Improved gene editing system

Abstract

The present invention provides a gene editing system for editing a target gene in the genome of a cell, comprising a CRISPR nuclease, a cytosine deaminase, an AP lyase, a guide RNA, and an optional uracil-DNA glycosylase. The present invention also provides a method for producing a genetically modified cell, and a kit comprising the gene editing system.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering. Specifically, the present invention relates to an improved gene editing system. More specifically, the present invention relates to a gene editing system capable of accurately editing the genome of eukaryotic cells, especially predictable and accurate polynucleotide deletion. Background of the Invention

[0003] In recent years, with the continuous development of genome editing technology, a large number of gene editing tools have been developed, improved and applied, including gene knockout tools mediated by SpCas9 to single-base editing tools mediated by nCas9 (D10A) fused with cytosine deaminase, etc. Under the guidance of guide RNA, SpCas9 binds and cuts double-stranded DNA to form double-strand breaks (DSBs), which often introduce insertions and / or deletions of different fragment lengths during the repair process of the body, but these insertions and / or deletions are random, imprecise and unpredictable (Wang et al., 2014; Zhang et al., 2016). et al. (2017) used Cas9 fused to 3' repair exonuclease 2 (Trex2), which significantly increased the frequency of deletion mutations and the deletion fragments were longer, but the mutation type was still imprecise and unpredictable; using a pair of sgRNAs for targeted deletion can obtain specific long-fragment deletions, but it will also produce inversions, small fragments InDel, etc., which also greatly reduces the efficiency of the former ( et al., 2017). In order to obtain precise fragment deletion, Wolfs et al. (2016) fused Cas9 with TevI nuclease, which recognizes the restriction site and cuts the double-stranded DNA. The cut and the DSB cut by Cas9 together form a 33-36bp deletion, but the efficiency of the system is low due to the restriction of the restriction site. So far, no tool has been developed that can perform efficient, precise and predictable short fragment deletion within the protospacer sequence.

[0004] Therefore, there is still a need in the art for a gene editing system that can accurately edit the genome of eukaryotic cells, especially predictable and accurate polynucleotide deletions.

[0005] Brief description of the invention

[0006] In one aspect, the present invention provides a gene editing system for editing a target sequence in a cell genome, comprising:

[0007] i) a first polypeptide and / or an expression construct comprising a nucleotide sequence encoding the first polypeptide;

[0008] ii) a second polypeptide and / or an expression construct comprising a nucleotide sequence encoding the second polypeptide; and

[0009] iii) a guide RNA and / or an expression construct comprising a nucleotide sequence encoding the guide RNA,

[0010] The first polypeptide comprises a CRISPR nuclease, a cytosine deaminase, and optionally a uracil-DNA glycosylase (UDG), and the second polypeptide comprises an AP lyase, wherein the guide RNA is capable of targeting the first polypeptide to a target sequence in the genome of a cell.

[0011] In one aspect, the present invention provides a gene editing system for editing a target sequence in a cell genome, comprising:

[0012] i) a polypeptide and / or an expression construct comprising a nucleotide sequence encoding the polypeptide; and

[0013] ii) a guide RNA and / or an expression construct comprising a nucleotide sequence encoding the guide RNA,

[0014] The polypeptide comprises a CRISPR nuclease, a cytosine deaminase, an AP lyase, and optionally a uracil-DNA glycosylase (UDG), wherein the guide RNA is capable of targeting the polypeptide to a target sequence in the genome of a cell.

[0015] In one aspect, the invention provides a method for producing a genetically modified cell, comprising introducing the gene editing system of the invention into a cell.

[0016] In one aspect, the invention provides a kit comprising the gene editing system of the invention, and instructions for use. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 .Shows the working mode of the ACD system.

[0019] Figure 2 .Shows a comparative analysis of InDel production efficiency between SpCas9 and ACD systems at different target sites.

[0020] Figure 3 The graph shows the types and efficiency of deletion mutations formed by the ACD system at the sgF3HT4 site.

[0021] Figure 4 Shown are the types and efficiency of Deletion mutations formed by the ACD system at the sgLART4 site.

[0022] Figure 5Shown are the types and efficiency of deletion mutations formed by the ACD system at the sgMYBT2 site.

[0023] Figure 6 Shown are the types and efficiency of Deletion mutations formed by the ACD system at the sgPMKT1 site.

[0024] Figure 7 The graph shows the types and efficiency of deletion mutations formed by the ACD system at the sgVRN1T1 site.

[0025] Figure 8 The figure shows the types and efficiency of deletion mutations formed by the ACD system at the sgGS6T2 site.

[0026] Fig. 9 .Shows the differences in deamination activity and deamination window of different cytosine deaminases.

[0027] Fig.10 . Schematic diagram showing the vector construction of two different types of AFID systems.

[0028] Fig.11 .Shows the deletion efficiency of Cas9, AFID-3, and eAFID-3 at different endogenous target sites in rice.

[0029] Fig.12 .Shows the deletion efficiency of Cas9, AFID-3, and eAFID-3 at different endogenous targets in wheat.

[0030] Fig.13 .Shows the types and proportions of deletion mutations of AFID-3 and eAFID-3 at endogenous target sites in rice.

[0031] Fig.14 .Shows the types and proportions of deletion mutations of AFID-3 and eAFID-3 at endogenous target sites in wheat.

[0032] Fig.15 . Shows the preference of AFID-3 and eAFID-3 for the cytosine base that predicts the start of a deletion.

[0033] Fig.16 .Shows the mutation types and their proportions of Cas9, AFID-3, and eAFID-3 producing the desired predictable in-frame deletions at the miR396h binding site of the rice OsGRF1 gene and the miR156 binding site of the OsIPA1 gene, respectively.

[0034] Fig.17 .Shows a schematic diagram of the construction of AFID-3 vector for rice Agrobacterium infection.

[0035] Fig.18.Shows the types of regenerated plant mutants produced by Cas9 and AFID-3 on the rice OsCDC48 gene. DETAILED DESCRIPTION OF THE INVENTION

[0037] I. Definition

[0038] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology related terms and laboratory operation procedures used herein are terms and routine procedures widely used in the corresponding fields. For example, the standard recombinant DNA and molecular cloning techniques used in the present invention are well known to those skilled in the art and are more fully described in the following documents: Sambrook, J., Fritsch, EF and Maniatis, T., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter referred to as "Sambrook"). At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0039] As used herein, the term "and / or" encompasses all combinations of items connected by the term, and each combination should be considered to have been listed separately herein. For example, "A and / or B" encompasses "A," "A and B," and "B." For example, "A, B, and / or C" encompasses "A," "B," "C," "A and B," "A and C," "B and C," and "A and B and C."

[0040] When the term "comprising" is used herein to describe a protein or nucleic acid sequence, the protein or nucleic acid may consist of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the activity described in the present invention. In addition, it is clear to those skilled in the art that the methionine encoded by the start codon at the N-terminus of the polypeptide may be retained in certain practical situations (for example, when expressed in a specific expression system), but it does not substantially affect the function of the polypeptide. Therefore, when describing a specific polypeptide amino acid sequence in the specification and claims of this application, although it may not contain a methionine encoded by a start codon at the N-terminus, a sequence containing the methionine is also covered at this time, and accordingly, its encoding nucleotide sequence may also contain a start codon; and vice versa.

[0041] "Genome" as used herein encompasses not only the chromosomal DNA present in the cell nucleus, but also the organellar DNA present in subcellular components of the cell (eg, mitochondria, plastids).

[0042] As used herein, "organism" includes any organism suitable for genome editing, preferably a eukaryotic organism. Examples of organisms include, but are not limited to, mammals such as humans, mice, rats, monkeys, dogs, pigs, sheep, cattle, cats; poultry such as chickens, ducks, geese; plants including monocots and dicots, such as rice, corn, wheat, sorghum, barley, soybeans, peanuts, Arabidopsis, etc.

[0043] "Genetically modified organism" or "genetically modified cell" means an organism or cell that contains an exogenous polynucleotide or a modified gene or expression control sequence in its genome. For example, the exogenous polynucleotide can be stably integrated into the genome of the organism or cell and inherited for consecutive generations. The exogenous polynucleotide can be integrated into the genome alone or as part of a recombinant DNA construct. The modified gene or expression control sequence is a sequence in the genome of the organism or cell that contains single or multiple deoxynucleotide substitutions, deletions and additions.

[0044] "Exogenous" with respect to a sequence refers to a sequence that is from a foreign species, or, if from the same species, a sequence that has been significantly altered in composition and / or locus from its native form through deliberate human intervention.

[0045] "Polynucleotide", "nucleic acid sequence", "nucleotide sequence" or "nucleic acid fragment" are used interchangeably and are single-stranded or double-stranded RNA or DNA polymers that optionally may contain synthetic, non-natural or altered nucleotide bases. Nucleotides are referred to by their single letter names as follows: "A" is adenosine or deoxyadenosine (RNA or DNA, respectively), "C" represents cytidine or deoxycytidine, "G" represents guanosine or deoxyguanosine, "U" represents uridine, "T" represents deoxythymidine, "R" represents purine (A or G), "Y" represents pyrimidine (C or T), "K" represents G or T, "H" represents A or C or T, "I" represents inosine, and "N" represents any nucleotide.

[0046] "Polypeptide", "peptide", and "protein" are used interchangeably herein to refer to polymers of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. The terms "polypeptide", "peptide", "amino acid sequence" and "protein" may also include modified forms, including but not limited to glycosylation, lipid attachment, sulfation, gamma carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation.

[0047] Sequence "identity" has a meaning recognized in the art, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using published techniques. Sequence identity can be measured along the full length of a polynucleotide or polypeptide or along a region of the molecule. (See, for example: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). Although there are many methods to measure the identity between two polynucleotides or polypeptides, the term "identity" is well known to those of skill (Carrillo, H. & Lipman, D., SIAM J Applied Math 48: 1073 (1988)).

[0048] In peptides or proteins, suitable conservative amino acid substitutions are known to those skilled in the art, and generally can be carried out without changing the biological activity of the resulting molecule. Generally, those skilled in the art recognize that single amino acid substitutions in non-essential regions of a polypeptide do not substantially change biological activity (see, e.g., Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub.co., p. 224).

[0049] As used herein, "expression construct" refers to a vector such as a recombinant vector suitable for expressing a nucleotide sequence of interest in an organism. "Expression" refers to the production of a functional product. For example, the expression of a nucleotide sequence can refer to the transcription of the nucleotide sequence (such as transcription to generate mRNA or functional RNA) and / or the translation of RNA into a precursor or mature protein.

[0050] The "expression construct" of the present invention can be a linear nucleic acid fragment, a circular plasmid, a viral vector, or, in some embodiments, can be a translatable RNA (such as mRNA).

[0051] An "expression construct" of the present invention may comprise regulatory sequences and a nucleotide sequence of interest from different sources, or regulatory sequences and a nucleotide sequence of interest from the same source but arranged in a manner different from that normally found in nature.

[0052] "Regulatory sequence" and "regulatory element" are used interchangeably and refer to nucleotide sequences located upstream (5' non-coding sequence), in the middle or downstream (3' non-coding sequence) of a coding sequence and affecting the transcription, RNA processing or stability or translation of the relevant coding sequence. Regulatory sequences may include, but are not limited to, promoters, translation leader sequences, introns and polyadenylation recognition sequences.

[0053] "Promoter" refers to a nucleic acid fragment that can control the transcription of another nucleic acid fragment. In some embodiments of the present invention, a promoter is a promoter that can control the transcription of a gene in a cell, whether or not it is derived from the cell. A promoter can be a constitutive promoter or a tissue-specific promoter or a developmentally regulated promoter or an inducible promoter.

[0054] "Constitutive promoter" refers to a promoter that will generally cause a gene to be expressed in most cell types under most circumstances. "Tissue-specific promoter" and "tissue-preferred promoter" are used interchangeably and refer to a promoter that is expressed primarily, but not necessarily exclusively, in one tissue or organ, and may also be expressed in one specific cell or cell type. "Developmentally regulated promoter" refers to a promoter whose activity is determined by developmental events. "Inducible promoter" selectively expresses an operably linked DNA sequence in response to endogenous or exogenous stimuli (environmental, hormonal, chemical signals, etc.).

[0055] Examples of promoters include, but are not limited to, polymerase (pol) I, pol II or pol III promoters. Examples of pol I promoters include chicken RNA pol I promoters. Examples of pol II promoters include, but are not limited to, cytomegalovirus immediate early (CMV) promoters, Rous sarcoma virus long terminal repeat (RSV-LTR) promoters, and simian virus 40 (SV40) immediate early promoters. Examples of pol III promoters include U6 and H1 promoters. Inducible promoters such as metallothionein promoters can be used. Other examples of promoters include T7 phage promoters, T3 phage promoters, β-galactosidase promoters, and Sp6 phage promoters. When used for plants, the promoter can be a cauliflower mosaic virus 35S promoter, a corn Ubi-1 promoter, a wheat U6 promoter, a rice U3 promoter, a corn U3 promoter, a rice actin promoter.

[0056] As used herein, the term "operably linked" refers to the connection of a regulatory element (e.g., but not limited to, a promoter sequence, a transcription termination sequence, etc.) to a nucleic acid sequence (e.g., a coding sequence or an open reading frame) such that transcription of the nucleotide sequence is controlled and regulated by the transcription regulatory element. Techniques for operably linking a regulatory element region to a nucleic acid molecule are known in the art.

[0057] "Introducing" a nucleic acid molecule (e.g., a plasmid, a linear nucleic acid fragment, RNA, etc.) or a protein into an organism refers to transforming an organism cell with the nucleic acid or protein so that the nucleic acid or protein can function in the cell. "Transformation" as used in the present invention includes stable transformation and transient transformation.

[0058] "Stable transformation" refers to the introduction of an exogenous nucleotide sequence into the genome, resulting in stable inheritance of the exogenous gene. Once stably transformed, the exogenous nucleic acid sequence is stably integrated into the genome of the organism and any successive generations thereof.

[0059] "Transient transformation" refers to the introduction of a nucleic acid molecule or protein into a cell to perform its function without the foreign gene being stably inherited. In transient transformation, the foreign nucleic acid sequence is not integrated into the genome.

[0060] 2. Improved gene editing system

[0061] The inventors surprisingly found that by targeting CRISPR nuclease to a target sequence in the cell genome through a guide RNA to form a double-strand break (DSB), and by converting C in the target sequence or its complementary sequence into U through a cytosine deaminase fused to the CRISPR nuclease, and then through the combined action of endogenous or exogenous uracil-DNA glycosylase (UDG) and AP lyase, precise deletion from the DSB site in the target sequence to the C nucleotide site can be achieved.

[0062] Therefore, the present invention provides a gene editing system for editing a target sequence in a cell genome, comprising:

[0063] i) a first polypeptide and / or an expression construct comprising a nucleotide sequence encoding the first polypeptide;

[0064] ii) a second polypeptide and / or an expression construct comprising a nucleotide sequence encoding the second polypeptide; and

[0065] iii) a guide RNA and / or an expression construct comprising a nucleotide sequence encoding the guide RNA,

[0066] Wherein the first polypeptide comprises cytosine deaminase, CRISPR nuclease and optionally uracil-DNA glycosylase (UDG), and the second polypeptide comprises AP lyase, wherein the guide RNA is capable of targeting the first polypeptide to a target sequence in the cell genome. In some embodiments, the expression construct comprising a nucleotide sequence encoding the first polypeptide, the expression construct encoding the second polypeptide, and / or the expression construct comprising a nucleotide sequence encoding the guide RNA can be different expression constructs, or any two or all of them are the same expression construct. In some embodiments, the first polypeptide is an isolated polypeptide, the second polypeptide is an isolated polypeptide, and / or the guide RNA is an isolated RNA.

[0067] As used herein, "gene editing system" refers to a combination of components required for gene editing of a genome in a cell. The individual components of the system, such as polypeptides, gRNAs, etc., may exist independently of each other, or may exist in any combination as a composition.

[0068] In some embodiments, the gene editing system comprises at least an expression construct comprising a nucleotide sequence encoding the first polypeptide, a nucleotide sequence encoding a self-cleaving peptide, and a nucleotide sequence encoding the second polypeptide connected in frame. In some embodiments, the nucleotide sequence encoding the first polypeptide, the nucleotide sequence encoding the self-cleaving peptide, and the nucleotide sequence encoding the second polypeptide are arranged in a 5' to 3' direction.

[0069] As used herein, "self-cleaving peptide" means a peptide that can achieve self-cleavage in a cell. For example, the self-cleaving peptide may include a protease recognition site, thereby being recognized and specifically cleaved by a protease in the cell.

[0070] Alternatively, the self-cleaving peptide can be a 2A polypeptide. 2A polypeptides are a class of short peptides from viruses, and their self-cleavage occurs during translation. When two different target polypeptides are expressed in the same reading frame using a 2A polypeptide, two target polypeptides are generated at almost a 1:1 ratio. Commonly used 2A polypeptides can be P2A from porcine techovirus-1, T2A from Thosea asigna virus, E2A from equine rhinitisA virus, and F2A from foot-and-mouth disease virus. Among them, P2A has the highest cleavage efficiency and is therefore preferred. A variety of functional variants of these 2A polypeptides are also known in the art, and these variants can also be used in the present invention. In some embodiments, the self-cleaving peptide is P2A shown in SEQ ID NO:9.

[0071] In some embodiments, the gene editing system comprises at least an expression construct comprising a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:10 or SEQ ID NO:11.

[0072] In another aspect, the present invention also provides a gene editing system for editing a target sequence in a cell genome, comprising:

[0073] i) a polypeptide and / or an expression construct comprising a nucleotide sequence encoding the polypeptide; and

[0074] ii) a guide RNA and / or an expression construct comprising a nucleotide sequence encoding the guide RNA,

[0075] Wherein the polypeptide comprises cytosine deaminase, CRISPR nuclease, AP lyase and optionally uracil-DNA glycosylase (UDG), wherein the guide RNA is capable of targeting the polypeptide to a target sequence in the cell genome. In some embodiments, the expression construct comprising a nucleotide sequence encoding a polypeptide and the expression construct comprising a nucleotide sequence encoding a guide RNA may be different expression constructs or may be the same expression construct. In some embodiments, the polypeptide is an isolated polypeptide and / or the guide RNA is an isolated RNA. In some embodiments, the polypeptide comprises the amino acid sequence shown in SEQ ID NO: 10 or SEQ ID NO: 11.

[0076] As used herein, the term "CRISPR nuclease" generally refers to a nuclease present in a naturally occurring CRISPR system, as well as a modified form thereof, a variant thereof, or a catalytically active fragment thereof. CRISPR nucleases can recognize, bind to and / or cut a target nucleic acid structure by interacting with a guide RNA. The term encompasses any nuclease or a functional variant thereof that is capable of achieving gene editing in a cell based on a CRISPR system. In some embodiments, the functional variant retains its double-strand cleavage activity, i.e., the ability to form a double-strand break (DSB) in a target sequence.

[0077] The CRISPR nuclease used in the gene editing system of the present invention can be selected from, for example, Cas3, Cas8a, Cas5, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, GSU0054, Cas10, Csm2, Cmr5, Cas10, Csx11, Csx10, Csf1, Cas9, Csn2, Cas4, Cpf1, C2c1, C2c3 or C2c2 proteins, or functional variants of these nucleases.

[0078] In some embodiments, the CRISPR nuclease includes a Cas9 nuclease or a variant thereof. The Cas9 nuclease can be a Cas9 nuclease from a different species, such as spCas9 from Streptococcus pyogenes (S. pyogenes). The Cas9 nuclease variant can, for example, include a highly specific variant of the Cas9 nuclease, such as the Cas9 nuclease variants eSpCas9 (1.0) (K810A / K1003A / R1060A) and eSpCas9 (1.1) (K848A / K1003A / R1060A) of Feng Zhang et al., and the Cas9 nuclease variant SpCas9-HF1 (N497A / R661A / Q695A / Q926A) developed by J. Keith Joung et al. In some specific embodiments, the CRISPR nuclease has an amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the CRISPR nuclease comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO:1, or having one or more conservative amino acid substitutions relative to SEQ ID NO:1.

[0079] In some embodiments, the CRISPR nuclease may also include a Cpf1 nuclease or a variant thereof, such as a high-specificity variant. The Cpf1 nuclease may be a Cpf1 nuclease from a different species, such as a Cpf1 nuclease from Francisella novicida U112, Acidaminococcus sp. BV3L6, and Lachnospiraceae bacterium ND2006.

[0080] As used herein, the "cytosine deaminase" refers to a deaminase that can accept single-stranded DNA as a substrate and can catalyze the deamination of cytidine or deoxycytidine to uracil or deoxyuracil, respectively. Examples of cytosine deaminases include, but are not limited to, for example, APOBEC1 deaminase, activation-induced cytidine deaminase (AID), APOBEC3G, CDA1, human APOBEC3A deaminase, and truncated APOBEC3B deaminase. In some embodiments, the cytosine deaminase is human APOBEC3A deaminase, for example, its amino acid sequence is shown in SEQ ID NO: 2. In some specific embodiments, the cytosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with SEQ ID NO: 2, or has one or more conservative amino acid substitutions relative to SEQ ID NO: 2. In some embodiments, the cytosine deaminase is a truncated APOBEC3B deaminase (APOBEC3Bctd), for example, whose amino acid sequence is shown in SEQ ID NO: 7. In some specific embodiments, the cytosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO: 7, or has one or more conservative amino acid substitutions relative to SEQ ID NO: 7.

[0081] As used herein, uracil-DNA glycosylase (UDG) or uracil-N-glycosylase (UNG) refers to an enzyme that can recognize the U base and remove the N-glycosidic bond of the base to form an apurinic or apyrimidinic site. The UDG may have different sources, such as from Escherichia coli. In some specific embodiments, UDG has the amino acid sequence shown in SEQ ID NO: 3. In some specific embodiments, UDG comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with SEQ ID NO: 3, or has one or more conservative amino acid substitutions relative to SEQ ID NO: 3.

[0082] "AP lyase", "AP lyase", AP endonuclease and "apurinic pyrimidine lyase" are used interchangeably herein and refer to an enzyme that can recognize apurinic or apyrimidinic sites on nucleic acids and cleave nucleic acids. The AP lyase can be from different sources, such as from Escherichia coli. In some specific embodiments, the AP lyase has the amino acid sequence shown in SEQ ID NO:4. In some specific embodiments, the AP lyase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with SEQ ID NO:4, or has one or more conservative amino acid substitutions relative to SEQ ID NO:4.

[0083] As used herein, "gRNA" and "guide RNA" are used interchangeably and refer to RNA molecules that can form a complex with a CRISPR nuclease and can target the complex to a target sequence due to a certain complementarity with the target sequence. For example, in a gene editing system based on Cas9, gRNA is generally composed of crRNA and tracrRNA molecules that are partially complementary to form a complex, wherein crRNA contains a sequence that is sufficiently complementary to the target sequence so as to hybridize with the target sequence and guide the CRISPR complex (Cas9+crRNA+tracrRNA) to specifically bind to the target sequence sequence. However, it is known in the art that a single guide RNA (sgRNA) can be designed, which simultaneously contains the features of crRNA and tracrRNA. In a genome editing system based on Cpf1, gRNA is generally composed of only mature crRNA molecules, wherein the sequence contained in crRNA has sufficient homology with the target sequence so as to hybridize with the complementary sequence of the target sequence and guide the complex (Cpf1+crRNA) to specifically bind to the target sequence sequence. It is within the capabilities of those skilled in the art to design a suitable gRNA based on the CRISPR nuclease used and the target sequence to be edited.

[0084] As used herein, a "target sequence" is a sequence that is complementary or identical (depending on the different CRISPR nucleases) to a guide sequence of about 20 nucleotides contained in a guide RNA. The guide RNA targets the target sequence by base pairing with the target sequence or its complementary strand.

[0085] In some embodiments of the present invention, the gene editing results in the deletion of one or more nucleotides in the target sequence, preferably resulting in the deletion of multiple consecutive nucleotides in the target sequence. The type and length of the deletion depends on the double-strand break (DSB) position caused by the CRISPR nuclease and the number and position of cytosine (C) bases present in the target sequence or its complementary sequence. In some embodiments, the length of the deletion does not exceed the length of the target sequence. For example, the deletion can be about 1-17 nucleotides, such as 10-17 nucleotides, such as 10, 11, 12, 13, 14, 15, 16, 17 nucleotides.

[0086] In some embodiments of the invention, the cytosine deaminase is fused to the N-terminus of the CRISPR nuclease.

[0087] In some embodiments of the invention, the cytosine deaminase, the CRISPR nuclease, the UDG and / or the AP lyase are directly linked to each other.

[0088] In some embodiments of the present invention, the cytosine deaminase, the CRISPR nuclease, the UDG and / or the AP lyase are connected by a linker. The linker can be a non-functional amino acid sequence with a length of 1-50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 20-25, 25-50) or more amino acids and no secondary structure or above. For example, the linker can be a flexible linker, such as GGGGS, GS, GAP, (GGGGS) x 3, GGS and (GGS) x 7, etc. In some embodiments, the linker comprises the amino acid sequence shown in SEQ ID NO: 8.

[0089] In some embodiments of the present invention, the polypeptide of the present invention further comprises a nuclear localization sequence (NLS). In general, one or more NLS in the polypeptide should have sufficient strength to drive the polypeptide to accumulate in the nucleus of the cell in an amount that can achieve its gene editing function. In general, the intensity of nuclear localization activity is determined by the number, position, one or more specific NLS used, or a combination of these factors of the NLS in the polypeptide.

[0090] In some embodiments of the present invention, the NLS of the polypeptide of the present invention may be located at the N-terminus and / or the C-terminus. In some embodiments of the present invention, the NLS of the polypeptide of the present invention may be located between the cytosine deaminase, the CRISPR nuclease, the UDG and / or the AP lyase. In some embodiments, the polypeptide comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLSs. In some embodiments, the polypeptide 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 polypeptide 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 to be independent of the other NLSs.

[0091] In general, NLS consists of one or more short sequences of positively charged lysine or arginine exposed on the surface of the protein, but other types of NLS are also known. Non-limiting examples of NLS include: KKRKV (nucleotide sequence 5'-AAGAAGAGAAAGGTC-3'), PKKKRKV (nucleotide sequence 5'-CCCAAGAAGAAGAGGAAGGTG-3' or CCAAAGAAGAAGAGGAAGGTT), or SGGSPKKKRKV (nucleotide sequence 5'-TCGGGGGGGAGCCCAAAGAAGAAGCGGAAGGTG-3').

[0092] In addition, depending on the DNA location to be edited, the polypeptide of the present invention may also include other localization sequences, such as cytoplasmic localization sequences, chloroplast localization sequences, mitochondrial localization sequences, etc.

[0093] In some specific embodiments of the present invention, the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 5. In some specific embodiments of the present invention, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 6.

[0094] In order to obtain efficient expression in cells, in some embodiments of the present invention, the nucleotide sequence encoding the polypeptide is codon-optimized for the organism from which the cell to be gene-edited comes.

[0095] Codon optimization refers to a method of modifying a nucleic acid sequence to enhance expression in a host cell of interest by replacing at least one codon of the native sequence (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons) with codons that are more frequently or most frequently used in the genes of the host cell while maintaining the native amino acid sequence. Different species exhibit specific preferences for certain codons for specific amino acids. Codon bias (differences in codon usage between organisms) is often correlated with the efficiency of translation of messenger RNA (mRNA), which in turn is believed to depend on the nature of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell generally reflects the codons that are most frequently used for peptide synthesis. Therefore, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, in the Codon Usage Database available at www.kazusa.orjp / codon / , and these tables can be adapted for use in different ways. See, Nakamura et al., 2001. Y. et al., "Codon usage tabulated from the international DNAsequence databases: status for the year 2000. Nucl. Acids Res., 28:292 (2000).

[0096] The organisms from which the cells that can be gene-edited by the system of the present invention come are preferably eukaryotic organisms, including but not limited to mammals such as humans, mice, rats, monkeys, dogs, pigs, sheep, cattle, and cats; poultry such as chickens, ducks, and geese; and plants including monocotyledonous and dicotyledonous plants, such as rice, corn, wheat, sorghum, barley, soybeans, peanuts, Arabidopsis, and the like.

[0097] 3. Methods for modifying target sequences in cell genomes

[0098] In another aspect, the present invention provides a method for modifying a target sequence in a cell genome, comprising introducing the gene editing system of the present invention into the cell.

[0099] In some embodiments, the modification results in the deletion of one or more nucleotides, preferably multiple consecutive nucleotides in the target sequence. In the present invention, the type and length of the deletion caused by the modification depends on the double-strand break (DSB) position caused by the CRISPR nuclease and the number and position of cytosine (C) bases present in the target sequence or its complementary sequence. In some embodiments, the deletion is located within the target sequence. In some embodiments, the modification does not include insertion and / or substitution mutations.

[0100] In another aspect, the present invention also provides a method for producing genetically modified cells, comprising introducing the gene editing system of the present invention into the cells.

[0101] In another aspect, the present invention also provides a genetically modified organism comprising a genetically modified cell produced by the method of the present invention or a progeny cell thereof.

[0102] In the present invention, the target sequence to be modified can be located at any position in the genome, for example, in a functional gene such as a protein coding gene, or, for example, in a gene expression regulatory region such as a promoter region or an enhancer region, thereby achieving modification of the gene function or modification of gene expression. The modification in the cell target sequence can be detected by T7EI, PCR / RE or sequencing methods.

[0103] In the method of the present invention, the gene editing system can be introduced into cells by various methods well known to those skilled in the art.

[0104] Methods that can be used to introduce the gene editing system of the present invention into cells include, but are not limited to, calcium phosphate transfection, protoplast fusion, electroporation, liposome transfection, microinjection, viral infection (such as baculovirus, vaccinia virus, adenovirus, adeno-associated virus, lentivirus and other viruses), gene gun technique, PEG-mediated protoplast transformation, and soil Agrobacterium-mediated transformation.

[0105] Cells that can be gene-edited by the methods of the present invention can come from, for example, mammals such as humans, mice, rats, monkeys, dogs, pigs, sheep, cattle, and cats; poultry such as chickens, ducks, and geese; plants, including monocots and dicots, such as rice, corn, wheat, sorghum, barley, soybeans, peanuts, Arabidopsis, and the like.

[0106] In some embodiments, the methods of the invention are performed in vitro. For example, the cells are isolated cells, or cells in isolated tissues or organs.

[0107] In other embodiments, the method of the present invention can also be performed in vivo. For example, the cell is a cell in an organism, and the system of the present invention can be introduced into the cell in vivo by, for example, a virus or Agrobacterium-mediated method.

[0108] IV. Test Kit

[0109] The present invention also includes a kit for the method of the present invention, the kit comprising the gene editing system of the present invention, and instructions for use. The kit generally includes a label indicating the intended use and / or method of use of the contents of the kit. The term label includes any written or recorded material provided on or with the kit or otherwise provided with the kit. Example

[0110] Materials and Methods

[0111] 1. Vector construction

[0112] In order to construct pA3A-Cas9-UDG and pJIT163-Ubi-AP vectors, UDG and AP lyase sequences from Escherichia coli were obtained from NCBI (accession numbers AMB53293.1 and WP_115209270.1, respectively), and rice codon optimization and gene synthesis were carried out at Suzhou GeneWeizhi Company. Finally, the APOBEC3A, Cas9 and UDG fusion protein gene fragments and the APlyase gene fragment were introduced into the pJIT163 vector backbone, respectively, to obtain pA3A-SpCas9-UDG and pJIT163-Ubi-AP vectors.

[0113] In addition, APOBEC3A was fused to the N-terminus of Cas9 using an XTEN linker, UDG was fused to the C-terminus of Cas9, and AP lyase was fused to the C-terminus of UDG using a self-cleaving 2A polypeptide (P2A), and finally the fusion protein gene fragments were introduced into the pJIT163 vector backbone to construct the transient transformation vector AFID-3. Then, APOBEC3Bctd (APOBC3B sequence from humans (accession number NM_004900.5), and the C-terminal functional catalytic domain of APOBEC3B was truncated (APOBEC3Bctd)) was used to replace APOBEC3 in AFID-3 to construct eAFID-3. In addition, the fusion gene fragment with APOBEC3A was integrated into the pHUE411 backbone together with the sgRNA expression component using the Gibson method to construct a stable transformation vector pH-AFID-3 for Agrobacterium infection-mediated rice genetic transformation.

[0114] The gene coding sequences of key enzyme genes in the synthesis of wheat seed coat pigment (flavanone-3-hydroxylase gene, TaF3H-A1 / B1 / D1; colorless anthocyanin reductase gene TaLAR-A1 / B1 / D1) and their regulatory genes (TaMYB10-A1 / B1 / D1), plasma membrane kinase related to plant disease resistance (TaPMK-A1 / B1 / D1), vernalization response-related genes (TaVRN1-A1 / B1 / D1), and gibberellin-stimulated regulatory factor genes related to growth and development (TaGASR6-A1 / B1 / D1) were obtained. The targeting site sequences (sgF3HT4, sgLART4, sgMYBT2, sgPMKT1, sgVRN1T1 and sgGS6T2, see Table 1 for specific sequences) were compiled, sgRNA targeting site primers were synthesized, annealed and ligated into the pTaU6-sgRNA vector using T4 ligase to obtain pTaU6-sgF3HT4, pTaU6-sgLART4, pTaU6-sgMYBT2, pTaU6-sgPMKT1, pTaU6-sgVRN1T1 and pTaU6-sgGS6T2 vectors, respectively.

[0115] Table 1. sgRNA targeting primers

[0116]

[0117]

[0118] Nine endogenous target sites were selected from seven rice genes (OsAAT, OsACC, OsCDC48, OsNRT1.1B, OsPDS, OsGRF1, and OsSPL14 / OsIPA1) for the construction of pOsU3-sgRNA vectors, and four endogenous target sites were selected from four wheat genes (TaF3H, TaGASR6, TaMYB10, and TamiR396) for the construction of pTaU6-sgRNA vectors. All target site sequences are shown in Table 2. sgRNA target site primers were synthesized, annealed, and ligated to the sgRNA vector using T4 ligase.

[0119] Table 2. sgRNA targeting sites and sequences

[0120]

[0121] PAM sequence is shown in bold

[0122] 2. Protoplast isolation and transformation (4 biological replicates)

[0123] 2.1 Rice or wheat seedling culture

[0124] The Zhonghua 11 rice seeds were first rinsed with 75% ethanol for 1 minute, then treated with 4% sodium hypochlorite for 30 minutes, washed with sterile water for more than 5 times, and cultured on M6 medium for 3-4 weeks at 26°C in the dark.

[0125] The wheat seeds were potted and planted in a culture room, and cultured for about 1-2 weeks (about 10 days) at a temperature of 25±2°C, a light intensity of 1000Lx, and a light intensity of 14-16h / d.

[0126] 2.2 Protoplast isolation

[0127] (1) Take young leaves of rice or wheat, cut the middle part into 0.5-1 mm strips with a blade, put them into 0.6 M Mannitol solution and keep them away from light for 10 min, then filter them with a filter, put them into 50 mL of enzymatic solution (filtered with 0.45 μm filter membrane), evacuate (pressure about 15 KPa) for 30 min, take them out and place them on a shaker (10 rpm) for enzymatic hydrolysis at room temperature for 5 h; (2) Add 30-50 mL of W5 to dilute the enzymatic hydrolysis product, filter the enzymatic hydrolysis solution with a 75 μm nylon filter membrane and place it in a round-bottom centrifuge tube (50 mL); (3) Centrifuge at 23°C, 100 g (rcf), up 3 times and down 3 times, and discard the supernatant; (4) Gently suspend with 10 mL of W5 and place on ice for 30 min; the protoplasts gradually settle and the supernatant is discarded; (5) Add an appropriate amount of MMG to suspend, place on ice and wait for transformation.

[0128] 2.3 Protoplast transformation

[0129] (1) Add 10 μg of each desired transformation vector to a 2 mL centrifuge tube and mix well. Use a sharpened pipette tip to draw up 200 μL of protoplasts and flick to mix well. Immediately add 250 μL of PEG4000 solution and flick to mix well. Induce transformation at room temperature in the dark for 20-30 min. (2) Add 800 μL of W5 (room temperature) and gently invert to mix. Centrifuge at 100 g (rcf), up 3 times and down 3 times for 3 minutes and discard the supernatant. (3) Add 1 mL of W5 and gently invert to mix well. Gently transfer to a 6-well plate that has been pre-added with 1 mL of W5. Wrap the 6-well plate with tin foil and culture at 23°C in the dark for 48 h.

[0130] 3. Protoplast DNA extraction and amplicon sequencing analysis

[0131] 3.1 Protoplast DNA extraction

[0132] The protoplasts were collected in 2 mL centrifuge tubes, and the protoplast DNA (~30 μL) was extracted using the CTAB method. The concentration (30-60 ng / μL) was determined using a NanoDrop ultra-micro spectrophotometer and stored at -20°C.

[0133] 3.2 Amplicon sequencing analysis

[0134] (1) PCR amplification of protoplast DNA template was performed using universal genomic primers. The 20 μL amplification system contained 4 μL 5×Fastpfu buffer, 1.6 μL dNTPs (2.5 mM), 0.4 μL Forward primer (10 μM), 0.4 μL Reverseprimer (10 μM), 0.4 μL FastPfu polymerase (2.5 U / μL), and 2 μL DNA template (~60 ng). Amplification conditions: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 50-64°C for 30 s, extension at 72°C for 30 s, 35 cycles; full extension at 72°C for 5 min, storage at 12°C;

[0135] (2) The above amplification product was diluted 10 times, and 1 μL was taken as the template for the second round of PCR amplification. The amplification primer was a sequencing primer containing a barcode. The 50 μL amplification system contained 10 μL 5×Fastpfu buffer, 4 μL dNTPs (2.5 mM), 1 μL Forward primer (10 μM), 1 μL Reverse primer (10 μM), 1 μL FastPfu polymerase (2.5 U / μL), and 1 μL DNA template. The amplification conditions were the same as above, and the number of amplification cycles was 38 cycles.

[0136] (3) The PCR products were separated by 2% agarose gel electrophoresis, and the target fragments were recovered by gel extraction using AxyPrepTM DNA Gel Extraction kit. The recovered products were quantitatively analyzed using NanoDrop ultra-micro spectrophotometer; 100 ng of the recovered products were mixed and sent to Genewise Biotechnology Co., Ltd. for amplicon sequencing library construction and amplicon sequencing analysis.

[0137] (4) After sequencing is completed, the raw data are split according to the sequencing primers. The sgRNA sequence and its flanking sequences are used as reference sequences, and the WT is used as a control to compare and analyze the type and efficiency of gene editing at different gene targeting sites in four repeated experiments.

[0138] Example 1: Construction of a gene editing system for precise short fragment deletion (ACD)

[0139] The single-base editing system was established in 2016 (Komor et al., 2016; Ma et al., 2016; Nishida et al., 2016). The system uses nCas9 (D10A) to guide cytosine deaminase to act on the non-complementary strand of the DNA target site and deaminate cytosine (C) in a specific region into uracil (U). Uracil (U) will be replaced by thymine (T) during DNA replication, thereby achieving precise single-base replacement of C-to-T. In the repair process of plant and animal organisms, uracil-DNA glycosylase (UDG) will preferentially recognize the U base and remove the N-glycosidic bond of the base to form an apurinic or apyrimidinic site (AP site), and then the U base will be repaired to the original C base through base excision repair under the action of AP lyase (AP lyase). Therefore, uracil-DNA glycosylase inhibitor (UGI) is often introduced into the single-base editing system to improve the C-to-T editing efficiency.

[0140] The inventors surprisingly found that replacing the nCas9 of the fusion protein in the single-base editing system with wild-type Cas9 enables the fusion protein to regain the ability to break the DNA double strand, and at the same time replaces UGI with UDG to recognize the U base and remove its glycosidic bond to form an AP site, which is then recognized by AP lyase and the glycosylated U base is removed, and finally efficient, accurate and predictable short fragment deletion can be achieved in cells. . The inventors thus constructed an efficient, accurate and predictable short fragment deletion system (APOBEC3A Coupled Deletion, ACD) composed of Cas9, APOBEC3A, UDG and AP lyase, wherein Cas9 mediates the generation of DSB at the DNA target site, and APOBEC3A, UDG and AP lyase mediate the generation of multiple gaps at the non-complementary chain C base upstream of the DSB, thereby causing the deletion of single-stranded DNA fragments on the non-complementary chain, and finally forming a short fragment deletion of the DNA double strand under the action of DNA repair in the body ( Figure 1Without wishing to be bound by any theory, APOBEC3A can efficiently mediate the C-to-U replacement of the non-targeted strand upstream of DSB, while UDG and AP lyase mediate the formation of a gap at the U base, resulting in the loss of single-stranded DNA fragments on the non-targeted strand. At this time, the targeted strand forms a 5' overhanging end, which will first be recognized and removed by the Artemis-DNA-PK complex during the body's non-homologous end repair process, and then form a short-fragment missing DNA double-strand under the action of a ligation complex composed of DNA ligase IV, XRCC4, XRCC4-like factor (XLF) and its paralogs (PAXX) (Chang et al. 2017).

[0141] The efficiency of insertion and deletion produced by SpCas9 and ACD at the targeted editing sites of sgF3HT4, sgLART4, sgMYBT2, sgPMKT1, sgVRN1T1 and sgGS6T2 was compared and analyzed. The results showed that compared with SpCas9, the insertion mutation rate of the ACD system was significantly reduced, while the deletion mutation rate was significantly increased, and the deletion mutation rate was 1.5-23.6 times that of SpCas9, which fully demonstrated the high efficiency of the ACD system ( Figure 2 ).

[0142] Example 2: Analysis of Deletion Types Generated by the ACD System

[0143] Sequence analysis of the Deletion mutations generated by the ACD system at different target sites ( Figure 3-8 ), except for a few types, most mutation types are in line with expectations, all of which are deletions between the APOBEC3A action base (NGG (PAM) corresponds to the C base; CCN (PAM) corresponds to the G base) and the Cas9 cutting site. However, due to the asymmetry of Cas9 when cutting double strands, Cas9 will cut between the 3rd and 4th positions or the 4th and 5th positions near the PAM end. In addition, the bases where APOBEC3A acts on the non-targeted chain will use the targeted chain as a template to form 1-2 bases that pair with the complementary chain during the repair process. Therefore, 1-2 bases that complement the targeted chain may also be introduced.

[0144] The ACD system has a very low efficiency in producing insertions, but a very high efficiency in producing deletions, and deletions only occur within the 20-bp pre-spacer sequence. In these targeted sites, most deletions are 10 to 17 nt in length, and different types of deletions can be stably detected in more than three biological repeat experiments, which is something that SpCas9 and other tools cannot do, which fully demonstrates the accuracy and predictability of the ACD system.

[0145] Example 3: Construction of AFID (APOBEC-Cas9 Fusion-Induced Deletion) system

[0146] The present invention selects human APOBEC3A with high deamination activity and wide deamination window to construct the AFID-3 system, and selects an APOBEC3Bctd with higher deamination activity and narrow window to replace APOBEC3A to construct the eAFID-3 system ( Fig. 9 and Fig.10 ). A comparative analysis of the deletion efficiency of Cas9, AFID-3 and eAFID-3 at endogenous gene targets in rice and wheat showed that the efficiency of deletion mutations produced by Cas9, AFID-3 and eAFID-3 increased significantly, with their average deletion mutation rates being 2.2 and 2.6 times that of Cas9, respectively, which fully demonstrates the high efficiency of the AFID system.

[0147] Example 4: Analysis of mutation types generated by the AFID system

[0148] The mutation types and proportions generated by AFID-3 and eAFID-3 at different endogenous target sites were analyzed. The results showed that the length of the deletion fragment mainly depends on the position of the deamination C nucleotide and its deamination activity. At target sites with strong deamination activity, deletion mutations are the main mutation type; but at target sites with weak deamination activity, a certain proportion of insertion mutations will appear. The mutation types with a larger proportion are predictable multi-nucleotide deletion mutations (from the C nucleotide where the deaminase acts to the Cas9 cleavage site (Cas9 cuts the double strands asymmetric, resulting in the Cas9 cleavage site appearing between the 3rd and 4th or 4th and 5th positions near the PAM end) Fig.13 , 14 ). In addition, it was also found that during the NHEJ repair process, templated insertion of C nucleotides occurred at the deaminated C nucleotides ( Fig.13 , 14 ), which is mainly due to the fact that during the excision process of the 5' protruding end on the targeted chain, the DNA polymerase can easily use this 5' protruding end as a template to perform base repair on the non-targeted chain.

[0149] In order to detect the preference of AFID-3 and eAFID-3 for the C base at the start of the predictable deletion, the proportion of deletion mutations from the AC, TC, CC and GC motifs to DSBs at different targets was counted. The results showed that AFID-3 could mediate predictable deletion mutations from the AC, TC, CC and GC motifs to DSBs. Compared with AFID-3, eAFID-3 showed an enhanced preference for the TC base, and most of its predictable deletion mutations were deletion mutations from the TC motif to DSBs ( Fig.15 ). In addition, the types and proportions of mutations produced by Cas9, AFID-3 and eAFID-3 at the miR396h binding site of the rice OsGRF1 gene and the miR156 binding site of the OsIPA1 gene were analyzed. The results showed that Cas9 was almost unable to produce such predictable in-frame deletion mutations; while both AFID-3 and eAFID-3 could produce such predictable deletion mutations, but the proportion produced by eAFID-3 was significantly higher than that of AFID-3 ( Fig.16 ). This also fully demonstrates the accuracy and predictability of the AFID system.

[0150] Example 5: AFID system mediates predictable polynucleotide deletion mutations in plants

[0151] To determine whether the AFID system can mediate predictable multinucleotide deletion mutations in plants, two targets (TamiR396 and TaGASR6) were selected in wheat, and Cas9 and AFID-3 were delivered to immature embryos of wheat by gene gun bombardment. Three targets (OsCDC48-T2, OsSPL14, and OsPDS) were selected in rice to construct the corresponding pH-Cas9 and pH-AFID-3 Agrobacterium vectors ( Fig.17 ) and transformed rice callus using Agrobacterium infection. The results showed that among the tested targets, Cas9 did not produce predictable polynucleotide deletion mutants, and its mutation types were mainly 1-bp insertion and 1-3bp deletion; while AFID-3 produced mostly polynucleotide deletion mutants, of which the predictable proportion accounted for 25.0-55.5% (Table 3, Fig.18 ). It can be seen that the AFID system can mediate predictable multi-nucleotide deletion mutations in plants.

[0152] Table 3 Statistics of predictable deletion plant mutants generated by AFID-3

[0153]

[0154] Sequence Listing

[0155] SEQ ID NO:1 SpCas9

[0156]

[0157] SEQ ID NO:2 APOBEC3A

[0158] MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGFLHNQAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGEVRAFLQENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIMTYDEFKHCWDTFVDHQGCPFQPWDGLDEHSQALSGRLRAILQNQGNSGSETPGTSESATPES

[0159] SEQ ID NO:3 UDG

[0160] ANELTWHDVLAEEKQQPYFLNTLQTVASERQSGVTIYPPQKDVFNAFRFTELGDVKVVILGQDPYHGPGQAHGLAFSVRPGIAIPPSLLNMYKELENTIPGFTRPNHGYLESWARQGVLLLNTVLTVRAGQAHSHASLGWETFTDKVISLINQHREGVVFLLWGSHAQKKGAIIDKQRHHVLKAPHPSPLSAHRGFFGCNHFVLANQWLEQRGETPIDWMPVLPAESE

[0161] SEQ ID NO:4 AP lyase

[0162] MPEGPEIRRAADNLEAAIKGKPLTDVWFAFPQLKPYQSQLIGQHVTHVETRGKALLTHFSNDLTLYSHNQLYGVWRVVDTGEEPQTTRVLRVKLQTADKTILLYSASDIEMLTPEQLTTHPFLQRVGPDVLDPNLTPEVVKERLLSPRFRNRQFAGLLLDQAFLAGLGNYLRVEILWQVGLTGNHKAKDLNAAQLDALAHALLEIPRFSYATRGQVDENKHHGALFRFKVFHRDGEPCERCGSIIEKTTLSSRPFYWCPGCQH

[0163] SEQ ID NO:5 Exemplary first polypeptide

[0164]

[0165] SEQ ID NO:6 Exemplary second polypeptide

[0166] MPEGPEIRRAADNLEAAIKGKPLTDVWFAFPQLKPYQSQLIGQHVTHVETRGKALLTHFSNDLTLYSHNQLYGVWRVVDTGEEPQTTRVLRVKLQTADKTILLYSASDIEMLTPEQLTTHPFLQRVGPDVLDPNLTPEVVKERLLSPRFRNRQFAGLLLDQAFLAGLGNYLRVEILWQVGLTGNHKAKDLNAAQLDALAHALLEIPRFSYATRGQVDENKHHGALFRFKVFHRDGEPCERCGSIIEKTTLSSRPFYWCPGCQHPKKKRKV

[0167] SEQ ID NO:7 APOBEC3B ctd

[0168] MEILRYLMDPDTFTFNFNNDPLVLRRRQTYLCYEVERLDNGTWVLMDQHMGFLCNEAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGEVRAFLQENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIMTYDEFEYCWDTFVYRQGCPFQPWDGLEEHSQALSGRLRAILQNQGN

[0169] SEQ ID NO:8 XTEN linker

[0170] SGSETPGTSESATPES

[0171] SEQ ID NO:9 P2A

[0172] GSGATNFSLLKQAGDVEENPGPPE

[0173] SEQ ID NO:10 AFID-3

[0174]

[0175] SEQ ID NO:11 eAFID-3

[0176] Sequence Listing <110> Institute of Genetics and Developmental Biology, Chinese Academy of Sciences <120> Improved gene editing system <130> TC6229 <150> 201910375061.9 <151> 2019-05-07 <160> 11 <170> PatentIn version 3.5 <210> 1 <211> 1368 <212> PRT <213> Streptococcus pyogenes <400> 1 Lys Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val 1 5 10 15 Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 20 25 30 Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 35 40 45 Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60 Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys 65 70 75 80 Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser 85 90 95 Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 100 105 110 His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 115 120 125 His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp 130 135 140 Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 145 150 155 160 Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165 170 175 Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 180 185 190 Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 195 200 205 Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 210 215 220 Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 225 230 235 240 Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 245 250 255 Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270 Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280 285 Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295 300 Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 305 310 315 320 Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 325 330 335 Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 340 345 350 Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 355 360 365 Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 370 375 380 Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 385 390 395 400 Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405 410 415 Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 420 425 430 Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 435 440 445 Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 450 455 460 Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 465 470 475 480 Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 485 490 495 Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510 Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520 525 Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530 535 540 Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 545 550 555 560 Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 565 570 575 Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 580 585 590 Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 595 600 605 Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 610 615 620 Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 625 630 635 640 His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655 Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 660 665 670 Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 675 680 685 Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 690 695 700 Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 705 710 715 720 His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735 Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 740 745 750 Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 755 760 765 Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780 Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 785 790 795 800 Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 805 810 815 Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 820 825 830 Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys 835 840 845 Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860 Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 865 870 875 880 Arg Tyr Asn To Lys Arg Asn With Thr Gln Arg Lys 885,890,895 Phe Asp Asp With Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu With Asp 900 905 910 Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915,920,925 Lys Tyr Asp Ser Arg Met Asn Thr Lys Tyr Asp 930,935,940 Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Lys Ser 945 950 955 960 Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965,970,975 Glu Ile Asn Asn Tyr His Ala His Asp Ala Tyr Leu Asn Ala Val 980,985,990 Val Gly Thr Ala Leu Ile Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005 Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1010 1015 1020 Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe 1025 1030 1035 Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala 1040 1045 1050 Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu 1055 1060 1065 Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val 1070 1075 1080 Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr 1085 1090 1095 Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1100 1105 1110 Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1115 1120 1125 Light Light Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val 1130 1135 1140 Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys 1145 1150 1155 Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser 1160 1165 1170 Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys 1175 1180 1185 Glu Val Lys Asp Leu Ile Ile Leu Pro Lys Tyr Ser Leu 1190 1195 1200 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215 Leu Glu Gln Lys Gly Asn Leu Glu Wing Pro Ser Tyr Val 1220 1225 1230 Asn Phe Leu Tyr Leu Ala Ser is Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245 Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 1250 1255 1260 His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys 1265 1270 1275 Arg Val Leu Valley Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1280 1285 1290 Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn 1295 1300 1305 Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala 1310 1315 1320 Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser 1325 1330 1335 Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1340 1345 1350 Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp 1355 1360 1365 <210> 2 <211> 215 <212> PRT <213> Homo sapiens <400> 2 Met Glu Ala Ser Pro Ala Ser Gly Pro Arg His Leu Met Asp Pro His 1 5 10 15 Ile Phe Thr Ser Asn Phe Asn Asn Gly Ile Gly Arg His Lys Thr Tyr 20 25 30 Leu Cys Tyr Glu Val Glu Arg Leu Asp Asn Gly Thr Ser Val Lys Met 35 40 45 Asp Gln His Arg Gly Phe Leu His Asn Gln Ala Lys Asn Leu Leu Cys 50 55 60 Gly Phe Tyr Gly Arg His Ala Glu Leu Arg Phe Leu Asp Leu Val Pro 65 70 75 80 Ser Leu Gln Leu Asp Pro Ala Gln Ile Tyr Arg Val Thr Trp Phe Ile 85 90 95 Ser Trp Ser Pro Cys Phe Ser Trp Gly Cys Ala Gly Glu Val Arg Ala 100 105 110 Phe Leu Gln Glu Asn Thr His Val Arg Leu Arg Ile Phe Ala Ala Arg 115 120 125 Ile Tyr Asp Tyr Asp Pro Leu Tyr Lys Glu Ala Leu Gln Met Leu Arg 130 135 140 Asp Ala Gly Ala Gln Val Ser Ile Met Thr Tyr Asp Glu Phe Lys His 145 150 155 160 Cys Trp Asp Thr Phe Val Asp His Gln Gly Cys Pro Phe Gln Pro Trp 165 170 175 Asp Gly Leu Asp Glu His Ser Gln Ala Leu Ser Gly Arg Leu Arg Ala 180 185 190 Ile Leu Gln Asn Gln Gly Asn Ser Gly Ser Glu Thr Pro Gly Thr Ser 195 200 205 Glu Ser Ala Thr Pro Glu Ser 210 215 <210> 3 <211> 228 <212> PRT <213> Escherichia coli <400> 3 Ala Asn Glu Leu Thr Trp His Asp Val Leu Ala Glu Glu Lys Gln Gln 1 5 10 15 Pro Tyr Phe Leu Asn Thr Leu Gln Thr Val Ala Ser Glu Arg Gln Ser 20 25 30 Gly Val Thr Ile Tyr Pro Pro Gln Lys Asp Val Phe Asn Ala Phe Arg 35 40 45 Phe Thr Glu Leu Gly Asp Val Lys Val Val Ile Leu Gly Gln Asp Pro 50 55 60 Tyr His Gly Pro Gly Gln Ala His Gly Leu Ala Phe Ser Val Arg Pro 65 70 75 80 Gly Ile Ala Ile Pro Pro Ser Leu Leu Asn Met Tyr Lys Glu Leu Glu 85 90 95 Asn Thr Ile Pro Gly Phe Thr Arg Pro Asn His Gly Tyr Leu Glu Ser 100 105 110 Trp Ala Arg Gln Gly Val Leu Leu Leu Asn Thr Val Leu Thr Val Arg 115 120 125 Ala Gly Gln Ala His Ser His Ala Ser Leu Gly Trp Glu Thr Phe Thr 130 135 140 Asp Lys Val Ile Ser Leu Ile Asn Gln His Arg Glu Gly Val Val Phe 145 150 155 160 Leu Leu Trp Gly Ser His Ala Gln Lys Lys Gly Ala Ile Ile Asp Lys 165 170 175 Gln Arg His His Val Leu Lys Ala Pro His Pro Ser Pro Leu Ser Ala 180 185 190 His Arg Gly Phe Phe Gly Cys Asn His Phe Val Leu Ala Asn Gln Trp 195 200 205 Leu Glu Gln Arg Gly Glu Thr Pro Ile Asp Trp Met Pro Val Leu Pro 210 215 220 Ala Glu Ser Glu 225 <210> 4 <211> 263 <212> PRT <213> escherichia coli <400> 4 Met Pro Glu Gly Pro Glu Ile Arg Arg Ala Ala Asp Asn Leu Glu Ala 1 5 10 15 Ala Ile Lys Gly Lys Pro Leu Thr Asp Val Trp Phe Ala Phe Pro Gln 20 25 30 Leu Lys Pro Tyr Gln Ser Gln Leu Ile Gly Gln His Val Thr His Val 35 40 45 Glu Thr Arg Gly Lys Ala Leu Leu Thr His Phe Ser Asn Asp Leu Thr 50 55 60 Leu Tyr Ser His Asn Gln Leu Tyr Gly Val Trp Arg Val Val Asp Thr 65 70 75 80 Gly Glu Glu Pro Gln Thr Thr Arg Val Leu Arg Val Lys Leu Gln Thr 85 90 95 Ala Asp Lys Thr Ile Leu Leu Tyr Ser Ala Ser Asp Ile Glu Met Leu 100 105 110 Thr Pro Glu Gln Leu Thr Thr His Pro Phe Leu Gln Arg Val Gly Pro 115 120 125 Asp Val Leu Asp Pro Asn Leu Thr Pro Glu Val Val Lys Glu Arg Leu 130 135 140 Leu Ser Pro Arg Phe Arg Asn Arg Gln Phe Ala Gly Leu Leu Leu Asp 145 150 155 160 Gln Ala Phe Leu Ala Gly Leu Gly Asn Tyr Leu Arg Val Glu Ile Leu 165 170 175 Trp Gln Val Gly Leu Thr Gly Asn His Lys Ala Lys Asp Leu Asn Ala 180 185 190 Ala Gln Leu Asp Ala Leu Ala His Ala Leu Leu Glu Ile Pro Arg Phe 195 200 205 Ser Tyr Ala Thr Arg Gly Gln Val Asp Glu Asn Lys His His Gly Ala 210 215 220 Leu Phe Arg Phe Lys Val Phe His Arg Asp Gly Glu Pro Cys Glu Arg 225 230 235 240 Cys Gly Ser Ile Ile Glu Lys Thr Thr Leu Ser Ser Arg Pro Phe Tyr 245 250 255 Trp Cys Pro Gly Cys Gln His 260 <210> 5 <211> 1842 <212> PRT <213> Artificial Sequence <220> <223> Exemplary first polypeptide <400> 5 Met Glu Ala Ser Pro Ala Ser Gly Pro Arg His Leu Met Asp Pro His 1 5 10 15 Ile Phe Thr Ser Asn Phe Asn Asn Gly Ile Gly Arg His Lys Thr Tyr 20 25 30 Leu Cys Tyr Glu Val Glu Arg Leu Asp Asn Gly Thr Ser Val Lys Met 35 40 45 Asp Gln His Arg Gly Phe Leu His Asn Gln Ala Lys Asn Leu Leu Cys 50 55 60 Gly Phe Tyr Gly Arg His Ala Glu Leu Arg Phe Leu Asp Leu Val Pro 65 70 75 80 Ser Leu Gln Leu Asp Pro Ala Gln Ile Tyr Arg Val Thr Trp Phe Ile 85 90 95 Ser Trp Ser Pro Cys Phe Ser Trp Gly Cys Ala Gly Glu Val Arg Ala 100 105 110 Phe Leu Gln Glu Asn Thr His Val Arg Leu Arg Ile Phe Ala Ala Arg 115 120 125 Ile Tyr Asp Tyr Asp Pro Leu Tyr Lys Glu Ala Leu Gln Met Leu Arg 130 135 140 Asp Ala Gly Ala Gln Val Ser Ile Met Thr Tyr Asp Glu Phe Lys His 145 150 155 160 Cys Trp Asp Thr Phe Val Asp His Gln Gly Cys Pro Phe Gln Pro Trp 165 170 175 Asp Gly Leu Asp Glu His Ser Gln Ala Leu Ser Gly Arg Leu Arg Ala 180 185 190 Ile Leu Gln Asn Gln Gly Asn Ser Gly Ser Glu Thr Pro Gly Thr Ser 195 200 205 Glu Ser Ala Thr Pro Glu Ser Leu Lys Asp Lys Lys Tyr Ser Ile Gly 210 215 220 Leu Asp Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu 225 230 235 240 Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg 245 250 255 His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly 260 265 270 Glu Thr Ala Glu Ala Thr Arg Leu Lys Thr Ala Arg Arg Tyr 275 280 285 Three Arg Lys Asn Arg With Cys Tyr Leading To Gln Glu With Phe Ser Asn 290,295,300 Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser 305 310 315 320 Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly 325 330 335 Asn Ile Val Asp Glue Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr 340 345 350 His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg 355 360 365 Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe 370 375 380 Leu Has Glu Gly Asp Leu Asn Pro Asp Ser Asp Val Asp Lys Leu 385 390 395 400 Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro 405 410 415 The Asn Protein Is Gly Val Asp Protein Lys Protein Leu Protein Is Arg Leu Protein 420 425 430 Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu 435 440 445 Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu 450 455 460 Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu 465 470 475 480 Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala 485 490 495 Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu 500 505 510 Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile 515 520 525 Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His 530 535 540 His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro 545 550 555 560 Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala 565 570 575 Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile 580 585 590 Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys 595 600 605 Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly 610 615 620 Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg 625 630 635 640 Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile 645 650 655 Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala 660 665 670 Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr 675 680 685 Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala 690 695 700 Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn 705 710 715 720 Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val 725 730 735 Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys 740 745 750 Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu 755 760 765 Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr 770 775 780 Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu 785 790 795 800 Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile 805 810 815 Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu 820 825 830 Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile 835 840 845 Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met 850 855 860 Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg 865 870 875 880 Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu 885 890 895 Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu 900 905 910 Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln 915 920 925 Val Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala 930 935 940 Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val 945 950 955 960 Asp Glu Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val 965 970 975 Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn 980 985 990 Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly 995 1000 1005 Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln 1010 1015 1020 Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met 1025 1030 1035 Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp 1040 1045 1050 Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile 1055 1060 1065 Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser 1070 1075 1080 Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr 1085 1090 1095 Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe 1100 1105 1110 Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 1115 1120 1125 Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile 1130 1135 1140 Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys 1145 1150 1155 Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr 1160 1165 1170 Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe 1175 1180 1185 Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala 1190 1195 1200 Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro 1205 1210 1215 Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp 1220 1225 1230 Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala 1235 1240 1245 Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys 1250 1255 1260 Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu 1265 1270 1275 Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly 1280 1285 1290 Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val 1295 1300 1305 Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys 1310 1315 1320 Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg 1325 1330 1335 Light Light Asp Trp Asp Pro Light Light Tyr Gly Gly Phe Asp Ser Pro 1340 1345 1350 Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly 1355 1360 1365 Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr 1370 1375 1380 Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu 1385 1390 1395 Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys 1400 1405 1410 Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg 1415 1420 1425 Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala 1430 1435 1440 Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr 1445 1450 1455 Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu 1460 1465 1470 Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln 1475 1480 1485 Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu 1490 1495 1500 Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile 1505 1510 1515 Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn 1520 1525 1530 Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp 1535 1540 1545 Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu 1550 1555 1560 Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu 1565 1570 1575 Ser Gln Leu Gly Gly Asp Lys Arg Pro Ala Ala Thr Lys Lys Ala 1580 1585 1590 Gly Gln Ala Lys Lys Lys Lys Thr Arg Asp Ser Gly Gly Ser Ala 1595 1600 1605 Asn Glu Leu Thr Trp His Asp Val Leu Ala Glu Glu Lys Gln Gln 1610 1615 1620 Pro Tyr Phe Leu Asn Thr Leu Gln Thr Val Ala Ser Glu Arg Gln 1625 1630 1635 Ser Gly Val Thr Ile Tyr Pro Pro Gln Lys Asp Val Phe Asn Ala 1640 1645 1650 Phe Arg Phe Thr Glu Leu Gly Asp Val Lys Val Val Ile Leu Gly 1655 1660 1665 Gln Asp Pro Tyr His Gly Pro Gly Gln Ala His Gly Leu Ala Phe 1670 1675 1680 Ser Val Arg Pro Gly Ile Ala Ile Pro Pro Ser Leu Leu Asn Met 1685 1690 1695 Tyr Lys Glu Leu Glu Asn Thr Ile Pro Gly Phe Thr Arg Pro Asn 1700 1705 1710 His Gly Tyr Leu Glu Ser Trp Ala Arg Gln Gly Val Leu Leu Leu 1715 1720 1725 Asn Thr Val Leu Thr Val Arg Ala Gly Gln Ala His Ser His Ala 1730 1735 1740 Ser Leu Gly Trp Glu Thr Phe Thr Asp Lys Val Ile Ser Leu Ile 1745 1750 1755 Asn Gln His Arg Glu Gly Val Val Phe Leu Leu Trp Gly Ser His 1760 1765 1770 Ala Gln Lys Lys Gly Ala Ile Ile Asp Lys Gln Arg His His Val 1775 1780 1785 Leu Lys Ala Pro His Pro Ser Pro Leu Ser Ala His Arg Gly Phe 1790 1795 1800 Phe Gly Cys Asn His Phe Val Leu Ala Asn Gln Trp Leu Glu Gln 1805 1810 1815 Arg Gly Glu Thr Pro Ile Asp Trp Met Pro Val Leu Pro Ala Glu 1820 1825 1830 Ser Glu Pro Lys Lys Lys Arg Lys Val 1835 1840 <210> 6 <211> 270 <212> PRT <213> Artificial Sequence <220> <223> Exemplary second polypeptide <400> 6 Met Pro Glu Gly Pro Glu Ile Arg Arg Ala Ala Asp Asn Leu Glu Ala 1 5 10 15 Ala Ile Lys Gly Lys Pro Leu Thr Asp Val Trp Phe Ala Phe Pro Gln 20 25 30 Leu Lys Pro Tyr Gln Ser Gln Leu Ile Gly Gln His Val Thr His Val 35 40 45 Glu Thr Arg Gly Lys Ala Leu Leu Thr His Phe Ser Asn Asp Leu Thr 50 55 60 Leu Tyr Ser His Asn Gln Leu Tyr Gly Val Trp Arg Val Val Asp Thr 65 70 75 80 Gly Glu Glu Pro Gln Thr Thr Arg Val Leu Arg Val Lys Leu Gln Thr 85 90 95 Ala Asp Lys Thr Ile Leu Leu Tyr Ser Ala Ser Asp Ile Glu Met Leu 100 105 110 Thr Pro Glu Gln Leu Thr Thr His Pro Phe Leu Gln Arg Val Gly Pro 115 120 125 Asp Val Leu Asp Pro Asn Leu Thr Pro Glu Val Val Lys Glu Arg Leu 130 135 140 Leu Ser Pro Arg Phe Arg Asn Arg Gln Phe Ala Gly Leu Leu Leu Asp 145 150 155 160 Gln Ala Phe Leu Ala Gly Leu Gly Asn Tyr Leu Arg Val Glu Ile Leu 165 170 175 Trp Gln Val Gly Leu Thr Gly Asn His Lys Ala Lys Asp Leu Asn Ala 180 185 190 Ala Gln Leu Asp Ala Leu Ala His Ala Leu Leu Glu Ile Pro Arg Phe 195 200 205 Ser Tyr Ala Thr Arg Gly Gln Val Asp Glu Asn Lys His His Gly Ala 210 215 220 Leu Phe Arg Phe Lys Val Phe His Arg Asp Gly Glu Pro Cys Glu Arg 225 230 235 240 Cys Gly Ser Ile Ile Glu Lys Thr Thr Leu Ser Ser Arg Pro Phe Tyr 245 250 255 Trp Cys Pro Gly Cys Gln His Pro Lys Lys Lys Arg Lys Val 260 265 270 <210> 7 <211> 197 <212> PRT <213> artificial sequence <220> <223> APOBEC3Bctd <400> 7 Met Glu Ile Leu Arg Tyr Leu Met Asp Pro Asp Thr Phe Thr Phe Asn 1 5 10 15 Phe Asn Asn Asp Pro Leu Val Leu Arg Arg Arg Gln Thr Tyr Leu Cys 20 25 30 Tyr Glu Val Glu Arg Leu Asp Asn Gly Thr Trp Val Leu Met Asp Gln 35 40 45 His Met Gly Phe Leu Cys Asn Glu Ala Lys Asn Leu Leu Cys Gly Phe 50 55 60 Tyr Gly Arg His Ala Glu Leu Arg Phe Leu Asp Leu Val Pro Ser Leu 65 70 75 80 Gln Leu Asp Pro Ala Gln Ile Tyr Arg Val Thr Trp Phe Ile Ser Trp 85 90 95 Ser Pro Cys Phe Ser Trp Gly Cys Ala Gly Glu Val Arg Ala Phe Leu 100 105 110 Gln Glu Asn Thr His Val Arg Leu Arg Ile Phe Ala Ala Arg Ile Tyr 115 120 125 Asp Tyr Asp Pro Leu Tyr Lys Glu Ala Leu Gln Met Leu Arg Asp Ala 130 135 140 Gly Ala Gln Val Ser Ile Met Thr Tyr Asp Glu Phe Glu Tyr Cys Trp 145 150 155 160 Asp Thr Phe Val Tyr Arg Gln Gly Cys Pro Phe Gln Pro Trp Asp Gly 165 170 175 Leu Glu Glu His Ser Gln Ala Leu Ser Gly Arg Leu Arg Ala Ile Leu 180 185 190 Gln Asn Gln Gly Asn 195 <210> 8 <211> 16 <212> PRT <213> artificial sequence <220> <223> XTEN linker <400> 8 Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser 1 5 10 15 <210> 9 <211> 24 <212> PRT <213> artificial sequence <220> <223> P2A <400> 9 Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val 1 5 10 15 Glu Glu Asn Pro Gly Pro Pro Glu 20 <210> 10 <211> 2135 <212> PRT <213> artificial sequence <220> <223> AFID-3 <400> 10 Met Glu Ala Ser Pro Ala Ser Gly Pro Arg His Leu Met Asp Pro His 1 5 10 15 Ile Phe Thr Ser Asn Phe Asn Asn Gly Ile Gly Arg His Lys Thr Tyr 20 25 30 Leu Cys Tyr Glu Val Glu Arg Leu Asp Asn Gly Thr Ser Val Lys Met 35 40 45 Asp Gln His Arg Gly Phe Leu His Asn Gln Ala Lys Asn Leu Leu Cys 50 55 60 Gly Phe Tyr Gly Arg His Ala Glu Leu Arg Phe Leu Asp Leu Val Pro 65 70 75 80 Ser Leu Gln Leu Asp Pro Ala Gln Ile Tyr Arg Val Thr Trp Phe Ile 85 90 95 Ser Trp Ser Pro Cys Phe Ser Trp Gly Cys Ala Gly Glu Val Arg Ala 100 105 110 Phe Leu Gln Glu Asn Thr His Val Arg Leu Arg Ile Phe Ala Ala Arg 115 120 125 Ile Tyr Asp Tyr Asp Pro Leu Tyr Lys Glu Ala Leu Gln Met Leu Arg 130 135 140 Asp Ala Gly Ala Gln Val Ser Ile Met Thr Tyr Asp Glu Phe Lys His 145 150 155 160 Cys Trp Asp Thr Phe Val Asp His Gln Gly Cys Pro Phe Gln Pro Trp 165 170 175 Asp Gly Leu Asp Glu His Ser Gln Ala Leu Ser Gly Arg Leu Arg Ala 180 185 190 Ile Leu Gln Asn Gln Gly Asn Ser Gly Ser Glu Thr Pro Gly Thr Ser 195 200 205 Glu Ser Ala Thr Pro Glu Ser Leu Lys Asp Lys Lys Tyr Ser Ile Gly 210 215 220 Leu Asp Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu 225 230 235 240 Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg 245 250 255 His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly 260 265 270 Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr 275 280 285 Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn 290 295 300 Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser 305 310 315 320 Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly 325 330 335 Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr 340 345 350 His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg 355 360 365 Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe 370 375 380 Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu 385 390 395 400 Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro 405 410 415 Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu 420 425 430 Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu 435 440 445 Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu 450 455 460 Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu 465 470 475 480 Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala 485 490 495 Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu 500 505 510 Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile 515 520 525 Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His 530 535 540 His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro 545 550 555 560 Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala 565 570 575 Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile 580 585 590 Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys 595 600 605 Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly 610 615 620 Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg 625 630 635 640 Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile 645 650 655 Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala 660 665 670 Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr 675 680 685 Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala 690 695 700 Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn 705 710 715 720 Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val 725 730 735 Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys 740 745 750 Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu 755 760 765 Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr 770 775 780 Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu 785 790 795 800 Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile 805 810 815 Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu 820 825 830 Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile 835 840 845 Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met 850 855 860 Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg 865 870 875 880 Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu 885 890 895 Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu 900 905 910 Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln 915 920 925 Val Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala 930 935 940 Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val 945 950 955 960 Asp Glu Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val 965 970 975 Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn 980 985 990 Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly 995 1000 1005 Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln 1010 1015 1020 Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met 1025 1030 1035 Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp 1040 1045 1050 Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile 1055 1060 1065 Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser 1070 1075 1080 Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr 1085 1090 1095 Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe 1100 1105 1110 Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 1115 1120 1125 Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile 1130 1135 1140 Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys 1145 1150 1155 Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr 1160 1165 1170 Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe 1175 1180 1185 Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala 1190 1195 1200 Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro 1205 1210 1215 Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp 1220 1225 1230 Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala 1235 1240 1245 Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys 1250 1255 1260 Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu 1265 1270 1275 Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly 1280 1285 1290 Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val 1295 1300 1305 Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys 1310 1315 1320 Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg 1325 1330 1335 Light Light Asp Trp Asp Pro Light Light Tyr Gly Gly Phe Asp Ser Pro 1340 1345 1350 Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly 1355 1360 1365 Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr 1370 1375 1380 Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu 1385 1390 1395 Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys 1400 1405 1410 Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg 1415 1420 1425 Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala 1430 1435 1440 Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr 1445 1450 1455 Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu 1460 1465 1470 Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln 1475 1480 1485 Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu 1490 1495 1500 Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile 1505 1510 1515 Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn 1520 1525 1530 Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp 1535 1540 1545 Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu 1550 1555 1560 Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu 1565 1570 1575 Ser Gln Leu Gly Gly Asp Lys Arg Pro Ala Ala Thr Lys Lys Ala 1580 1585 1590 Gly Gln Ala Lys Lys Lys Lys Thr Arg Asp Ser Gly Gly Ser Ala 1595 1600 1605 Asn Glu Leu Thr Trp His Asp Val Leu Ala Glu Glu Lys Gln Gln 1610 1615 1620 Pro Tyr Phe Leu Asn Thr Leu Gln Thr Val Ala Ser Glu Arg Gln 1625 1630 1635 Ser Gly Val Thr Ile Tyr Pro Pro Gln Lys Asp Val Phe Asn Ala 1640 1645 1650 Phe Arg Phe Thr Glu Leu Gly Asp Val Lys Val Val Ile Leu Gly 1655 1660 1665 Gln Asp Pro Tyr His Gly Pro Gly Gln Ala His Gly Leu Ala Phe 1670 1675 1680 Ser Val Arg Pro Gly Ile Ala Ile Pro Pro Ser Leu Leu Asn Met 1685 1690 1695 Tyr Lys Glu Leu Glu Asn Thr Ile Pro Gly Phe Thr Arg Pro Asn 1700 1705 1710 His Gly Tyr Leu Glu Ser Trp Ala Arg Gln Gly Val Leu Leu Leu 1715 1720 1725 Asn Thr Val Leu Thr Val Arg Ala Gly Gln Ala His Ser His Ala 1730 1735 1740 Ser Leu Gly Trp Glu Thr Phe Thr Asp Lys Val Ile Ser Leu Ile 1745 1750 1755 Asn Gln His Arg Glu Gly Val Val Phe Leu Leu Trp Gly Ser His 1760 1765 1770 Ala Gln Lys Lys Gly Ala Ile Ile Asp Lys Gln Arg His His Val 1775 1780 1785 Leu Lys Ala Pro His Pro Ser Pro Leu Ser Ala His Arg Gly Phe 1790 1795 1800 Phe Gly Cys Asn His Phe Val Leu Ala Asn Gln Trp Leu Glu Gln 1805 1810 1815 Arg Gly Glu Thr Pro Ile Asp Trp Met Pro Val Leu Pro Ala Glu 1820 1825 1830 Ser Glu Pro Lys Lys Lys Arg Lys Val Ser Ala Gly Ser Gly Ala 1835 1840 1845 Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn 1850 1855 1860 Pro Gly Pro Pro Glu Gly Pro Glu Ile Arg Arg Ala Ala Asp Asn 1865 1870 1875 Leu Glu Ala Ala Ile Lys Gly Lys Pro Leu Thr Asp Val Trp Phe 1880 1885 1890 Ala Phe Pro Gln Leu Lys Pro Tyr Gln Ser Gln Leu Ile Gly Gln 1895 1900 1905 His Val Thr His Val Glu Thr Arg Gly Lys Ala Leu Leu Thr His 1910 1915 1920 Phe Ser Asn Asp Leu Thr Leu Tyr Ser His Asn Gln Leu Tyr Gly 1925 1930 1935 Val Trp Arg Val Val Asp Thr Gly Glu Glu Pro Gln Thr Thr Arg 1940 1945 1950 Val Leu Arg Val Lys Leu Gln Thr Ala Asp Lys Thr Ile Leu Leu 1955 1960 1965 Tyr Ser Ala Ser Asp Ile Glu Met Leu Thr Pro Glu Gln Leu Thr 1970 1975 1980 Thr His Pro Phe Leu Gln Arg Val Gly Pro Asp Val Leu Asp Pro 1985 1990 1995 Asn Leu Thr Pro Glu Val Val Lys Glu Arg Leu Leu Ser Pro Arg 2000 2005 2010 Phe Arg Asn Arg Gln Phe Ala Gly Leu Leu Leu Asp Gln Ala Phe 2015 2020 2025 Leu Ala Gly Leu Gly Asn Tyr Leu Arg Val Glu Ile Leu Trp Gln 2030 2035 2040 Val Gly Leu Thr Gly Asn His Lys Ala Lys Asp Leu Asn Ala Ala 2045 2050 2055 Gln Leu Asp Ala Leu Ala His Ala Leu Leu Glu Ile Pro Arg Phe 2060 2065 2070 Ser Tyr Ala Thr Arg Gly Gln Val Asp Glu Asn Lys His His Gly 2075 2080 2085 Ala Leu Phe Arg Phe Lys Val Phe His Arg Asp Gly Glu Pro Cys 2090 2095 2100 Glu Arg Cys Gly Ser Ile Ile Glu Lys Thr Thr Leu Ser Ser Arg 2105 2110 2115 Pro Phe Tyr Trp Cys Pro Gly Cys Gln His Pro Lys Lys Lys Arg 2120 2125 2130 Lys Val 2135 <210> 11 <211> 2133 <212> PRT <213> artificial sequence <220> <223> eAFID-3 <400> 11 Met Glu Ile Leu Arg Tyr Leu Met Asp Pro Asp Thr Phe Thr Phe Asn 1 5 10 15 Phe Asn Asn Asp Pro Leu Val Leu Arg Arg Arg Gln Thr Tyr Leu Cys 20 25 30 Tyr Glu Val Glu Arg Leu Asp Asn Gly Thr Trp Val Leu Met Asp Gln 35 40 45 His Met Gly Phe Leu Cys Asn Glu Ala Lys Asn Leu Leu Cys Gly Phe 50 55 60 Tyr Gly Arg His Ala Glu Leu Arg Phe Leu Asp Leu Val Pro Ser Leu 65 70 75 80 Gln Leu Asp Pro Ala Gln Ile Tyr Arg Val Thr Trp Phe Ile Ser Trp 85 90 95 Ser Pro Cys Phe Ser Trp Gly Cys Ala Gly Glu Val Arg Ala Phe Leu 100 105 110 Gln Glu Asn Thr His Val Arg Leu Arg Ile Phe Ala Ala Arg Ile Tyr 115 120 125 Asp Tyr Asp Pro Leu Tyr Lys Glu Ala Leu Gln Met Leu Arg Asp Ala 130 135 140 Gly Ala Gln Val Ser Ile Met Thr Tyr Asp Glu Phe Glu Tyr Cys Trp 145 150 155 160 Asp Thr Phe Val Tyr Arg Gln Gly Cys Pro Phe Gln Pro Trp Asp Gly 165 170 175 Leu Glu Glu His Ser Gln Ala Leu Ser Gly Arg Leu Arg Ala Ile Leu 180 185 190 Gln Asn Gln Gly Asn Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser 195 200 205 Ala Thr Pro Glu Ser Leu Lys Asp Lys Lys Tyr Ser Ile Gly Leu Asp 210 215 220 Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys 225 230 235 240 Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser 245 250 255 Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr 260 265 270 Only Glu Only Thr Arg Leu Only Thr Arg Only On Arg Arg Tyr Thr Arg 275 280 285 Arg Lys Asn Arg With Cys Tyr Leu Gln Glu With Phe Served Asn Glu Met 290,295,300 Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu 305 310 315 320 Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile 325 330 335 Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu 340 345 350 Arg Lys Lys Leu Val Asp Serves Thr Asp Ala Asp Leu Arg Leu Ile 355 360 365 Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile 370 375 380 Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile 385 390 395 400 Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn 405 410 415 Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys 420 425 430 Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys 435 440 445 Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro 450 455 460 Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu 465 470 475 480 Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile 485 490 495 Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp 500 505 510 Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys 515 520 525 Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln 530 535 540 Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys 545 550 555 560 Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr 565 570 575 Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro 580 585 590 Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn 595 600 605 Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile 610 615 620 Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln 625 630 635 640 Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys 645 650 655 Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly 660 665 670 Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr 675 680 685 Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser 690 695 700 Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys 705 710 715 720 Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn 725 730 735 Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala 740 745 750 Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys 755 760 765 Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys 770 775 780 Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg 785 790 795 800 Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys 805 810 815 Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp 820 825 830 Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu 835 840 845 Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln 850 855 860 Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu 865 870 875 880 Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe 885 890 895 Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His 900 905 910 Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser 915 920 925 Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser 930 935 940 Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu 945 950 955 960 Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu 965 970 975 Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg 980 985 990 Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln 995 1000 1005 Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu 1010 1015 1020 Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val 1025 1030 1035 Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp 1040 1045 1050 His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn 1055 1060 1065 Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn 1070 1075 1080 Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg 1085 1090 1095 Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn 1100 1105 1110 Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala 1115 1120 1125 Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys 1130 1135 1140 His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 1145 1150 1155 Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys 1160 1165 1170 Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys 1175 1180 1185 Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu 1190 1195 1200 Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu 1205 1210 1215 Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg 1220 1225 1230 Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala 1235 1240 1245 Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu 1250 1255 1260 Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu 1265 1270 1275 Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp 1280 1285 1290 Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile 1295 1300 1305 Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser 1310 1315 1320 Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys 1325 1330 1335 Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val 1340 1345 1350 Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser 1355 1360 1365 Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met 1370 1375 1380 Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala 1385 1390 1395 Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro 1400 1405 1410 Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu 1415 1420 1425 Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro 1430 1435 1440 Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys 1445 1450 1455 Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val 1460 1465 1470 Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser 1475 1480 1485 Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys 1490 1495 1500 Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu 1505 1510 1515 Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly 1520 1525 1530 Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys 1535 1540 1545 Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His 1550 1555 1560 Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln 1565 1570 1575 Leu Gly Gly Asp Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln 1580 1585 1590 Ala Lys Lys Lys Lys Thr Arg Asp Ser Gly Gly Ser Ala Asn Glu 1595 1600 1605 Leu Thr Trp His Asp Val Leu Ala Glu Glu Lys Gln Gln Pro Tyr 1610 1615 1620 Phe Leu Asn Thr Leu Gln Thr Val Ala Ser Glu Arg Gln Ser Gly 1625 1630 1635 Val Thr Ile Tyr Pro Pro Gln Lys Asp Val Phe Asn Ala Phe Arg 1640 1645 1650 Phe Thr Glu Leu Gly Asp Val Lys Val Val Ile Leu Gly Gln Asp 1655 1660 1665 Pro Tyr His Gly Pro Gly Gln Ala His Gly Leu Ala Phe Ser Val 1670 1675 1680 Arg Pro Gly Ile Ala Ile Pro Pro Ser Leu Leu Asn Met Tyr Lys 1685 1690 1695 Glu Leu Glu Asn Thr Ile Pro Gly Phe Thr Arg Pro Asn His Gly 1700 1705 1710 Tyr Leu Glu Ser Trp Ala Arg Gln Gly Val Leu Leu Leu Asn Thr 1715 1720 1725 Val Leu Thr Val Arg Ala Gly Gln Ala His Ser His Ala Ser Leu 1730 1735 1740 Gly Trp Glu Thr Phe Thr Asp Lys Val Ile Ser Leu Ile Asn Gln 1745 1750 1755 His Arg Glu Gly Val Val Phe Leu Leu Trp Gly Ser His Ala Gln 1760 1765 1770 Lys Lys Gly Ala Ile Ile Asp Lys Gln Arg His His Val Leu Lys 1775 1780 1785 Ala Pro His Pro Ser Pro Leu Ser Ala His Arg Gly Phe Phe Gly 1790 1795 1800 Cys Asn His Phe Val Leu Ala Asn Gln Trp Leu Glu Gln Arg Gly 1805 1810 1815 Glu Thr Pro Ile Asp Trp Met Pro Val Leu Pro Ala Glu Ser Glu 1820 1825 1830 Pro Lys Lys Lys Arg Lys Val Ser Ala Gly Ser Gly Ala Thr Asn 1835 1840 1845 Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly 1850 1855 1860 Pro Pro Glu Gly Pro Glu Ile Arg Arg Ala Ala Asp Asn Leu Glu 1865 1870 1875 Ala Ala Ile Lys Gly Lys Pro Leu Thr Asp Val Trp Phe Ala Phe 1880 1885 1890 Pro Gln Leu Lys Pro Tyr Gln Ser Gln Leu Ile Gly Gln His Val 1895 1900 1905 Thr His Val Glu Thr Arg Gly Lys Ala Leu Leu Thr His Phe Ser 1910 1915 1920 Asn Asp Leu Thr Leu Tyr Ser His Asn Gln Leu Tyr Gly Val Trp 1925 1930 1935 Arg Val Val Asp Thr Gly Glu Glu Pro Gln Thr Thr Arg Val Leu 1940 1945 1950 Arg Val Lys Leu Gln Thr Ala Asp Lys Thr Ile Leu Leu Tyr Ser 1955 1960 1965 Ala Ser Asp Ile Glu Met Leu Thr Pro Glu Gln Leu Thr Thr His 1970 1975 1980 Pro Phe Leu Gln Arg Val Gly Pro Asp Val Leu Asp Pro Asn Leu 1985 1990 1995 Thr Pro Glu Val Val Lys Glu Arg Leu Leu Ser Pro Arg Phe Arg 2000 2005 2010 Asn Arg Gln Phe Ala Gly Leu Leu Leu Asp Gln Ala Phe Leu Ala 2015 2020 2025 Gly Leu Gly Asn Tyr Leu Arg Val Glu Ile Leu Trp Gln Val Gly 2030 2035 2040 Leu Thr Gly Asn His Lys Ala Lys Asp Leu Asn Ala Ala Gln Leu 2045 2050 2055 Asp Ala Leu Ala His Ala Leu Leu Glu Ile Pro Arg Phe Ser Tyr 2060 2065 2070 Ala Thr Arg Gly Gln Val Asp Glu Asn Lys His His Gly Ala Leu 2075 2080 2085 Phe Arg Phe Lys Val Phe His Arg Asp Gly Glu Pro Cys Glu Arg 2090 2095 2100 Cys Gly Ser Ile Ile Glu Lys Thr Thr Leu Ser Ser Arg Pro Phe 2105 2110 2115 Tyr Trp Cys Pro Gly Cys Gln His Pro Lys Lys Lys Arg Lys Val 2120 2125 2130

Claims

1. A gene editing system for editing a target sequence in a cell genome, comprising: i) a first polypeptide, and / or an expression construct comprising a nucleotide sequence encoding the first polypeptide; ii) a second polypeptide, and / or an expression construct comprising a nucleotide sequence encoding the second polypeptide; and iii) a guide RNA, and / or an expression construct comprising a nucleotide sequence encoding the guide RNA, wherein the first polypeptide is a fusion polypeptide of cytosine deaminase, CRISPR nuclease and uracil-DNA glycosylase connected in sequence, the second polypeptide is AP lyase, wherein the guide RNA can target the first polypeptide to a target sequence in the cell genome, wherein the CRISPR nuclease is a wild-type Cas9 nuclease, The wild-type Cas9 nuclease has double-stranded DNA cleavage activity. wherein the cytosine deaminase is APOBEC3A deaminase, The amino acid sequence of the APOBEC3A deaminase is SEQ ID NO: 2, The amino acid sequence of the uracil-DNA glycosylase is SEQ ID NO: 3, The amino acid sequence of the AP lyase is SEQ ID NO:

4.

2. A gene editing system for editing a target sequence in a cell genome, comprising: i) a polypeptide, and / or an expression construct comprising a nucleotide sequence encoding the polypeptide; and ii) a guide RNA, and / or an expression construct comprising a nucleotide sequence encoding the guide RNA, The polypeptides include cytosine deaminase, CRISPR nuclease, uracil-DNA glycosylase and AP lyase, and the enzymes are connected in sequence. wherein the guide RNA is capable of targeting the polypeptide to a target sequence in the cell genome, wherein the CRISPR nuclease is a wild-type Cas9 nuclease, The wild-type Cas9 nuclease has double-stranded DNA cleavage activity. wherein the cytosine deaminase is APOBEC3A deaminase, The amino acid sequence of the APOBEC3A deaminase is SEQ ID NO: 2, The amino acid sequence of the uracil-DNA glycosylase is SEQ ID NO: 3, The amino acid sequence of the AP lyase is SEQ ID NO:

4.

3. The gene editing system of claim 1 or 2, wherein the wild-type Cas9 nuclease is spCas9.

4. The gene editing system of claim 1, wherein the amino acid sequence of the first polypeptide is SEQ ID NO:5, and the amino acid sequence of the second polypeptide is SEQ ID NO:

6.

5. The gene editing system of claim 2, wherein the amino acid sequence of the polypeptide is SEQ ID NO:

10.

6. A method for producing a genetically modified plant cell, comprising introducing the gene editing system of any one of claims 1-5 into a plant cell, wherein the genetic modification is a deletion of 2-18 consecutive nucleotides in the target sequence.

7. The method of claim 6, wherein the genetic modification is a deletion of 10-17 consecutive nucleotides in the target sequence.

8. The method of claim 6 or 7, wherein the plants include monocots and dicots.

9. The method of claim 8, wherein the plant is selected from the group consisting of rice, corn, wheat, sorghum, barley, soybean, peanut, and Arabidopsis.

10. A kit comprising the gene editing system of any one of claims 1-5.

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