MSEPPRAETFVFLDLEATGLPNMDPEIAEISLFAVHRSSLENPERDDSGSLVLPRVLDKL

[0997] TLCMCPERPFTAKASEITGLSSESLMHCGKAGFNGAVVRTLQGFLSRQEGPICLVAHN

[0998] GFDYDFPLLCTELQRLGAHLPQDTVCLDTLPALRGLDRAHSHGTRAQGRKSYSLASLF

[0999] HRYFQAEPSAAHSAEGDVHTLLLIFLHRAPELLAWADEQARSWAHIEPMYVPPDGPSL

[1000] EAMKRTADGSEFESPKKKRKV

[1001] (SEQ ID NO. 126)

[1002] OsBADH2-NLS-A3A-TALE-L-FokI-L-T2A-TALE-R-FokI-R D450A ( Figure 16C

[1003] OsBADH2-NLS-A3A-TALE-L-FokI-L D450A -T2A-TALE-R-FokI-R( Figure 16C

[1004] mExoI-NLS( Figure 16C )

[1005] MGIQGLLQFI QEASEPVNVKK YKGQAVAVDT YCWLHKGAI ACAEKLAKGE PTDRYV

[1006] GFCMKFVNML LS YGVKPILIF DGCTLPSKKE VERSRRERRQ SNLLKGKQLL REGKVSE

[1007] ARDCFARSINITHAMAHKVIKAARALGVDCLVAPYEADAQLAYLNKAGIVQAVITED

[1008] SDLLAFGCKKVILKMDQFGNGLEVDQARLGMCKQLGDVFTEEKFRYMCILSGCDYLA

[1009] SLRGIGLAKACKVLRLANNPDIVKVIKKIGHYLRMNITVPEDYITGFIRANNTFLYQLV

[1010] FDPIQRKLVPLNAYGDDVNPETLTYAGQYVGDSVALQIALGNRDVNTFEQIDDYSPDT

[1011] MPAHSRSHSWNEKAGQKPPGTNSIWHKNYCPRLEVNSVSHAPQLKEKPSTLGLKQVIS

[1012] TKGLNLPRKSCVLKRPRNEALAEDDLLSQYSSVSKKIKENGCGDGTSPNSSKMSKSCP

[1013] DSGTAHKTDAHTPSKMRNKFATFLQRRNEESGAVVVPGTRSRFFCSSQDFDNFIPKKE

[1014] SGQPLNETVATGKATTSLLGALDCPDTEGHKPVDANGTHNLSSQIPGNAAVSPEDEAQ

[1015] SSETSKLLGAMSPPSLGTLRSCFSWSGTLREFSRTPSPSASTTLQQFRRKSDPPACLPEA

[1016] SAVVTDRCDSKSEMLGETSQPLHELGCSSRSQESMDSSCGLNTSSLSQPSSRDSGSEES

[1017] DCNNKSLDNQGEQNSKQHLPHFSKKDGLRRNKVPGLCRSSSMDSFSTTKIKPLVPARV

[1018] SGLSKKSGSMQTRKHHDVENKPGLQTKISELWKNFGFKKDSEKLPSCKKPLSPVKDNI

[1019] QLTPETEDEIFNKPECVRAQRAIFHMKRTADGSEFESPKKKRKV (SEQ ID NO. 129)

[1020] Trex2-NLS( Figure 16C )

[1021] MSEPPRAETFVFLDLEATGLPNMDPEIAEISLFAVHRSSLENPERDDSGSLVLPRVLDKL

[1022] TLCMCPERPFTAKASEITGLSSESLMHCGKAGFNGAVVRTLQGFLSRQEGPICLVAHN

[1023] GFDYDFPLLCTELQRLGAHLPQDTVCLDTLPALRGLDRAHSHGTRAQGRKSYSLASLF

[1024] HRYFQAEPSAAHSAEGDVHTLLLIFLHRAPELLAWADEQARSWAHIEPMYVPPDGPSL

[1025] EAMKRTADGSEFESPKKKRKV

[1026] (SEQ ID NO. 130)

[1027] UGI-NLS( Figure 16C )

[1028] MKRTADGSEFESPKKKRKVTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDIL

[1029] VHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML

[1030] (SEQ ID NO. 131)

[1031] OsBADH2-NLS-A3A-TALE-L-FokI-L-T2A-TALE-R-FokI-R D450A -UGI( Figure 16D

[1032] (SEQ ID NO. 132)

[1033] OsBADH2-NLS-A3A-TALE-L-FokI-L D450A -T2A-TALE-R-FokI-R-UGI( Figure 16D

[1034] (SEQ ID NO. 133)

[1035] mExoI-NLS( Figure 16D )

[1036] MGIQGLLQFI QEASEPVNVKKYKGQAVAVDTYCWLHKGAIACAEKLAKGEPTDRYV

[1037] GFCMKFVNMLLSYGVKPILIFDGCTLPSKKEVERSRRERRQSNLLKGKQLLREGKVSE

[1038] ARDCFARSINITHAMAHKVIKAARALGVDCLVAPYEADAQLAYLNKAGIVQAVITED

[1039] SDLLAFGCKKVILKMDQFGNGLEVDQARLGMCKQLGDVFTEEKFRYMCILSGCDYLA

[1040] SLRGIGLAKACKVLRLANNPDIVKVIKKIGHYLRMNITVPEDYITGFIRANNTFLYQLV

[1041] FDPIQRKLVPLNAYGDDVNPETLTYAGQYVGDSVALQIALGNRDVNTFEQIDDYSPDT

[1042] MPAHSRSHSWNEKAGQKPPGTNSIWHKNYCPRLEVNSVSHAPQLKEKPSTLGLKQVIS

[1043] TKGLNLPRKSCVLKRPRNEALAEDDLLSQYSSVSKKIKENGCGDGTSPNSSKMSKSCP

[1044] DSGTAHKTDAHTPSKMRNKFATFLQRRNEESGAVVVPGTRSRFFCSSQDFDNFIPKKE

[1045] SGQPLNETVATGKATTSLLGALDCPDTEGHKPVDANGTHNLSSQIPGNAAVSPEDEAQ

[1046] SSETSKLLGAMSPPSLGTLRSCFSWSGTLREFSRTPSPSASTTLQQFRRKSDPPACLPEA

[1047] SAVVTDRCDSKSEMLGETSQPLHELGCSSRSQESMDSSCGLNTSSLSQPSSRDSGSEES

[1048] DCNNKSLDNQGEQNSKQHLPHFSKKDGLRRNKVPGLCRSSSMDSFSTTKIKPLVPARV

[1049] SGLSKKSGSMQTRKHHDVENKPGLQTKISELWKNFGFKKDSEKLPSCKKPLSPVKDNI

[1050] QLTPETEDEIFNKPECVRAQRAIFHMKRTADGSEFESPKKKRKV (SEQ ID NO. 134)

[1051] Trex2-NLS( Figure 16D )

[1052] MSEPPRAETFVFLDLEATGLPNMDPEIAEISLFAVHRSSLENPERDDSGSLVLPRVLDKL

[1053] TLCMCPERPFTAKASEITGLSSESLMHCGKAGFNGAVVRTLQGFLSRQEGPICLVAHN

[1054] GFDYDFPLLCTELQRLGAHLPQDTVCLDTLPALRGLDRAHSHGTRAQGRKSYSLASLF

[1055] HRYFQAEPSAAHSAEGDVHTLLLIFLHRAPELLAWADEQARSWAHIEPMYVPPDGPSL

[1056] EAMKRTADGSEFESPKKKRKV

[1057] (SEQ ID NO. 135)

[1058] OsBADH2-NLS-A3A-TALE-L-FokI-L-T2A-TALE-R-FokI-R D450A -UGI--mExoI-NLS( Figure 16E )

[1059]

[1060] (SEQ ID NO. 136)

[1061] OsBADH2-NLS-A3A-TALE-L-FokI-L D450A -T2A-TALE-R-FokI-R-UGI--mExoI-NLS( Figure 16E )

[1062]

[1063] (SEQ ID NO. 137)

[1064] ND6-MTS-TALE-L-FokI-L Figure 17A )

[1065] MALSRAVCGTSRQLAPVLGYLGSRQKHSLPDYPYDVPDYAGYPYDVPDYAGYPYDV

[1066] PDYAMDIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAA

[1067] LGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQ

[1068] LLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNNGGKQALETVQRLLPV

[1069] LCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQ

[1070] ALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQV

[1071] VAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVL

[1072] CQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQA

[1073] LETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVV

[1074] AIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGSQL

[1075] VKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHL

[1076] GGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVKENQTRNKHINP

[1077] NEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHKTNCNGAVLSVEELLIGGEMI

[1078] KAGTLTLEEVRRKFNNGEINF

[1079] (SEQ ID NO. 138)

[1080] ND6-MTS-TALE-R-FokI-RD 450A ( Figure 17B )

[1081] MASVLTPLLLRGLTGSARRLPVPRAKIHSLDYKDHDGDYKDHDIDYKDDDDKMDIAD

[1082] LRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQ

[1083] DMIAALPEATHEAIVGVGKRGAGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGG

[1084] VTAVEAVHAWRNALTGAPLNLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTP

[1085] EQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLP

[1086] VLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGK

[1087] QALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQ

[1088] VLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVL

[1089] VVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQ

[1090] VVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQ

[1091] VVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQ

[1092] VVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQ

[1093] VVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQ

[1094] VVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQ

[1095] VVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQ D450A ( Figure 17A )

[1096] VVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQ

[1097] VVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQ

[1098] VVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQ

[1099] VVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQ

[1100] LCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQ

[1101] ALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQV

[1102] VAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVL

[1103] CQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQA

[1104] LETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVV

[1105] AIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGSQL

[1106] VKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHL

[1107] GGSRKPAGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVKENQTRNKHINP

[1108] NEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHKTNCNGAVLSVEELLIGGEMI

[1109] KAGTLTLEEVRRKFNNGEINF

[1110] (SEQ ID NO. 140)

[1111] ND6-MTS-TALE-R-FokI-R( Figure 17B )

[1112] MASVLTPLLLRGLTGSARRLPVPRAKIHSLDYKDHDGDYKDHDIDYKDDDDKMDIAD

[1113] LRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQ

[1114] DMIAALPEATHEAIVGVGKRGAGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGG

[1115] VTAVEAVHAWRNALTGAPLNLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTP

[1116] EQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLP

[1117] VLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGK

[1118] QALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQ

[1119] VVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVL

[1120] CQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQA

[1121] LETVQRLLPVLCQAHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLV

[1122] ALACLGGRPALDAVKKGLGGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQ

[1123] DRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNL

[1124] PIGQADEMERYVEENQTRNKHLNPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTR

[1125] LNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF (SEQ ID NO. 141)

[1126] MTS-mExoI( Figure 17D )

[1127] MALSRAVCGTSRQLAPVLGYLGSRQKHSLPDMGIQGLLQFIQEASEPVNVKKYKGQA

[1128] VAVDTYCWLHKGAIACAEKLAKGEPTDRYVGFCMKFVNMLLSYGVKPILIFDGCTLP

[1129] SKKEVERSRRERRQSNLLKGKQLLREGKVSEARDCFARSINITHAMAHKVIKAARALG

[1130] VDCLVAPYEADAQLAYLNKAGIVQAVITEDSDLLAFGCKKVILKMDQFGNGLEVDQA

[1131] RLGMCKQLGDVFTEEKFRYMCILSGCDYLASLRGIGLAKACKVLRLANNPDIVKVIKK

[1132] IGHYLRMNITVPEDYITGFIRANNTFLYQLVFDPIQRKLVPLNAYGDDVNPETLTYAGQ

[1133] YVGDSVALQIALGNRDVNTFEQIDDYSPDTMPAHSRSHSWNEKAGQKPPGTNSIWHK

[1134] NYCPRLEVNSVSHAPQLKEKPSTLGLKQVISTKGLNLPRKSCVLKRPRNEALAEDDLL

[1135] SQYSSVSKKIKENGCGDGTSPNSSKMSKSCPDSGTAHKTDAHTPSKMRNKFATFLQRR

[1136] NEESGAVVVPGTRSRFFCSSQDFDNFIPKKESGQPLNETVATGKATTSLLGALDCPDTE

[1137] GHKPVDANGTHNLSSQIPGNAAVSPEDEAQSSETSKLLGAMSPPSLGTLRSCFSWSGTL

[1138] REFSRTPSPSASTTLQQFRRKSDPPACLPEASAVVTDRCDSKSEMLGETSQPLHELGCSS

[1139] RSQESMDSSCGLNTSSLSQPSSRDSGSEESDCNNKSLDNQGEQNSKQHLPHFSKKDGL

[1140] RRNKVPGLCRSSSMDSFSTTKIKPLVPARVSGLSKKSGSMQTRKHHDVENKPGLQTKI

[1141] SELWKNFGFKKDSEKLPSCKKPLSPVKDNIQLTPETEDEIFNKPECVRAQRAIFH (SEQ ID NO. 142)

[1142] MTS-Trex2( Figure 17D )

[1143] MALSRAVCGTSRQLAPVLGYLGSRQKHSLPDMSEPPRAETFVFLDLEATGLPNMDPEI

[1144] AEISLFAVHRSSLENPERDDSGSLVLPRVLDKLTLCMCPERPFTAKASEITGLSSESLMH

[1145] CGKAGFNGAVVRTLQGFLSRQEGPICLVAHNGFDYDFPLLCTELQRLGAHLPQDTVCL

[1146] DTLPALRGLDRAHSHGTRAQGRKSYSLASLFHRYFQAEPSAAHSAEGDVHTLLLIFLH

[1147] RAPELLAWADEQARSWAHIEPMYVPPDGPSLEA

[1148] (SEQ ID NO. 143)

[1149] MTS-A3A( Figure 17C )

[1150] MALSRAVCGTSRQLAPVLGYLGSRQKHSLPDEASPASGPRHLMDPHIFTSNFNNGIGR

[1151] HKTYLCYEVERLDNGTSVKMDQHRGFLHNQAKNLLCGFYGRHAELRFLDLVPSLQL

[1152] DPAQIYRVTWFISWSPCFSWGCAGEVRAFLQENTHVRLRIFAARIYDYDPLYKEALQM

[1153] LRDAGAQVSIMTYDEFKHCWDTFVDHQGCPFQPWDGLDEHSQALSGRLRAILQNQG

[1154] N

[1155] (SEQ ID NO. 144)

[1156] MTS-C57 / Sdd7( Figure 17C )

[1157] MALSRAVCGTSRQLAPVLGYLGSRQKHSLPDLEAVRARLIGEGGGPGAVPEGGDGPP

[1158] AVPAEEVERLRGELPPPVVPGTGQKTHGRWIGPDGRVRAIVSGRDEDAALVHAQLAA

[1159] KGIPDEPTRNSDVEQKLAAHMVANGIRHVTLVINHRPCRGFDDSCDTLVPIILPEGCTL

[1160] TVHGQTDKGMRVRVRYTGGARPWWS

[1161] (SEQ ID NO. 145)

[1162] MTS-UGI( Figure 17E )

[1163] MALSRAVCGTSRQLAPVLGYLGSRQKHSLPDGSSGGSTNLSDIIEKETGKQLVIQESIL

[1164] MLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKI

[1165] KML

[1166] (SEQ ID NO. 146)

[1167] ND6-MTS-A3A-TALE-L-FokI-L Figure 17F )

[1168] MALSRAVCGTSRQLAPVLGYLGSRQKHSLPDYPYDVPDYAGYPYDVPDYAGYPYDV

[1169] PDYAEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGFL

[1170] HNQAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGEVRA

[1171] FLQENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIMTYDEFKHCWDTFVDHQ

[1172] GCPFQPWDGLDEHSQALSGRLRAILQNQGNSGSETPGTSESATPESSGGSSGGSSGSET

[1173] PGTSESATPESSGGSSGGSMDIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGF

[1174] THAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVA

[1175] GELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNNG

[1176] GKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPE

[1177] QVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLP

[1178] VLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGK

[1179] QALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQ

[1180] VVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVL

[1181] CQAHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPAL

[1182] DAVKKGLGGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFF

[1183] MKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYV

[1184] KENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHKTNCNGAV

[1185] LSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF

[1186] (SEQ ID NO. 147)

[1187] ND6-MTS-Trex2-TALE-R-FokI-R D450A ( Figure 17G )

[1188] MASVLTPLLLRGLTGSARRLPVPRAKIHSLDYKDHDGDYKDHDIDYKDDDDKMSEPP

[1189] RAETFVFLDLEATGLPNMDPEIAEISLFAVHRSSLENPERDDSGSLVLPRVLDKLTLCM

[1190] CPERPFTAKASEITGLSSESLMHCGKAGFNGAVVRTLQGFLSRQEGPICLVAHNGFDY

[1191] DFPLLCTELQRLGAHLPQDTVCLDTLPALRGLDRAHSHGTRAQGRKSYSLASLFHRYF

[1192] QAEPSAAHSAEGDVHTLLLIFLHRAPELLAWADEQARSWAHIEPMYVPPDGPSLEASG

[1193] SETPGTSESATPESSGGSSGGSSGSETPGTSESATPESSGGSSGGSMDIADLRTLGYSQQ

[1194] QQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEA

[1195] THEAIVGVGKRGAGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHA

[1196] WRNALTGAPLNLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNI

[1197] GGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLT

[1198] PEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRL

[1199] LPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGG

[1200] GKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPE

[1201] QVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLP

[1202] VLCQAHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRP

[1203] ALDAVKKGLGGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVM

[1204] EFFMKVYGYRGKHLGGSRKPAGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEME

[1205] RYVEENQTRNKHLNPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNG

[1206] AVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF

[1207] (SEQ ID NO. 148)

[1208] ND6-MTS-UGI-Trex2-TALE-R-FokI-R D450A ( Figure 17H )

[1209] MASVLTPLLLRGLTGSARRLPVPRAKIHSLDYKDHDGDYKDHDIDYKDDDDKTNLSD

[1210] IIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYK

[1211] PWALVIQDSNGENKIKMLSGGSGGSGGSMSEPPRAETFVFLDLEATGLPNMDPEIAEIS

[1212] LFAVHRSSLENPERDDSGSLVLPRVLDKLTLCMCPERPFTAKASEITGLSSESLMHCGK

[1213] AGFNGAVVRTLQGFLSRQEGPICLVAHNGFDYDFPLLCTELQRLGAHLPQDTVCLDTL

[1214] PALRGLDRAHSHGTRAQGRKSYSLASLFHRYFQAEPSAAHSAEGDVHTLLLIFLHRAP

[1215] ELLAWADEQARSWAHIEPMYVPPDGPSLEASGSETPGTSESATPESSGGSSGGSSGSET

[1216] PGTSESATPESSGGSSGGSMDIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGF

[1217] THAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKRGAGARALEALLTVA

[1218] GELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNNG

[1219] GKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPE

[1220] QVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLP

[1221] VLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGK

[1222] QALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQ

[1223] VVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVL

[1224] CQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGRPA

[1225] LESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGSQLVKSELEEKKSE

[1226] LRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPAGAIY

[1227] TVGSPIDYGVIVDTKAYSGGYNLPIGQADEMERYVEENQTRNKHLNPNEWWKVYPSS

[1228] VTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVR

[1229] RKFNNGEINF

[1230] (SEQ ID NO. 149)

[1231] In the examples, exemplary amino acid sequences of the elements or fusion proteins are shown below, and the corresponding fusion proteins can be constructed using the construct schematics shown below, and the exemplary sequences shown below, in combination with the sequences disclosed herein. Figure 16A to Figure 16E and Figure 17A to 17H In the examples, exemplary amino acid sequences of the elements or fusion proteins are shown below, and the corresponding fusion proteins can be constructed using the construct schematics shown below, and the exemplary sequences shown below, in combination with the sequences disclosed herein.

[1232] In the examples, exemplary amino acid sequences of the elements or fusion proteins are shown below, and the corresponding fusion proteins can be constructed using the construct schematics shown below, and the exemplary sequences shown below, in combination with the sequences disclosed herein.

[1233] TALEN WT (SEQ ID NO. 154)

[1234]

[1235] TALE-FokI-R nickase(D450A) or TALE-FokI-R nickase (SEQ ID NO. 155)

[1236]

[1237] TALE-FokI-R nickase(D467A) (SEQ ID NO. 156)

[1238]

[1239] The nicking enzyme used in the OsDEP1 editing experiment:

[1240] TALEN WT (SEQ ID NO. 157)

[1241]

[1242] TALE-FokI-R nickase(D450A) or TALE-FokI-R nickase (SEQ ID NO. 158)

[1243]

[1244] TALE-FokI-R nickase(D467A) (SEQ ID NO. 159)

[1245]

[1246] The nicking enzyme used in the OsCKX2 editing experiment:

[1247] TALEN WT (SEQ ID NO. 160)

[1248]

[1249] TALE-FokI-R nickase (SEQ ID NO. 161)

[1250]

[1251] TALE-FokI-L nickase (SEQ ID NO. 162)

[1252]

[1253] In Examples 1-6, mExol: i.e., the foregoing mExol-NLS( Figure 16B ), SEQ ID NO. 125; A3A-UGI: i.e., the foregoing: NLS-A3A-XTEN-UGI( Figure 16B ), SEQ ID NO. 123; Trex2: i.e., the foregoing Trex2-NLS( Figure 16B ), SEQ ID NO. 126.

[1254] In Examples 1-6, the amino acid sequence of UGI is the foregoing NLS-UGI( Figure 16B )(SEQ ID NO.

[1255] 163).

[1256] In Example 4, the amino acid sequence of APOBEC1-UGI is the foregoing NLS- rAPOBEC1-XTEN-UGI( Figure 16B )(SEQ ID NO. 164).

[1257] In Example 1, the amino acid sequence of ExoV (ExoV-NLS) (SEQ ID NO. 165):

[1258] MAETGEEETASAEASGFSDLSDSELVEFLDLEEAKESAVSLSKPGPSAELPGKDDKPVSLQNWKGGLDVLSPMERFHLKYLYVTDLCTQNWCELQMVYGKELPGSLTPEKAAVLDTGASIHLAKELELHDLVTVPIATKEDAWAVKFLNILAMIPALQSEGRVREFPVFGEVEGIFLVGVIDELHYTSKGELELAELKTRRRPVLPLPAQKKKDYFQVSLYKYIFDAMVQGKVTPASLIHHTKLCLDKPLGPSVLRHARQGGVSVKSLGDLMELVFLSLTLSDLPAIDTLKLEYIHQETATILGTEIVAFEEKEVKSKVQHYVAYWMGHRDPQGVDVEEAWKCRTCDYVDICEWRRGSGVLSSSWEPKAKKFKMKRTADGSEFESPKKKRKV

[1259] In Example 5, the amino acid sequence of TadA-8e is the foregoing TadA8e-NLS( Figure 16B )(SEQ ID NO. 166).

[1260] In Example 6:

[1261] Amino acid sequence of mExoI-16aa-A3A-UGI (SEQ ID NO. 167):

[1262]

[1263] Amino acid sequence of mExo I-48aa-A3A-UGI (SEQ ID NO. 168):

[1264]

[1265] A3A-TALE-FokI-R nickase (SEQ ID NO. 169)

[1266]

[1267] APOBEC1-TALE-FokI-R nickase (SEQ ID NO. 170)

[1268]

[1269] A3A-TALE-FokI-L nickase (SEQ ID NO. 171)

[1270]

[1271] APOBEC1-TALE-FokI-L nickase (SEQ ID NO. 172)

[1272]

[1273] SIRT6-NLS-TALE-L-DddA N -UGI (SEQ ID NO. 173)

[1274]

[1275] SIRT6-NLS-TALE-R-DddA C -UGI (SEQ ID NO. 174)

[1276] MASVLTPLLLRGLTGSARRLPVPRAKIHSLDYKDHDGDYKDHDIDYKDDDDKMDIADLRTLGYSQ

[1277] QQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVG

[1278] VGKRGAGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPD

[1279] QVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGL

[1280] TPDQVVAIANNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIANNNGGKQALETVQRLLPVLCQD

[1281] HGLTPDQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLC

[1282] QAHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIANNNGGKQALETVQRLLP

[1283] VLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQR

[1284] LLPVLCQAHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALET

[1285] VQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQAL

[1286] ETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGK

[1287] QALETVQRLLPVLCQDHGLTPEQVVAIASNNGGRPALESIVAQLSRPDPALAALTNDHLVALACLG

[1288] GRPALDAVKKGLGGSAIPVKRGATGETKVFTGNSNSPKSPTKGGCSGGSTNLSDIIEKETGKQLVIQ

[1289] ESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML

[1290] In Examples 11, 14 and 15:

[1291] ND6-MTS-TALE-L-DddA N -UGI (SEQ ID NO. 175)

[1292] MALSRAVCGTSRQLAPVLGYLGSRQKHSLPDYPYDVPDYAGYPYDVPDYAGYPYDVPDYAMDIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGSGSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML

[1293] ND6-MTS-TALE-R-DddA C -UGI (SEQ ID NO. 176)

[1294] MASVLTPLLLRGLTGSARRLPVPRAKIHSLDYKDHDGDYKDHDIDYKDDDDKMDIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKRGAGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGSAIPVKRGATGETKVFTGNSNSPKSPTKGGCSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML

[1295] ND1.2-MTS-TALE-L-DddA N -UGI (SEQ ID NO. 177)

[1296] MALSRAVCGTSRQLAPVLGYLGSRQKHSLPDYPYDVPDYAGYPYDVPDYAGYPYDVPDYAMDIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPQQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNNGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGSGSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML

[1297] ND1.2-MTS-TALE-R-DddA C -UGI (SEQ ID NO. 178)

[1298] MASVLTPLLLRGLTGSARRLPVPRAKIHSLDYKDHDGDYKDHDIDYKDDDDKMDIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKRGAGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGSIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGSAIPVKRGATGETKVFTGNSNSPKSPTKGGCSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML

[1299] ND1.3-MTS-TALE-L-DddA N -UGI (SEQ ID NO. 179)

[1300] MALSRAVCGTSRQLAPVLGYLGSRQKHSLPDYPYDVPDYAGYPYDVPDYAGYPYDVPDYAMDIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNNGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGSGSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML

[1301] ND1.3-MTS-TALE-R-DddA C -UGI (SEQ ID NO. 180)

[1302] MASVLTPLLLRGLTGSARRLPVPRAKIHSLDYKDHDGDYKDHDIDYKDDDDKMDIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKRGAGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPQQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPQQVVAIASHDGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGSAIPVKRGATGETKVFTGNSNSPKSPTKGGCSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLND6.2-MTS-TALE-L-DddA N -UGI (SEQ ID NO. 181)

[1303] MALSRAVCGTSRQLAPVLGYLGSRQKHSLPDYPYDVPDYAGYPYDVPDYAGYPYDVPDYAMDIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPQQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPQQVVAIASNIGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGSGSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML

[1304] ND6.2-MTS-TALE-R-DddA C -UGI (SEQ ID NO. 182)

[1305] MASVLTPLLLRGLTGSARRLPVPRAKIHSLDYKDHDGDYKDHDIDYKDDDDKMDIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKRGAGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGLTPQQVVAIASHDGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGSAIPVKRGATGETKVFTGNSNSPKSPTKGGCSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML

[1306] ND3-MTS-TALE-L-DddA N -UGI (SEQ ID NO. 183)

[1307] MALSRAVCGTSRQLAPVLGYLGSRQKHSLPDYPYDVPDYAGYPYDVPDYAGYPYDVPDYAMDIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGLTPQQVVAIASNIGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGSGSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML

[1308] ND3-MTS-TALE-R-DddA C -UGI (SEQ ID NO. 184)

[1309] MASVLTPLLLRGLTGSARRLPVPRAKIHSLDYKDHDGDYKDHDIDYKDDDDKMDIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKRGAGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQLETVQRLLPVLCQAHGLTPQQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGLTPQQVVAIASHDGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGSAIPVKRGATGETKVFTGNSNSPKSPTKGGCSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML

[1310] The target sequences in the following examples and the figures referred to in relation to them are as follows:

[1311] OsBADH2 target A strand in the figure

[1312] SEQ ID NO. 188

[1313] GCTGGATGCTTTGAGTACTTTGCAGATCTTGCAGAATCCTTGGACAAAAGGC

[1314] OsBADH2 target B strand in the figure

[1315] SEQ ID NO. 189

[1316] CGACCTACGAAACTCATGAAACGTCTAGAACGTCTTAGGAACCTGTTTTCCG

[1317] OsDEP1 target A strand in the figure

[1318] SEQ ID NO. 190

[1319] GCAAAAGACCAAGGTGCCTCAATTGTTCTTGCAGCTCATGCTGCGACGAGCC

[1320] OsDEP1 target B strand in the figure

[1321] SEQ ID NO. 191

[1322] CGTTTTCTGGTTCCACGGAGTTAACAAGAACGTCGAGTACGACGCTGCTCGG

[1323] OsCKX2 target A strand in the figure

[1324] SEQ ID NO. 192

[1325] CCTGGACCGCGTCCACGACGGCGAGCTCAAGCTCCGCGCCGCGGGGCTCTGGG

[1326] OsCKX2 target B strand in the figure

[1327] SEQ ID NO. 193

[1328] GGACCTGGCGCAGGTGCTGCCGCTCGAGTTCGAGGCGCGGCGCCCCGAGACCC

[1329] Human ND6 target A strand in the figure

[1330] SEQ ID NO. 194

[1331] CCCCTGACCCCCATGCCTCAGGATACTCCTCAATAGCCATCGCTGTA

[1332] Human ND6 target B strand in figure

[1333] SEQ ID NO. 195

[1334] GGGGACTGGGGGTACGGAGTCCTATGAGGAGTTATCGGTAGCGACAT

[1335] OsSD1 target A strand in figure

[1336] SEQ ID NO. 196

[1337] CCAGGACGACGTCGGCGGCCTCGAGGTCCTCGTCGACGGCGAATGGCGCCCCGTC

[1338] OsSD1 target B strand in figure

[1339] SEQ ID NO. 197

[1340] GGTCCTGCTGCAGCCGCCGGAGCTCCAGGAGCAGCTGCCGCTTACCGCGGGGCAG

[1341] SIRT6 target A strand in figure

[1342] SEQ ID NO. 198

[1343] TACGCGGCGGGGCTGTCGCCGTACGCGGACAAGGGCAAGTGCGGCCTCCCGG

[1344] SIRT6 target B strand in figure

[1345] SEQ ID NO. 199

[1346] ATGCGCCGCCCCGACAGCGGCATGCGCCTGTTCCCGTTCACGCCGGAGGGCC

[1347] OsRbcL target A strand in figure

[1348] SEQ ID NO. 200

[1349] TTACCAAAGATGATGAAAACGTAAACTCACAACCATTTATGCGTTGG

[1350] OsRbcL target B strand in figure

[1351] SEQ ID NO. 201

[1352] AATGGTTTCTACTACTTTTGCATTTGAGTGTTGGTAAATACGCAACC

[1353] ND6.2 target A strand in figure

[1354] SEQ ID NO. 202

[1355] GACCCCCATGCCTCAGGATACTCCTCAATAGCCATCGCTGTAGTATATCCAA

[1356] ND6.2 target B strand in figure

[1357] SEQ ID NO. 203

[1358] CTGGGGGTACGGAGTCCTATGAGGAGTTATCGGTAGCGACATCATATAGGTT

[1359] ND1.2 target A strand in figure

[1360] SEQ ID NO. 204

[1361] CCTATTTATTCTAGCCACCTCTAGCCTAGCCGTTTACTCA

[1362] ND1.2 target B strand in figure

[1363] SEQ ID NO. 205

[1364] GGATAAATAAGATCGGTGGAGATCGGATCGGCAAATGAGT

[1365] ND1.3 target A strand in figure

[1366] SEQ ID NO. 206

[1367] TCTCCACACTAGCAGAGACCAACCGAACCCCCTTCGACCTTGCCGAAGGGG

[1368] ND1.3 target B strand in figure

[1369] SEQ ID NO. 207

[1370] AGAGGTGTGATCGTCTCTGGTTGGCTTGGGGGAAGCTGGAACGGCTTCCCC

[1371] Figure ND3 Target A strand

[1372] SEQ ID NO. 208

[1373] ACGAGTGCGGCTTCGACCCTATATCCCCCGCCCGCGTCCCTTTCTCCAT

[1374] Figure ND3 Target B strand

[1375] SEQ ID NO. 209

[1376] TGCTCACGCCGAAGCTGGGATATAGGGGGCGGGCGCAGGGAAAGAGGTA

[1377] Figure ND1 Target A strand

[1378] SEQ ID NO. 210

[1379] CTAGCCTAGCCGTTTACTCAATCCTCTCATCAGGGTGAGCATCAAACTC

[1380] Figure ND1 Target B strand

[1381] SEQ ID NO. 211

[1382] GATCGGATCGGCAAATGAGTTAGGAGACTAGTCCCACTCGTAGTTTGAG

[1383] Figure ND4 Target A strand

[1384] SEQ ID NO. 212

[1385] GCTAGTAACCACGTTCTCCTGATCAAATATCACTCTCCTACTTACAGG

[1386] Figure ND4 Target B strand

[1387] SEQ ID NO. 213

[1388] CGATCATTGGTGCAAGAGGACTAGTTTATAGTGAGAGGATGAATGTCC

[1389] Figure ND5.1 Target A strand

[1390] SEQ ID NO. 214

[1391] AGCATTAGCAGGAATACCTTTCCTCACAGGTTTCTACTCCAAAG

[1392] Figure ND5.1 Target B strand

[1393] SEQ ID NO. 215

[1394] TCGTAATCGTCCTTATGGAAAGGAGTGTCCAAAGATGAGGTTTC

[1395] Example 1: Synthesis and testing of base editors

[1396] The synthesis strategy of the base editor of the present application is shown in Figure 1 .

[1397] To verify the above strategy, a target site in the rice OsBADH2 gene was selected, and two groups of TALE coding vectors modified to target the site were constructed, and the components are listed in Table 3.

[1398] Table 3 Special examples of combinations of base editors

[1399]

[1400] FokI CD (or mutants) monomers were fused to the C-terminus of TALE-L and TALE-R, respectively, and wild-type FokI (without D450A or D467A mutations) was used as a control group Figure 16A ). The application of two exonucleases (Exonuclease I (rat Exonuclease I, referred to as mExoI) and Exonuclease V (referred to as ExoV)) and one deaminase (hAPOBEC3A, referred to as hA3A or A3A) in the new base editor was evaluated, wherein UGI was fused to the carboxyl terminus of the deaminase with an XTEN spacer peptide in each group Figure 16B ). A nuclear localization signal (NLS, i.e., SV40NLS in Table 2) was fused to the end of the protein.

[1401] Recombinant expression constructs encoding these components were transformed into rice protoplasts by PEG-mediated transformation. The constructs are as follows Figure 16A - Figure 16BThe different combinations of constructs were transformed into rice protoplasts to target the OsBADH2 site, and the C>T base editing frequency was determined using next generation sequencing technology (NGS). The sequencing results Figure 2A indicated that the highest cytosine base editing frequency could reach about 10% for the combination containing FokI nickase, deaminase, exonuclease and UGI. Importantly, the results also indicated that the new nucleic acid base editor only induced very low indel byproducts (as shown in Figure 2B ), which indicated that the new base editor had the feature of high product purity, which was important for precise genome editing.

[1402] In Figure 2A and Figure 2B , the experimental treatments or combinations of constructs involved in the figures and the associated vector schematics are shown as follows:

[1403]

[1404] Example 2: Single strand cleavage performance characterization of base editor

[1405] The base editing window of the base editor tested in Example 1 was analyzed. In the four C sites (C1, C6, C11 and C15, the first base adjacent to TALE-L in the spacer sequence of the two TALEs was counted as 1) in the A strand of the target gene (as shown in Figure 3A ), C6 and C11 cytosines were effectively edited Figure 3B ).

[1406] In Figure 3B , the experimental treatments or combinations of constructs involved in the figures and the associated vector schematics are shown as follows:

[1407]

[1408] These results show that the base editor containing FokI-R nickase (FokI-L mutation in the FokI-L of the dimeric nickase composed of FokI-L and FokI-R) is more inclined to cleave the B strand through the nickase, and then the exonuclease digests the cleaved single strand, and leaves a short ssDNA fragment in the A strand, and the direction of digestion depends on the enzyme catalytic direction of the exonuclease (5' to 3' or 3 to 5').

[1409] To validate the above results, the inventors evaluated the nucleic acid base editor at another site, OsDEP1, in the present example, which contains 5 C bases (C1, C9, C13, C16 and C18) in the A strand. Different combinations of constructs were transformed into rice protoplasts for NGS analysis of the targeted OsDEP1 site. The results showed that the base editing window was mainly located near the 5' region of the A strand (C9 and C1), while slight editing also occurred at C13 and C16 (as shown in Figure 4A ). Importantly, similar to the OsBADH2 site, only very low indel byproducts were observed in the marker products of the OsDEP1 site (as shown in Figure 4B ). The above results demonstrated that the novel base editor has the advantage of high product purity.

[1410] In Figure 4A and Figure 4B , the experimental treatments or combinations of constructs involved in the figures and the associated vector schematics are shown below:

[1411]

[1412] Example 3: Effect of exonuclease digestion direction and nicking enzyme single strand preference on editing results

[1413] For nucleic acid exonucleases with 5'→3' digestion directionality (e.g., rat nucleic acid exonuclease I (mExol)), cytosine residues located near the 5' region of the target site in the complementary strand are exposed and deaminated by the deaminase; while for 3' nucleic acid exonucleases, cytosine residues located near the 3' region of the target site in the complementary strand are exposed and deaminated by the deaminase. To verify that the base editor disclosed in the present application can produce the expected effect for different exonuclease digestion directions, the inventors tested both a 5' nucleic acid exonuclease mExol and a 3' nucleic acid exonuclease, Trex2 exonuclease of human origin, at the OsCKX2 target site, and analyzed the editing window of the resulting base editor by NGS. The experimental results showed that for FokI-R nickase mediated base editing, when a 5' nucleic acid exonuclease mExol was used, the editing window was mainly located in the 5' region of the A strand (C9 and C11) of the target site; in contrast, when a 3' nucleic acid exonuclease Trex2 was used, the editing window switched to the 3' adjacent region of the A strand (C11 and C15) of the OsCKX2 target site, while the cytosine residues of the B strand were not edited (as shown in Figure 5A , Figure 5B ). Further, the inventors evaluated the effect of the single strand preference of the nicking enzyme used on the single strand on which base editing can occur. FokI-R nickaseFokI-L which prefers to cleave A strand nickase As expected, the single strand where base editing occurs switched from A strand to B strand Figure 5A ); meanwhile, for the editing window, when 5' exonuclease mExoI is used, the editing window switches to the 5' adjacent region of B strand of OsCKX2 target site (C6 and C8), correspondingly, when 3' exonuclease Trex2 is used, the editing window can switch to the 3' adjacent region of B strand of OsCKX2 target site (C3 and C6), while the cytosine residue of A strand is not edited Figure 5A . It can be seen that the base editor of the present application can be used with different exonucleases in different digestion directions and exert the digestion effect of the corresponding exonuclease, so that the target site can be selectively edited.

[1414] Different construct combinations were transformed into rice protoplasts to target OsCKX2 site, and the C>T base editing efficiency and indel byproduct frequency detected by NGS are shown in the following table: Figure 5A and Figure 5B In

[1415]

[1416] Example 4: Influence of cytosine deaminase species

[1417] The novel base editor of the present application is independent of the species of deaminase and can be compatible with different species of deaminase. To exclude the dependence of the editing ability of the novel base editor on deaminase hAPOBEC3A (A3A), the inventors tested another cytosine deaminase rAPOBEC1 (APOBEC1) in this example. NGS analysis results show that, in the presence of exonucleases such as mExoI (as shown in Figure 6A ) and Trex2 (as shown in Figure 6B ), after replacing hAPOBEC3A with rAPOBEC1 at OsBADH2 site, high-purity targeted base editing is also achieved, indicating that different deaminase species are suitable for the base editor of the present application.

[1418] In Figure 6A , different construct combinations were transformed into rice protoplasts to target OsBADH2 site, and the C>T base editing efficiency and indel byproduct frequency detected by NGS are shown in the following table:

[1419]

[1420] InFigure 6B In the above-mentioned Example 1, different combinations of the constructs were transformed into rice protoplasts to target the OsDEP1 site, and the C>T base editing efficiency and indel byproduct frequency detected by NGS are shown in the following figures, in which the experimental treatments or combinations of constructs involved and the associated vector schematics are shown as follows:

[1421]

[1422] In analyzing the editing window of these base editors, cytosine residues located near the 5' region of the target site on the complementary strand of the single strand cleaved were efficiently edited in the group containing mExoI (as shown in Figure 7A ); while cytosine residues located near the 3' region of the target site on the complementary strand were efficiently edited in the group containing TREX2 (as shown in Figure 7B ), which is consistent with the results in the above-mentioned Example. These results show that the base editing method and base editors disclosed in the present application can be compatible with different cytosine deaminases.

[1423] In the above-mentioned Example 2, the base editing window of the base editors was analyzed according to the NGS results, in which the experimental treatments or combinations of constructs involved and the associated vector schematics are shown as follows: Figure 7A

[1424] In the above-mentioned Example 3, the base editing window of the base editors was analyzed according to the NGS results, in which the experimental treatments or combinations of constructs involved and the associated vector schematics are shown as follows:

[1425] Figure 7B

[1426]

[1427] Example 5: Base editors containing adenine deaminases

[1428] In order to expand the range of target sequences that can be edited by the base editors of the present application, in this example, a deoxyadenine (A) in single-stranded DNA as a substrate adenine deaminase TadA-8e was used as a deaminase to target A1, A7, A12, and A13 (as shown in Figure 8 ) of the OsCKX2 site. In this example, UGI is not an essential component of the base editors tested, as it is not necessary for adenine base editing. The adenine base editing window of the base editors was analyzed according to the NGS results, which showed that efficient targeted A to G conversion occurred at the target site (as shown in Figure 8 ​​), indicating that the base editor described in the application can be compatible with adenine deaminase for adenine base editing. As can be seen from Examples 4 and 5, the base editing method and base editor disclosed in the application can be compatible with different deaminases and exert their corresponding editing effects.

[1429] In Figure 8 , the experimental treatments or construct combinations involved in the figures and the associated vector schematics are shown as follows:

[1430]

[1431] Example 6: Base editor comprising fusion protein of base editing components

[1432] After the above examples demonstrate the function and effect of the base editor of the application, this example verifies whether the transformation efficiency (and the corresponding editing efficiency) can be improved by fusing the modular components into a single vector. The structures of two examples of such base editors comprising fusion components are shown in Figure 9 , in which the exonuclease is fused to the amino terminus of the deaminase-UGI fusion protein through an XTEN spacer peptide or a 48-amino acid spacer peptide (48aa) to target the OsDEP1 gene, i.e., the deaminase is fused to the exonuclease.

[1433] Different construct combinations were transformed into rice protoplasts to target the OsDEP1 site, and the C>T base editing efficiency and indel byproduct frequency detected by NGS. NGS analysis showed that targeted base editing could be achieved using exonuclease and deaminase fusion, but the efficiency under this vector structure was close to that when the exonuclease and deaminase were expressed alone (as shown in Figure 10A ). When using this base editor, the editing window is more inclined to C1 and C9 (as shown in Figure 10B ), which is consistent with the catalytic direction of mExoI exonuclease.

[1434] Figure 10A and Figure 10B , the experimental treatments or construct combinations involved in the figures and the associated vector schematics are shown as follows:

[1435]

[1436] In addition, the inventors also tested other fusion protein structures. The structures of the above base editors are shown in Figure 11A and Figure 11B , in which the deaminase (hAPOBEC3A or rAPOBEC1) is fused to TALE-L( Figure 11A ) or TALE-R( Figure 11B) are expressed by separate vectors, i.e. the deaminase, TALE protein, nickase are fused.

[1437] For deaminase-TALE-FokI-R nickase , OsDEP1 is selected as the target gene to be characterized (as shown in Figure 12A ), and for deaminase-TALE-FokI-L nickase , OsCKX2 is selected as the target gene to be characterized (as shown in Figure 12B ). NGS analysis shows that both deaminase-TALE-FokI-L / R nickase achieve the conversion of C to T at the target site. It shows that deaminase can form a fusion with TALE protein and nickase and does not interfere with the functions of each other. In addition, the experimental results further show that using deaminases hAPOBEC3A and rAPOBEC1 can both occur base editing (as shown in Figure 12A and Figure 12B ).

[1438] In Figure 12A , the experimental treatments or construct combinations involved in the figure and the associated vector schematic diagram are as follows:

[1439]

[1440] In Figure 12B , the experimental treatments or construct combinations involved in the figure and the associated vector schematic diagram are as follows:

[1441]

[1442] In order to investigate the effect of UGI or exonuclease fusion, in the deaminase-TALE-FokI-R nickase construct with the same target specificity as the present application, the base editor is connected to UGI at the carboxy terminus of FokI-L D450A (as shown in Figure 13A ) or at the amino terminus of the deaminase (as shown in Figure 13B ) through a 48-amino acid or 4-amino acid spacer peptide. NGS analysis shows that the effect of connecting UGI to the fusion protein is similar to that of the embodiment of expressing UGI alone ( Figure 14 ). In addition, in the deaminase-TALE-FokI-R nickase construct, the exonuclease is fused to the carboxy terminus of FokI-R through a 4-amino acid, 16-amino acid or 48-amino acid spacer peptide. The embodiment also has a similar editing efficiency ( Figure 14 ). Therefore, the separate expression of UGI / exonuclease and the fusion thereof to the vector for co-expression are both technical solutions that can be adopted by the present application.

[1443] In Figure 14 , different combinations of components were transformed into rice protoplasts to target the OsDEP1 site, and the DNA strand with base editing and the editing window were analyzed by high-throughput sequencing results. The experimental treatments or combinations of components involved in the figure and the associated vector schematic diagram are shown as follows:

[1444]

[1445] Based on the above results, the modular components of the base editor of the application can be expressed individually, or the components can form one or more fusion proteins.

[1446] Example 7: Base editing of plant nuclear genome

[1447] In the above examples, the function and characteristics of the base editor of the application were verified, i.e., the combination of the modular components containing deaminase, exonuclease, nickase, and DNA binding protein TALE can achieve efficient and accurate editing of DNA. For ease of description, the above base editor is named DENT (Deaminase-Exonuclease-Nickase-TALE), and is named CyDENT (Cytidine Deaminase-Exonuclease-Nickase-TALE) and AdDENT (Adenine Deaminase-Exonuclease-Nickase-TALE) according to the type of deaminase. In this example, the application environment and scenario of the base editor of the application were analyzed.

[1448] The inventors selected rice protoplast nuclear genome to evaluate the editing effect of the base editor of the application. In this example, four pairs of TALE proteins were designed for the endogenous gene sites OsDEP1, OsCKX2, OsBADH2, and OsSD1 of rice. Exonucleases with 5'→3' (mExol) or 3'→5' (Trex2) enzyme cutting preference were used to evaluate the effect of combining exonuclease and nickase to generate ssDNA intermediates. In this example, the efficient cytosine deaminase hAPOBEC3A (hA3A) was selected to deaminate cytosine in the ssDNA intermediate, and a uracil glycosylase inhibitor (UGI) peptide was fused to its C-terminus to minimize the impact of DNA base excision repair and further improve editing efficiency. Nuclear localization signal (NLS) was fused to the N-terminus of each component to directly edit the nuclear genome. This combination of base editor targeting the nuclear genome is called nuCyDENT, and the schematic diagram of an exemplary construct is shown as follows: Figure 18The nuCyDENT targeting rice OsDEP1, OsCKX2, OsBADH2 and OsSD1 sites were introduced into rice protoplasts, and the editing efficiency was evaluated after 2 days. Targeted cytosine base editing was evaluated within the 18 bp spacer region between the TALE binding sites at all four nuclear genomic sites using NGS analysis. We observed 3-18% editing efficiency and lower indel frequencies than the corresponding wild-type TALEN system Figure 19A and Figure 19B ). These results demonstrate that the base editor of the present application can achieve efficient base editing in the nuclear genome while only inducing low levels of indel byproducts.

[1449] In terms of single-strand editing performance, the inventors used nuCyDENT-L (containing FokI-L nickase structure) and nuCyDENT-R (containing FokI-R nickase structure) for base editing in rice genomic sites OsCKX2 and OsSD1, respectively, and the results showed that the top strand of DNA was edited when edited with nuCyDENT-R; the bottom strand of DNA was edited when edited using nuCyDENT-L Figure 20 ). This conclusion is the same as Example 2, and also shows the single-strand editing performance of CyDENT in the nuclear genome.

[1450] In Figure 19A , Figure 19B , Figure 20 , the experimental treatments or combinations of constructs involved in the figures are shown as follows:

[1451]

[1452] Example 8: Base editing of animal nuclear genomes

[1453] This example compares the base editing effect of CyDENT and DdCBE at the target site of human SIRT6 gene. The inventors designed TALE proteins for the SIRT6 target site, and designed nuCyDENT-L according to the method of Example 7, and designed DdCBE dependent on DddA according to the method in the prior art (Nakazato, I. et al. Targeted base editing in the mitochondrial genome of Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA. 119, e2121177119 (2022).). The experimental results show that nuCyDENT-L has higher base editing efficiency at the target site than DdCBE Figure 21). It is shown that the base editing system of the present application has good base editing performance in the animal cell nucleus genome.

[1454] In Figure 21 , the experimental treatments or combinations of constructs involved in the figures are shown as follows:

[1455]

[1456] Example 9: Base editing of DNA in organelles - chloroplast

[1457] The base editor of the present application can be used for mitochondrial DNA base editing and chloroplast DNA base editing, which has an advantage compared to CRISPR base editors that need to contain nucleic acid components. The protein components in the base editor described in the present application can be transferred into mitochondria and chloroplasts through mitochondrial targeting sequences (MTS) and chloroplast transit peptides (CTP), respectively. In these examples, MTS or CTP can be selected to replace NLS according to the type of target organelle.

[1458] First, we tried to use the CyDENT base editing strategy to edit plant chloroplast DNA. Plant chloroplast DNA is an important organelle specific to plants, with its own genomic DNA (cpDNA), which cannot be edited using CRISPR-derived base editors. The inventors replaced NLS with chloroplast transit peptide (CTP) in nuCyDENT designed according to the method of Reference Example 7 (Kang, B. C. et al. Chloroplast and mitochondrial DNA editing in plants. Nat Plants 7, 899-905 (2021).) Figure 22A ), which was named cpCyDENT. The inventors used cpCyDENT-L (containing FokI-L nickase ) and cpCyDENT-R (containing FokI-R nickase ) containing TALE proteins targeting the endogenous large subunit of ribulose-1,5-bisphosphate carboxylase / oxygenase (RuBisCO) gene (rbcL) to transform rice protoplasts. rbcL target base editing was detected in cpCyDENT-L treatment Figure 22B ). Notably, by regulating the types and directions of nickases and exonucleases of cpCyDENT, precise editing of specific bases can be achieved. For example, for G1 base (the nucleotide 5' most of the spacer region is counted as position 1, see Figure 22B), only when cpCyDENT-L (mExol) tool was used, i.e. containing FokI-Lnickase and 5'→3' mExol exonuclease, the base could be edited efficiently with an editing efficiency of about 1.67%. The result is consistent with the conclusion of the foregoing examples. These results show that cpCyDENT can selectively and accurately edit the DNA strand in the chloroplast genome.

[1459] In Figure 22B , the experimental treatments or combinations of constructs involved in the figures are shown as follows:

[1460]

[1461] Example 10: Base editing of DNA in organelles - mitochondria

[1462] In this example, the inventors evaluated the effect of CyDENT base editing on human cell mitochondrial DNA (mtDNA) base editing, replacing the NLS with a mitochondrial targeting sequence (MTS) and selecting a suitable promoter and terminator for expression in HEK293T cells, resulting in a base editor for mtDNA called mtCyDENT. The mtCyDENT constructs generated in this example are shown in Figure 15A TALE-FokI-R nickase and TALE-FokI-L nickase ).

[1463] First, a target site in the ND6 gene of human mitochondrial DNA was selected, and TALE-FokI-R nickase and TALE-FokI-L nickase expression vectors were constructed, in which the TALE protein was modified to target the site, and HEK293T cells were transfected with vectors expressing deaminases (hAPOBEC3A or C57), exonucleases (mExol or Trex2) and UGI, in which a mitochondrial targeting sequence (MTS) was fused to the end of the protein. NGS was used to determine the base editing frequency after transfection of the base editor. The results show that targeted cytosine base editing was achieved in the mitochondrial DNA target of human cells with an efficiency of about 6.0% ( Figure 15C ). The results show that the base editor of the present application can be used for base editing of organelle genomes.

[1464] In Figure 15C , different combinations of constructs were transfected into HEK293T cells to target the mitochondrial ND6 site, and the DNA strand and editing window in which base editing occurred were analyzed by high-throughput sequencing results. The experimental treatments or combinations of constructs involved in the figures are shown as follows:

[1465]

[1466] Example 11: Influence of base editor fusion status on mitochondrial DNA editing

[1467] Next, the inventors verified the influence of deaminase, exonuclease, UGI, TALE-FokI nickase expressed alone on mtDNA base editing efficiency.

[1468] To this end, the inventors used a small peptide called yb to fuse to the N-terminus of the domains of one or more modular components of mtCyDENT to drive recruitment of each protein component ( Figure 23A ). yb is an RNA silencing suppressor derived from Barley stripe mosaic virus (BSMV) with self-interaction (Jiang, Z., Yang, M., Zhang, Y., Jackson, A. O. & Li, D. in Encyclopedia of Virology 420-429 (2021).). The exonuclease the inventors selected in this experiment is Trex2. The inventors designed various fusion schemes of yb with each component to screen for the base editor composition with the best editing effect ( Figure 23B ). Considering the size of the protein components entering the mitochondria, this embodiment adopts the way of expressing the combination of 5 protein / fusion protein constructs as shown in Figure 23A , which are: the fusion protein of TALE-L and FokI-L (referred to as TALE-L-FokI-L, TALEL-FL or TALEL-FokI-L), the fusion protein of TALE-R and FokI-R (referred to as TALE-R-FokI-R, TALEL-FR or TALER-FokI-R), hA3A deaminase protein, Trex2 exonuclease protein, UGI protein. Among them, the tail mark D450A indicates its mutant, and WT indicates wild type.

[1469] The experimental results show that when yb is fused with UGI and Trex2 only, it has a higher editing effect, and the base editor composition with yb fused with UGI and Trex2 respectively is named mtCyDENT1b.

[1470] Next, we evaluated mtCyDENT and mtCyDENT1b on another 7 endogenous mtDNA genomic sites. We observed that the average editing frequency of mtCyDENT was 1.16-11.7%, and mtCyDENT1b further improved the average editing efficiency by 2.42-6.18 times, reaching 4.55-39.3% Figure 24) and mtCyDENT1b has higher editing efficiency than DdCBE on ND1.2, ND1.3, ND3 and ND6.2 targets with the same TALE sequence. In addition, we also noticed that using CyDENT base editing on mtDNA target sites has lower indel frequency than DdCBE Figure 25 In summary, both mtCyDENT and mtCyDENT1b can achieve efficient base editing in human mitochondrial DNA.

[1471] In Figure 23B , the experimental treatments or construct combinations involved in the figures are shown as follows (from top to bottom):

[1472]

[1473]

[1474] In Figure 24 - 27 , the experimental treatments or construct combinations involved in the figures are shown as follows:

[1475]

[1476]

[1477] Example 12: Improve the editing efficiency and precision of CyDENT

[1478] As described in the previous Example 4, the base editor of the present application can be self-assembled by multiple functional modules, which can be compatible with different types of deaminases. Therefore, the deaminase domain in the base editor can be replaced with a known deaminase in the prior art to take advantage of the unique characteristics of each deaminase, thereby enhancing activity or further improving the precision of the editing strand. A newly discovered single-stranded DNA (ssDNA) specific cytidine deaminase Sdd7 was found to have higher editing activity than other deaminases (Huang, J. et al. Discovery of new deaminase functions by structure-based protein clustering. bioRxiv (2023).). In this example, the inventors used Sdd7 as the deaminase of the editor, taking the mtCyDENT1b composition as an example, to evaluate the editing efficiency on mtDNA targets ND5.1, ND6, ND1.3. We observed that 87.5% of the base editing initiated by Sdd7-mtCyDENT1b-L occurred only on one DNA strand, and 93.0% of the base editing initiated by Sdd7-mtCyDENT1b-R occurred only on one DNA strand, which further proves that CyDENT has superior base editing strand specificityFigure 26 ) with an average editing efficiency of 4.88-9.13% on the target strand of DNA bottom ( Figure 27 ) These results further verified the ability of the base editor of the present application to replace the deaminase domain when modularly assembled.

[1479] Example 13: Improvement of base editor

[1480] In the foregoing examples, the inventors verified by experiments that the base editor composition of the present application has the technical advantages of single-strand editing specificity, modular assembly, high-efficiency precise controllable base editing, low indel frequency, etc. In subsequent examples, the inventors further optimized the base editor in order to obtain a base editor composition with more excellent functions.

[1481] In this embodiment, the deaminase and exonuclease domains were fused to the N-terminus of TALE-L and TALE-R using a 48-amino acid spacer peptide (flexible linker), and UGI was fused to the C-terminus and N-terminus of FokI-L and FokI-R, respectively. This construct structure is referred to as mtCyDENT2 ( Figure 28A ) in this embodiment. The base editing effect was detected on ND6 using mtCyDENT2-L (containing FokI-L nickase ) and mtCyDENT2-R (containing FokI-R Figure 28B ), and 94.5% of the base editing occurred only on the top strand, which demonstrated the good single-strand specific editing ability of the CyDENT system.

[1482] In Figures 28A-28B , the experimental treatments or construct combinations involved in the figures are shown as follows:

[1483]

[1484] Example 14: mtCyDNET base editing for GC motif

[1485] Since the DddA-dependent DdCBE system has strict T C motif context restriction for cytidine deamination, and it was found that the G CEditing in sequence context occurs at low frequency (Nakazato, I. et al. Targeted base editing in the mitochondrial genome of Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA. 119, e2121177119 (2022).). A bacteriophage-assisted non-continuous and continuous evolution was used to evolve “wild-type” DddA (Mok, B. Y. et al. CRISPR-free base editors with enhanced activity and expanded targeting scope in mitochondrial and nuclear DNA. Nat. Biotechnol. 40, 1378-1387 (2022).), resulting in DddA11 variants with better A C and C C sequence motif compatibility, but DddA11 editing on GC sequence motifs remained challenging. In this example, the modular replacement of the deaminase domain of CyDENT was utilized to achieve efficient and strand-selective G C sequence motif editing.

[1486] The inventors introduced a single-strand DNA-specific cytidine deaminase with G C sequence motif editing activity, thus developing a GC-compatible mtCyDENT base editor. Recently, a newly discovered single-strand DNA-specific, G C and A C compatible cytidine deaminase Sdd3, which exhibits higher editing activity than other deaminases on G C sequence motifs (Huang, J. et al. Discovery of new deaminase functions by structure-based protein clustering. bioRxiv (2023).).

[1487] Therefore, the present invention designed TALE arrays ( Figure 29 ) targeting ND1.2 and ND6.2 sites in HEK293T cells to evaluate the editing preference of sequence motifs that are difficult to edit by prior art. Notably, at ND1.2 and ND6.2 sites, G CThe efficiency of strand-specific cytosine base editing was observed to reach 21.0% and 20.0% under the sequence motif, respectively, which is the editing efficiency that the prior art DdCBE cannot reach under the same target; at the ND1.2 site, 96.9% of the editing occurred selectively on the top DNA strand, while at the ND6.2 site, 92.0% of the editing occurred selectively on the bottom DNA strand Figure 29

[1488] Subsequently, the inventors adjusted the TALE binding site and observed that Sdd3-mtCyDENT had an editing efficiency of 2.06% at the ND6.2 site Figure 30 It is reported that this particular mutation (m.14453G>A) is directly related to the development of Leigh syndrome, and the prior art DdCBE cannot achieve editing in this same target sequence background. Therefore, mtCyDENT and future optimized products can be used as an excellent base editing method to precisely edit pathogenic mutations on mtDNA.

[1489] In Figure 29 , 30 In the above-mentioned figures, the experimental treatments or combinations of constructs involved are as shown below:

[1490]

[1491] Example 15: Off-target analysis of mtCyDENT

[1492] The prior art DdCBE edits mitochondria and induces a large number of nuclear off-target editing. In order to evaluate the off-target rate of CyDENT in the whole genome of the cell nucleus and the whole genome of mitochondria, 2.25Tb of pure bases were obtained in this embodiment, with an average base of 281.13 Gb per sample, an average depth of mitochondrial genome sequencing of about 6362 times, and the human reference genome used was hg19.

[1493] In this embodiment, DdCBE plasmids targeting ND3 and mtCyDENT1b-R (hA3A) plasmids, and mtCyDENT2-L (Sdd3) plasmids targeting ND6.2 were designed to transfect HEK293T cells, and whole genome sequencing (WGS) and NGS analysis showed that these plasmids could edit G C on the sequence motif Figure 31A ​). Subsequently, the off-target rates of the whole mitochondrial genome and the whole nuclear genome were analyzed. The results showed that the average frequencies of C·G-to-T·A and G·C-to-A·T base conversion in the mitochondrial whole genome of the untreated negative control group, DdCBE, mtCyDENT1b-R(hA3A) and mtCyDENT2-L(Sdd3) treatment groups were 4.8%, 6.9%, 16.5% and 5.9%, respectively. Compared with the control group, we found an average of 32, 678 and 16 single nucleotide variations (SNVs) in the mitochondrial genome of the DdCBE, mtCyDENT1b-R(hA3A) and mtCyDENT2-L(Sdd3) treatment groups, respectively. By analyzing the 5bp region upstream and downstream of each potential off-target SNV, we found a conserved TC motif in the DdCBE and mtCyDENT1b-R(hA3A) groups, and a conserved GC / AC motif in the mtCyDENT2-L(Sdd3) group Figure 31B

[1494] In the nuclear genome, the inventors analyzed the TALE-dependent off-target effects. A total of 74963 potential off-target regions (containing 0-3 regions that do not match the ND3 and ND6.2 TALE binding sites) were identified. We observed no difference in the frequency of allelic SNVs and indels at the ND3 or ND6.2 sites between the control group, DdCBE, mtCyDENT1b-R(hA3A) and mtCyDENT2-L(Sdd3) treatment groups Figure 31C These results show that the modular assembly and optimization of CyDENTs can minimize off-target effects in the mitochondrial and nuclear genomes. mtCyDENTs are valuable tools for mitochondrial genome editing.

[1495] In Figures 31A-31C , the experimental treatments or combinations of constructs involved in the figures are shown as follows:

[1496]

[1497] The above description of the embodiments is only used to help understand the method of the present application and its core idea. It should be noted that, for those skilled in the art, without departing from the principles of the present application, some improvements and modifications can be made to the present application, and these improvements and modifications also fall within the protection scope of the claims of the present application.​

Claims

1. A nucleic acid base editor for organelles, characterized in that, The nucleic acid base editor comprises the following components: a) a sequence-specific DNA binding protein; b) a nickase; c) an exonuclease; d) a base-specific deaminase; e) an organelle targeting signal sequence; The sequence-specific DNA binding protein is a TALE protein; The nickase is a dimer of monomers of the cleavage functional domain of FokI or a mutant thereof, the dimer or the mutant thereof consists of a pair of interacting monomers of the cleavage functional domain of FokI, and only one of the monomers of the cleavage functional domain of FokI in the dimer or the mutant thereof has DNA endonuclease activity; The monomer of the cleavage functional domain of FokI with DNA endonuclease activity is selected from a FokI-L protein with an amino acid sequence as shown in SEQ ID No. 87, or a FokI-R protein with an amino acid sequence as shown in SEQ ID No. 88; a cleavage functional domain monomer of Fokl that has lost endonucleolytic activity is selected from the group consisting of Fokl-L having an amino acid sequence as shown in SEQ ID No. 60 D450A a protein selected from the group consisting of Fokl-L having an amino acid sequence as shown in SEQ ID No. 61 D467A a protein selected from the group consisting of Fokl-R having an amino acid sequence as shown in SEQ ID No. 62 D450A a protein selected from the group consisting of Fokl-R having an amino acid sequence as shown in SEQ ID No. 63 D467A a protein; The base-specific deaminase is selected from a cytosine-specific deaminase, or an adenine-specific deaminase; The exonuclease is selected from a 5' exonuclease or a 3' exonuclease; The organelle targeting signal sequence is selected from a mitochondrial targeting sequence MTS or a chloroplast transit peptide CTP.

2. The nucleic acid base editor of claim 1, wherein The components of the nucleic acid base editor exist individually, or form one or more fusion proteins.

3. The nucleic acid base editor of claim 1 or 2, wherein The amino acid sequence of the base-specific deaminase is selected from SEQ ID NO. 36-59, 80-86.

4. The nucleic acid base editor of claim 1 or 2, wherein The base-specific deaminase is a cytosine-specific deaminase.

5. The nucleic acid base editor of claim 4, wherein The cytosine-specific deaminase is one or more of hAPOBEC3A, rAPOBEC1, hAID, pmCDA1 or Sdd deaminase.

6. The nucleic acid base editor of claim 4, wherein The nucleic acid base editor further comprises: f) a uracil glycosylase inhibitor (UGI); and the uracil glycosylase inhibitor exists individually, or forms at least one fusion protein with other components of the nucleic acid base editor.

7. The nucleic acid base editor of claim 1 or 2, wherein The base-specific deaminase is an adenine-specific deaminase.

8. The nucleic acid base editor of claim 7, wherein The adenine-specific deaminase is TadA-8e.

9. The nucleic acid base editor of any one of claims 1, 2, 5-6, 8, wherein, The nucleic acid base editor further comprises: g) a gamma b; The gamma b forms at least one fusion protein with other components of the nucleic acid base editor.

10. The nucleic acid base editor of claim 1 or 2, wherein The amino acid sequence of the exonuclease is selected from SEQ ID NO. 64-67, 153.

11. A fusion protein as a nucleic acid base editor, characterized in that, The fusion protein comprises the protein domains of the base editor as described in any one of claims 1-10.

12. A recombinant expression construct for nucleic acid base editing, characterized in that, The recombinant expression construct is used to express the nucleic acid base editor as described in any one of claims 1-10 or the fusion protein as described in claim 11.

13. A method of nucleic acid base editing in a cell, comprising, The nucleic acid base editor as described in any one of claims 1-10 or the recombinant expression construct as described in claim 12 is introduced into a cell to edit a target gene.

14. The method of base editing of a nucleic acid of claim 13, wherein, The target gene is selected from mitochondrial genomic DNA, or chloroplast genomic DNA.

15. Use of a base editing technology in performing base editing. The base editing technology uses the base editor of any one of claims 1-10, the fusion protein of claim 11, the recombinant expression construct of claim 12, or the method of any one of claims 13 or 14 to base edit DNA in a cell, the cell being a mammalian cell, a bacterium, a protist, a fungus, an insect cell, a yeast, an unconventional yeast, or a plant cell.

16. The use according to claim 15, characterized in that, The plant cell is derived from a whole plant, seedling, meristem, ground tissue, vascular tissue, epidermal tissue, seed, leaf, root, shoot, stem, flower, fruit, stolon, bulb, tuber, corm, clonal ramet, bud, sprout, or tumor tissue of a monocotyledonous or dicotyledonous plant.

17. The use according to claim 15, characterized in that, The mammalian cell is selected from the group consisting of a human reproductive cell, neuron, muscle cell, endocrine / exocrine cell, epithelial cell, muscle cell, tumor cell, embryonic cell, hematopoietic cell, bone cell, germ line cell, somatic cell, stem cell, pluripotent stem cell, induced pluripotent stem cell, progenitor cell, meiotic cell, and mitotic cell.

18. A pharmaceutical composition for treating a disease in a subject in need thereof, characterized in that, The pharmaceutical composition comprises the base editor of any one of claims 1-10, the fusion protein of claim 11, or the recombinant expression construct of claim 12, and, optionally, a pharmaceutically acceptable carrier.

19. A method of producing a genetically modified plant, comprising, The method comprises introducing the base editor of any one of claims 1-10, the fusion protein of any one of claim 11, or the recombinant expression construct of claim 12 into at least one of the plants.

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