Modularized gene editing tool and application thereof

Abstract

The invention belongs to the field of gene engineering. Specifically, the invention relates to a modular gene editing tool and application thereof. Specifically, the invention provides a modularized gene editing tool protein capable of accurately editing a genome nucleotide sequence of an organism, a gene editing system comprising the protein and a method for performing gene editing by using the system. The modular gene editing tool disclosed by the invention can realize efficient, accurate and universal gene editing in cells of various eukaryotes such as animals and plants. The types of existing editing tools are enriched, and the method has a wide application prospect.

Description

[0001] Priority and related applications

[0002] The present invention claims priority to Chinese patent application No. 202311159790.3 filed on September 8, 2023, entitled “A modular gene editing tool and its application”, and all the contents of this application including the appendix are incorporated into the present invention by reference. Technical Field

[0003] The present invention belongs to the field of genetic engineering. Specifically, the present invention relates to a modular gene editing tool and its application. More specifically, the present invention provides a modular gene editing tool protein that can accurately edit the genomic nucleotide sequence of an organism, a base editing system comprising the modular gene editing tool protein, and a method for gene editing using the protein or system. The editing tool can achieve various types of precise nucleotide sequence editing. Background Art

[0004] Gene editing can precisely modify the genetic information of an organism and has become a disruptive technology in the field of life sciences. It has been applied to many areas, including biomedicine development, genetic engineering therapy, crop genetic improvement, and ecological and environmental protection.

[0005] Early gene editing technologies were represented by zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN), which achieve the positioning and editing of target genes through the interaction between proteins and nucleic acids. In 2012, gene editing technology based on clustered regularly interspaced short palindromic repeats (CRISPR) sequences was developed. Since then, the system has been rapidly applied by researchers in the field because of its simple operation and high editing efficiency. The principle is that under the guidance of a small RNA sequence called sgRNA, the endonuclease Cas protein locates to the target DNA site to perform cutting function and induce DNA double-strand breaks (DSB), thereby activating the role of DNA repair pathways such as non-homologous end joining (NHEJ) in the cell. The NHEJ pathway is not precise in performing DNA repair, which may result in the introduction of some base insertions or deletions (indels) in the repaired DNA, which in turn causes the target gene's expression frame to undergo a frameshift mutation and become inactive, ultimately achieving the purpose of gene knockout. Since NHEJ's indels are random, this type of gene editing system is random and imprecise.

[0006] In order to achieve more precise gene editing, base editing and base editing systems that do not rely on DSB were developed in 2016. Base editing is a type of gene editing technology, which refers to the process of replacing nucleotides at specific DNA sites through genetic engineering. The base editor is a fusion protein containing Cas protein and deaminase. When the specific Cas protein recognizes and opens the DNA double helix, the deaminase catalyzes the deamination reaction of the nucleotide base, thereby achieving base conversion. However, the current mainstream base editing systems only include cytosine base editor (CBE) and adenine base editor (ABE), which contain cytosine nucleotide deaminase and adenine nucleotide deaminase, respectively, and can only achieve C-to-T and A-to-G base conversion, and cannot achieve other types of base replacement, and the application fields and scenarios are limited.

[0007] In order to achieve more types of precise base replacements, a prime editor (PE) was developed in 2019. PE introduces target mutations into target DNA through reverse transcription of template RNA by reverse transcriptase, which can achieve replacement of all 12 types of bases, as well as insertion or deletion of any short nucleotide segments in a site-specific manner. It is worth noting that PE editing can achieve a lower rate of unexpected indels. Therefore, PE can achieve more precise targeted insertion, making editing applications that were previously impossible or impractical possible.

[0008] However, to date, most gene editing tools have been designed, improved and applied based on the two types of Cas systems that have been discovered, while there is little research on the structural optimization and modular improvement of editing tools, and their application scenarios and fields are limited. Summary of the invention

[0009] Problem that the invention aims to solve

[0010] In order to enrich the application scenarios of gene editing and provide a variety of gene editing tools for selection in the field, the inventors of the present application have artificially synthesized modular gene editing tools with nucleotide replacement, insertion or deletion functions through modular assembly after a lot of experiments and explorations.

[0011] Solutions for solving problems

[0012] The first aspect of the present invention provides a modular protein, wherein the modular protein comprises the following functional domains:

[0013] (a) comprising an amino acid sequence as set forth in SEQ ID NO:2, or a functional domain of an amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:2;

[0014] (b) a domain comprising the amino acid sequence as set forth in SEQ ID NO:3, or an amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:3;

[0015] (c) a domain comprising the amino acid sequence as set forth in SEQ ID NO:4, or an amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:4;

[0016] (d) a domain comprising the amino acid sequence as set forth in SEQ ID NO:5, or an amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:5;

[0017] (e) a domain comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:6; and

[0018] (f) a domain comprising the amino acid sequence as set forth in SEQ ID NO:7, or an amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:7;

[0019] The modular protein also includes:

[0020] (g) a DNA polymerase domain, which is located in the middle region of the modular protein at the non-terminal end and is connected to at least one of the functional domains in (a) to (f) above by terminal fusion or embedding.

[0021] In some embodiments, the DNA polymerase is an RNA-dependent DNA polymerase or a DNA-dependent DNA polymerase, preferably a reverse transcriptase.

[0022] In some specific embodiments, the DNA polymerase domain sequence comprises an amino acid sequence as shown in SEQ ID NO: 1, or an amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the DNA polymerase domain comprises (a) a mutation selected from any one of F156Y, F156V, F157Y, D525N, D201C, the amino acid position being referenced to SEQ ID NO: 1; (b) the connection sequence is deleted; and / or (c) the RNase H domain is mutated or deleted (see, patent WO2023030534A1).

[0023] In some embodiments, the modular protein further comprises:

[0024] (h) a functional domain comprising the amino acid sequence as set forth in SEQ ID NO:8, or an amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:8.

[0025] In some specific embodiments, the modular protein comprises one or more of the following sequences:

[0026] (i) an amino acid sequence as shown in any one of SEQ ID NOs: 9-35, 56-58;

[0027] (ii) an amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in any one of SEQ ID NOs: 9-35, 56-58;

[0028] (iii) an amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in any one of SEQ ID NOs: 9-35, 56-58; or,

[0029] (iv) an amino acid sequence encoded by a nucleotide sequence that hybridizes with a polynucleotide sequence encoding an amino acid sequence as shown in any one of SEQ ID NOs: 9-35, 56-58 under stringent conditions, wherein the stringent conditions are medium stringency conditions, medium-high stringency conditions, high stringency conditions or very high stringency conditions.

[0030] In some embodiments, the DNA polymerase domain is embedded in the functional domain (f)

[0031] In some embodiments, the DNA polymerase domain is embedded in the functional domain (f) at any position between amino acids 143-146 relative to SEQ ID NO:7.

[0032] In some specific embodiments, the modular protein comprises one or more of the following sequences:

[0033] (i) an amino acid sequence as shown in any one of SEQ ID NOs: 29-31, 56-58 and 13-15;

[0034] (ii) an amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 29-31, 56-58 and 13-15;

[0035] (iii) an amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in any one of SEQ ID NOs: 29-31, 56-58 and 13-15; or,

[0036] (iv) an amino acid sequence encoded by a nucleotide sequence that hybridizes with a polynucleotide sequence encoding the amino acid sequence shown in any one of SEQ ID NOs: 29-31, 56-58 and 13-15 under stringent conditions, wherein the stringent conditions are medium stringency conditions, medium-high stringency conditions, high stringency conditions or very high stringency conditions.

[0037] In some preferred embodiments, the modular protein comprises one or more of the following sequences:

[0038] (i) an amino acid sequence as shown in any one of SEQ ID NOs: 29-31, 56-58;

[0039] (ii) an amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 29-31, 56-58;

[0040] (iii) an amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in any one of SEQ ID NOs: 29-31, 56-58; or,

[0041] (iv) an amino acid sequence encoded by a nucleotide sequence that hybridizes with a polynucleotide sequence encoding the amino acid sequence shown in any one of SEQ ID NOs: 29-31, 56-58 under stringent conditions, wherein the stringent conditions are medium stringency conditions, medium-high stringency conditions, high stringency conditions or very high stringency conditions.

[0042] In some embodiments, the DNA polymerase domain is connected to the C-terminus of the functional domain (b) and the N-terminus of the functional domain (c) in a terminal fusion manner.

[0043] In some specific embodiments, the modular protein comprises one or more of the following sequences:

[0044] (i) the amino acid sequences shown in SEQ ID NOs: 11, 16-19 and 34-35;

[0045] (ii) an amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 11, 16-19 and 34-35;

[0046] (iii) an amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in SEQ ID NOs: 11, 16-19 and 34-35; or

[0047] (iv) an amino acid sequence encoded by a nucleotide sequence that hybridizes with a polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NOs: 11, 16-19 and 34-35 under stringent conditions, wherein the stringent conditions are medium stringency conditions, medium-high stringency conditions, high stringency conditions or very high stringency conditions.

[0048] In some preferred embodiments, the modular protein comprises one or more of the following sequences:

[0049] (i) the amino acid sequence shown in SEQ ID NO: 11;

[0050] (ii) an amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 11;

[0051] (iii) an amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in SEQ ID NO: 11; or,

[0052] (iv) an amino acid sequence encoded by a nucleotide sequence that hybridizes with a polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 11 under stringent conditions, wherein the stringent conditions are medium stringency conditions, medium-high stringency conditions, high stringency conditions or very high stringency conditions.

[0053] In some embodiments, the terminal fusion or insertion comprises direct fusion and / or connection through a linker.

[0054] In some specific embodiments, the linker comprises the amino acid sequence (GGGS)n(SEQ ID NO:43), (GGGGS)n(SEQ ID NO:41), (G)n, (EAAAK)n(SEQ ID NO:42), (GGS)n, (SGGS)n(SEQ ID NO:44), SGSETPGTSESATPES(SEQ ID NO:45), SGGSSGGSSGSETPGTSESATPESSGGSSGGS(SEQ ID NO:46) or (XP)n motif or a combination thereof, wherein n is independently an integer from 1-32, and wherein X is any amino acid.

[0055] A second aspect of the present invention provides a base editing system, the base editing system comprising:

[0056] (i) a modular protein as described in the first aspect of the present invention, or an expression construct of a nucleotide sequence encoding the modular protein; and

[0057] (ii) a modular protein guide RNA (mgRNA), or an expression construct of a nucleotide sequence encoding mgRNA.

[0058] In some embodiments, the mgRNA comprises, from 5' to 3' direction, a guide sequence, a scaffold sequence, a reverse transcription template (RT) sequence, and a primer binding site (PBS) sequence.

[0059] In some embodiments, the RT sequence is an exogenous donor DNA or RNA for inserting an exogenous nucleotide sequence.

[0060] In some embodiments, the PBS sequence is configured to be complementary to at least a portion of a target sequence of a nucleic acid molecule;

[0061] Preferably, the PBS sequence is complementary to at least a portion of the 3' free single strand caused by the nick in the DNA strand where the target sequence of the nucleic acid molecule is located.

[0062] In some embodiments, the modular protein binds to the nucleic acid molecule target sequence under the guidance of mgRNA, and replaces or inserts the RT sequence into the corresponding endogenous nucleotide sequence through DNA repair.

[0063] In some embodiments, the base editing system further comprises: (iii) a nuclear localization sequence (NLS).

[0064] The third aspect of the present invention provides a method for producing at least one genetically modified cell, the method comprising introducing a modular protein as described in the first aspect of the present invention or a base editing system as described in the second aspect of the present invention into at least one cell, thereby causing substitution, insertion or deletion of a nucleic acid sequence within a target nucleic acid region in the at least one cell.

[0065] In some embodiments, the modular protein or base editing system is introduced into cells by a method selected from the following: calcium phosphate transfection, protoplast fusion, electroporation, liposome transfection, microinjection, viral infection (such as baculovirus, vaccinia virus, adenovirus, adeno-associated virus, lentivirus or other virus), N-acetylgalactosamine (GalNAc) mediation, gene gun method, PEG-mediated protoplast transformation and / or soil Agrobacterium-mediated transformation.

[0066] In some embodiments, the cell is from a prokaryotic organism such as a bacterium, a eukaryotic organism such as a plant, a fungus, or a vertebrate.

[0067] In some embodiments, the vertebrate is a mammal such as a human, mouse, rat, monkey, dog, pig, sheep, cow, cat; the plant is a crop plant, such as wheat, rice, corn, soybean, sunflower, sorghum, rapeseed, alfalfa, cotton, barley, millet, sugarcane, tomato, tobacco, cassava or potato.

[0068] The fourth aspect of the present invention provides a polynucleotide, which encodes the modular protein as described in the first aspect of the present invention or the base editing system as described in the second aspect of the present invention.

[0069] The fifth aspect of the present invention provides an expression construct, which comprises the polynucleotide as described in the fourth aspect of the present invention.

[0070] The sixth aspect of the present invention provides a pharmaceutical composition, comprising the modular protein as described in the first aspect of the present invention, the polynucleotide as described in the fourth aspect of the present invention, the expression construct as described in the fifth aspect of the present invention, or the base editing system as described in the second aspect of the present invention;

[0071] Optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0072] The seventh aspect of the present invention provides a kit, which comprises the modular protein as described in the first aspect of the present invention, the polynucleotide as described in the fourth aspect of the present invention, the expression construct as described in the fifth aspect of the present invention, or the base editing system as described in the second aspect of the present invention.

[0073] The eighth aspect of the present invention provides a method for modifying a target nucleic acid, the method comprising the step of contacting the target nucleic acid with a modular protein as described in the first aspect of the present invention, a polynucleotide as described in the fourth aspect of the present invention, an expression construct as described in the fifth aspect of the present invention, a base editing system as described in the second aspect of the present invention, a pharmaceutical composition as described in the sixth aspect of the present invention, or a kit as described in the seventh aspect of the present invention.

[0074] The ninth aspect of the present invention provides the use of the modular protein as described in the first aspect of the present invention, the polynucleotide as described in the fourth aspect of the present invention, the expression construct as described in the fifth aspect of the present invention, or the base editing system as described in the second aspect of the present invention in the preparation of a reagent for modifying a target nucleic acid.

[0075] The tenth aspect of the present invention provides the use of the modular protein as described in the first aspect of the present invention, the polynucleotide as described in the fourth aspect of the present invention, the expression construct as described in the fifth aspect of the present invention, or the base editing system as described in the second aspect of the present invention in the preparation of a medicament for preventing and / or treating diseases related to or caused by gene mutations.

[0076] Effects of the Invention

[0077] The modular gene editing tool provided by the present invention can be used for all 12 possible base substitution types, and can also realize the insertion and deletion of DNA fragments. The modular base editing tool effectively reduces the frequency of indel occurrence and achieves editing efficiency comparable to or improved compared to traditional gene editing technology. In addition, the composition and structure of the modular gene editing tool are more streamlined, which helps the expression or delivery of the gene editing tool in the organism to adapt to more application scenarios. The modular gene editing tool of the present invention can realize efficient, accurate and universal gene editing in cells of various eukaryotic organisms including animals and plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 , DNA polymerase domain ( Figure 1 Schematic diagram of the assembly of reverse transcriptase (reverse transcriptase: RT) with protein functional domains in an embedded and terminal fusion manner.

[0079] Figure 2 , Schematic diagram of the structure of protein expression construct based on modular gene editing tools.

[0080] Figure 3 , the efficiency of base substitution by ME editor.

[0081] Figure 4 , the efficiency of base fragment insertion by ME editor.

[0082] Figure 5 , the efficiency of base fragment deletion by ME editor.

[0083] Figure 6 , preferred ME editor and outstanding effect in base substitution.

[0084] Figure 7 , preferred ME editors for indels in base substitution editing. DETAILED DESCRIPTION

[0085] I. Definition

[0086] 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.

[0087] 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."

[0088] 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.

[0089] "Gene", "genome" as used herein encompasses not only chromosomal DNA present in the cell nucleus, but also organelle DNA present in subcellular components of the cell (eg, mitochondria, plastids).

[0090] As used herein, "organism" includes any organism suitable for genome editing, such as prokaryotes, eukaryotes, preferably eukaryotes. 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 wheat, rice, corn, soybeans, sunflowers, sorghum, rapeseed, alfalfa, cotton, barley, millet, sugarcane, tomatoes, tobacco, cassava and potatoes, etc.

[0091] "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.

[0092] "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.

[0093] "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.

[0094] 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)).

[0095] 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).

[0096] The "RNA-dependent DNA polymerase" of the present invention refers to an enzyme that can synthesize a DNA chain using RNA as a template, recognize the base sequence on the RNA chain, and synthesize a complementary DNA sequence on the DNA chain.

[0097] The "domain" of the present invention refers to the part of a protein that has a relatively stable three-dimensional structure. Proteins are usually composed of one or more domains, each of which has its own spatial folding morphology. Domains can be continuous or discontinuous, and they are usually composed of one or more segments in an amino acid sequence. The presence of domains enables proteins to have functional diversity, and new functions can be achieved through the recombination and combination of domains. For example, the DNA polymerase domain shown in SEQ ID NO:1.

[0098] The "functional domain" of the present invention refers to the part of the protein that has a specific biological function or performs a specific task. The functional domain determines the specific function of the protein in the organism, such as enzyme activity, ligand binding, signal transduction, etc. Different functional domains can perform different biological functions in proteins. For example, the functional domains of the protein of the present invention include the RuvC functional domain (SEQID NO: 2-4), which is responsible for the cutting of the non-complementary strand in the double-stranded DNA; the REC functional domain (SEQ ID NO: 6), which is responsible for binding to the target double-stranded DNA and the mgRNA backbone; the PI functional domain (SEQ ID NO: 7), which is responsible for the recognition and interaction with the specific PAM sequence; the BH functional domain (SEQ ID NO: 5), which is involved in the formation and stabilization of the R-loop structure; and optionally, the HNH functional domain (SEQ ID NO: 8).

[0099] The "modular gene editing tool protein" and "modular protein" of the present invention can be used interchangeably, and refer to a non-natural protein with a new function obtained by connecting two or more functional domains or structural domains with specific functions.

[0100] As used herein, "expression construct" or "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, expression of a nucleotide sequence may refer to the transcription of the nucleotide sequence (such as transcription to generate mRNA or functional RNA) and / or translation of RNA into a precursor or mature protein.

[0101] 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).

[0102] 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.

[0103] "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.

[0104] "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.

[0105] "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.).

[0106] 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.

[0107] "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.

[0108] "Stable transformation" refers to the introduction of an exogenous nucleotide sequence into a genome, resulting in the stable inheritance of the exogenous nucleotide sequence. Once stably transformed, the exogenous nucleic acid sequence is stably integrated into the genome of the organism and any successive generations thereof.

[0109] "Transient transformation" refers to the introduction of a nucleic acid molecule or protein into a cell to perform its function without the stable inheritance of the exogenous nucleotide sequence. In transient transformation, the exogenous nucleic acid sequence is not integrated into the genome.

[0110] "Trait" refers to a physiological, morphological, biochemical, or physical characteristic of a cell or organism.

[0111] "Agronomic traits" specifically refer to measurable indicator parameters of crop plants, including but not limited to: leaf green, grain yield, growth rate, total biomass or accumulation rate, fresh weight at maturity, dry weight at maturity, fruit yield, seed yield, plant total nitrogen content, fruit nitrogen content, seed nitrogen content, plant vegetative tissue nitrogen content, plant total free amino acid content, fruit free amino acid content, seed free amino acid content, plant vegetative tissue free amino acid content, plant total protein content, fruit protein content, seed protein content, plant vegetative tissue protein content, herbicide resistance and drought resistance, nitrogen absorption, root lodging, harvest index, stem lodging, plant height, ear height, ear length, disease resistance, cold resistance, salt resistance and tillering number, etc.

[0112] 2. Modular gene editing tool proteins

[0113] In one aspect, the present invention provides a modular protein comprising the following functional domains:

[0114] (a) comprising an amino acid sequence as set forth in SEQ ID NO:2, or a functional domain of an amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:2;

[0115] (b) a domain comprising the amino acid sequence as set forth in SEQ ID NO:3, or an amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:3;

[0116] (c) a domain comprising the amino acid sequence as set forth in SEQ ID NO:4, or an amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:4;

[0117] (d) a domain comprising the amino acid sequence as set forth in SEQ ID NO:5, or an amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:5;

[0118] (e) a domain comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:6; and

[0119] (f) a functional domain comprising the amino acid sequence as set forth in SEQ ID NO:7, or an amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:7.

[0120] In some embodiments, the modular protein further includes (g) a DNA polymerase domain, which is located in the non-terminal middle region of the modular protein and is connected to at least one of the functional domains in (a) to (f) above by terminal fusion or embedding.

[0121] The modular protein can recognize and bind to the nucleic acid molecule target sequence through its functional domain, and replace or insert the template nucleotide sequence into the corresponding endogenous nucleotide sequence (i.e., the target nucleic acid region) through DNA repair mediated by the template nucleotide sequence; the template nucleotide sequence is an exogenous donor DNA or RNA.

[0122] As used herein, "non-terminal middle region" means that the DNA polymerase domain is located inside the overall modular protein and is not located at the N-terminus or C-terminus of the overall modular protein.

[0123] "Terminal fusion" means that the DNA polymerase domain is connected to the N-terminus or C-terminus of the above-mentioned single functional domain with or without a linker; "embedded" means that the DNA polymerase domain is connected to the inside of the above-mentioned single functional domain with or without a linker.

[0124] In some embodiments, the DNA polymerase is an RNA-dependent DNA polymerase or a DNA-dependent DNA polymerase, preferably a reverse transcriptase.

[0125] In some embodiments, the DNA polymerase domain comprises an amino acid sequence as shown in SEQ ID NO:1, or an amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in SEQ ID NO:1.

[0126] In some embodiments, the DNA polymerase domain comprises (a) a mutation selected from any one of F156Y, F156V, F157Y, D525N, D201C, the amino acid position referring to SEQ ID NO: 1; (b) the connection sequence is deleted; and / or (c) the RNase H domain is mutated or deleted (see, patent WO2023030534A1).

[0127] In some embodiments, the modular protein further comprises:

[0128] (h) a functional domain comprising the amino acid sequence as set forth in SEQ ID NO:8, or an amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:8.

[0129] In some embodiments, the terminal fusion or insertion comprises direct fusion and / or connection through a linker.

[0130] As used herein, a "linker" can be a non-functional amino acid sequence 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 in length, without a secondary or higher structure. For example, the linker can be a flexible linker. In some embodiments, the linker comprises the amino acid sequence (GGGS)n(SEQ ID NO:43), (GGGGS)n(SEQ ID NO:41), (G)n, (EAAAK)n(SEQ ID NO:42), (GGS)n, (SGGS)n(SEQ ID NO:44), SGSETPGTSESATPES(SEQ ID NO:45), SGGSSGGSSGSETPGTSESATPESSGGSSGGS(SEQ ID NO:46), or (XP)n motif, or a combination thereof, wherein n is independently an integer from 1-32, and wherein X is any amino acid.

[0131] In some embodiments, the modular protein comprises one or more of the following sequences:

[0132] (i) the amino acid sequence shown in any one of SEQ ID NOs: 9-35, 56-58;

[0133] (ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% sequence identity to the amino acid sequence shown in any one of SEQ ID NOs: 9-35, 56-58;

[0134] (iii) an amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in any one of SEQ ID NOs: 9-35, 56-58; or,

[0135] (iv) an amino acid sequence encoded by a nucleotide sequence that hybridizes with a polynucleotide sequence encoding an amino acid sequence as shown in any one of SEQ ID NOs: 9-35, 56-58 under stringent conditions, wherein the stringent conditions are medium stringency conditions, medium-high stringency conditions, high stringency conditions or very high stringency conditions.

[0136] In the present invention, "moderate stringency conditions", "moderate-high stringency conditions", "high stringency conditions" or "very high stringency conditions" describe conditions for nucleic acid hybridization and washing. Guidance for conducting hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6, which is incorporated herein by reference. Aqueous and non-aqueous methods are described in this document, and either can be used. For example, specific hybridization conditions are as follows: (1) low stringency hybridization conditions are in 6× sodium chloride / sodium citrate (SSC) at about 45°C, followed by two washes in 0.2×SSC, 0.1% SDS at at least 50°C (for low stringency conditions, the washing temperature can be increased to 55°C); (2) moderate stringency hybridization conditions are in 6×SSC at about 45°C, followed by one or more washes in 0.2×SSC, 0.1% SDS at 60°C; (3) high stringency hybridization conditions are in 6×SSC at about 45°C, followed by one or more washes in 0.2×SSC, 0.1% SDS at 65°C and preferably; (4) very high stringency hybridization conditions are 0.5 M sodium phosphate, 7% SDS at 65°C, followed by one or more washes in 0.2×SSC, 1% SDS at 65°C.

[0137] In some preferred embodiments, the modular protein comprises an amino acid sequence shown in any one of SEQ ID NOs: 11, 13-19, 29-31, 56-58 and 33-35.

[0138] In some more preferred embodiments, the modular protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 11, 17-19, 29-31 and 56-58.

[0139] In some further preferred embodiments, the modular protein comprises an amino acid sequence shown in any one of SEQ ID NOs: 11, 29-31 and 56-58.

[0140] In some specific embodiments, the DNA polymerase domain is embedded in the functional domain (f) (SEQ ID NO: 7).

[0141] In some preferred embodiments, the DNA polymerase domain is embedded in the functional domain (f) at any position between amino acids 143-146 relative to SEQ ID NO:7, such as a modular protein having an amino acid sequence shown in any one of SEQ ID NOs: 13-15, 29-31 and 56-58 (i.e., ME5-7, 21-23 and 28-30).

[0142] In other specific embodiments, the DNA polymerase domain is connected to the C-terminus of the functional domain (b) (SEQ ID NO: 3) and the N-terminus of the functional domain (c) (SEQ ID NO: 4) in the form of terminal fusion, such as modular proteins with amino acid sequences shown in SEQ ID NOs: 11, 16-19 and 34-35 (i.e., ME3, 8-11, 26-27).

[0143] In another aspect, the present invention also provides a polynucleotide encoding the above modular protein.

[0144] In another aspect, the present invention also provides an expression construct comprising the above-mentioned polynucleotide.

[0145] 3. Base editing system containing modular gene editing tool proteins

[0146] In another aspect, the present invention provides a base editing system comprising:

[0147] (i) the modular protein described in “II. Modular gene editing tool protein” above, or an expression construct of a nucleotide sequence encoding the modular protein; and

[0148] (ii) a modular protein guide RNA (mgRNA), or an expression construct of a nucleotide sequence encoding mgRNA.

[0149] On the other hand, the present invention also provides a polynucleotide encoding the above-mentioned base editing system.

[0150] In another aspect, the present invention also provides an expression construct comprising the above-mentioned polynucleotide.

[0151] As used herein, "mgRNA" comprises a guide sequence, a scaffold sequence, an optional reverse transcription template (RT) sequence, and a primer binding site (PBS) sequence. The mgRNA is used to guide the modular protein described in the present invention to recognize the PAM sequence, so that it forms an incision at a specific position of the target DNA chain, which can cause the target DNA chain to form a free single strand with a 3' end (3' free single strand) and a free single strand with a 5' end (5' free single strand). The PBS sequence of the mgRNA then binds to the cut target DNA chain, and the RT sequence is read by the reverse transcriptase to write the required changes into the new DNA chain. Subsequently, the cell's own DNA repair mechanism will replace the original chain with the newly synthesized DNA chain, thereby completing precise gene editing.

[0152] In some embodiments, the mgRNA contains some additional modifications that can stabilize the mgRNA or otherwise prevent it from being degraded. For example, synonymous mutations are regularly introduced into the mgRNA; or the mgRNA backbone is optimized, for example, the base pairs in its hairpin structure are modified; or a structural motif is attached to the 3' end of the mgRNA. Suitable structural motifs include, but are not limited to, aptamers, stem loops, hairpins, toe loops (e.g., 3' toe loops) or RNA-protein recruitment domains (e.g., MS2 hairpins), G-quadruplexes, tRNAs, riboswitches, and ribozymes. In some embodiments, non-limiting examples of structural motifs include evopreQ1 and mpknot motifs.

[0153] In some embodiments, the RT sequence is present in isolation or contained in the mgRNA.

[0154] In some embodiments, the RT sequence is an exogenous donor DNA or RNA for inserting an exogenous nucleotide sequence.

[0155] In some embodiments, the PBS sequence is configured to be complementary to at least a portion of a target nucleic acid sequence. Preferably, the PBS sequence is complementary to at least a portion of a 3' free single strand caused by a nick in a DNA chain (i.e., a target DNA chain) where the target nucleic acid sequence is located.

[0156] In some embodiments, the modular protein binds to the nucleic acid molecule target sequence under the guidance of mgRNA, and replaces or inserts the RT sequence into the corresponding endogenous nucleotide sequence through DNA repair.

[0157] In some embodiments, the base editing system further includes: (iii) a nuclear localization sequence (NLS). In general, one or more NLSs in the protein should have sufficient strength to drive the modular protein to accumulate in the nucleus of the cell in an amount that can achieve its base editing function. In general, the strength of the nuclear localization activity is determined by the number, position, one or more specific NLSs used, or a combination of these factors in the modular protein.

[0158] In some embodiments, the NLS of the modular protein 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 modular protein of the present invention may be located between each functional domain or domain. In some embodiments, the modular protein comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLSs. In some embodiments, the modular protein comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLSs at or near the N-terminus. In some embodiments, the modular protein comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLSs at or near the C-terminus. In some embodiments, the polypeptide comprises a combination of these, such as one or more NLSs at the N-terminus and one or more NLSs at the C-terminus. When there is more than one NLS, each can be selected to be independent of other NLSs.

[0159] 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 (SEQ ID NO: 51), PKKKRKV (SEQ ID NO: 49), or KRPAATKKAGQAKKKK (SEQ ID NO: 50).

[0160] In addition, depending on the DNA location to be edited, the modular protein of the present invention may also include other localization sequences, such as cytoplasmic localization sequences, chloroplast localization sequences, mitochondrial localization sequences, etc.

[0161] IV. Methods for Producing Genetically Modified Cells

[0162] In another aspect, the present invention provides a method for producing at least one genetically modified cell, comprising introducing the modular protein or nucleic acid construct into at least one of the cells, thereby causing substitution, insertion or deletion of a nucleic acid sequence within a target nucleic acid region in the at least one cell.

[0163] In some embodiments, the method further comprises the step of screening the at least one cell for cells having the desired one or more nucleotide substitutions.

[0164] 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.

[0165] In another aspect, the present invention also provides a genetically modified organism comprising a genetically modified cell or a progeny cell thereof produced by the method of the present invention. Preferably, the genetically modified cell or a progeny cell thereof has a desired one or more nucleotide substitutions.

[0166] In the present invention, the target nucleic acid region to be modified can be located at any position of 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. In some embodiments, the desired nucleotide substitution results in a desired gene function modification or gene expression modification.

[0167] In some embodiments, the target nucleic acid region is related to the proterties of the cell or organism. In some embodiments, the mutation in the target nucleic acid region causes a change in the proterties of the cell or organism. In some embodiments, the target nucleic acid region is located in the coding region of protein. In some embodiments, the function-related motif or domain of the target nucleic acid region encoding protein. In some preferred embodiments, one or more nucleotides in the target nucleic acid region replace the amino acid replacement in the amino acid sequence of the protein. In some embodiments, the one or more nucleotides replace the change in the function of the protein.

[0168] In some embodiments, the modular protein or base editing system is introduced into cells by a method selected from the following: calcium phosphate transfection, protoplast fusion, electroporation, liposome transfection, microinjection, viral infection (such as baculovirus, vaccinia virus, adenovirus, adeno-associated virus, lentivirus or other virus), N-acetylgalactosamine (GalNAc) mediation, gene gun method, PEG-mediated protoplast transformation, soil Agrobacterium-mediated transformation.

[0169] In some embodiments, the cell is from a prokaryotic organism such as a bacterium, a eukaryotic organism such as a plant, a fungus, or a vertebrate.

[0170] In some embodiments, organisms that can be subjected to genome modification by the methods of the present invention include any organisms suitable for base editing, preferably eukaryotic organisms. 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, for example, the plants are crop plants, including but not limited to wheat, rice, corn, soybeans, sunflowers, sorghum, rapeseed, alfalfa, cotton, barley, millet, sugarcane, tomatoes, tobacco, cassava and potatoes.

[0171] 5. Test kit

[0172] The present invention also includes a kit for the method of the present invention, the kit comprising a modular protein provided by the present invention, a polynucleotide provided by the present invention, an expression construct provided by the present invention, or a base editing system provided by 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.

[0173] In some specific embodiments, the kit may be a disease treatment kit. In other specific embodiments, the kit may be a kit for target nucleic acid modification or for generating genetically modified cells.

[0174] VI. Pharmaceutical Composition

[0175] The present disclosure provides a pharmaceutical composition, which comprises the modular protein provided by the present invention, the polynucleotide provided by the present invention, the expression construct provided by the present invention, or the base editing system provided by the present invention.

[0176] In some optional embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0177] In some embodiments, the pharmaceutically acceptable carrier may be a delivery carrier, such as a lipid, a cationic lipid or other polymers having drug delivery function.

[0178] VII. Methods and uses of modifying target nucleic acids

[0179] The present invention provides a method for modifying a target nucleic acid, the method comprising contacting the target nucleic acid with a modular protein provided by the present invention, a polynucleotide provided by the present invention, an expression construct provided by the present invention, a base editing system provided by the present invention, a pharmaceutical composition provided by the present invention, or a kit provided by the present invention. In some embodiments, the contacting results in modification of the target nucleic acid by the modular protein or base editing system.

[0180] The present invention also provides the use of the modular protein provided by the present invention, the polynucleotide provided by the present invention, the expression construct provided by the present invention, or the base editing system provided by the present invention in the preparation of a reagent for modifying a target nucleic acid.

[0181] In some specific embodiments, the modification is a change in a nucleotide in the target nucleic acid. In some specific embodiments, the nucleotide change can be a nucleotide substitution, deletion or insertion.

[0182] 8. Methods and uses of treating diseases

[0183] The present invention provides a method for preventing and / or treating a disease associated with or caused by a gene mutation, comprising administering to a subject a therapeutically effective amount of a modular protein, polynucleotide, expression construct, base editing system, pharmaceutical composition, or kit of the present invention. The gene mutation can be corrected by, for example, a modular protein or base editing system provided herein.

[0184] The present invention also provides the use of the modular protein, polynucleotide, expression construct, base editing system, or kit described in the present invention in the preparation of a drug for preventing and / or treating diseases related to or caused by gene mutations.

[0185] The present invention also provides the modular protein, polynucleotide, expression construct, base editing system, pharmaceutical composition, or kit of the present invention, which is used to prevent and / or treat diseases related to or caused by gene mutations.

[0186] Modular proteins or base editing systems provided herein produce nucleotide changes that correct disease-related genes. Exemplary, disease-related genes may be associated with a monogenic disorder selected from the group consisting of adenosine deaminase (ADA) deficiency; alpha-1 antitrypsin deficiency; cystic fibrosis; Duchenne muscular dystrophy; galactosemia; hemochromatosis; Huntington's disease; maple syrup urine disease; Marfan syndrome; neurofibromatosis type 1; congenital pachyonychia; phenylketonuria; severe combined immunodeficiency; sickle cell disease; Stevens-Lee-Ou syndrome; and Tay-Sachs disease. In other embodiments, disease-related genes may be associated with polygenic disorders selected from the group consisting of heart disease; hypertension; Alzheimer's disease; arthritis; diabetes; cancer; and obesity.

[0187] Example

[0188] Example 1: Acquisition of new modular gene editing tools

[0189] The inventors conducted a modular analysis of the functional domain / domain SEQ ID NO: 1-8 by bioinformatics and divided the DNA polymerase domain ( Figure 1 Reverse transcriptase: RT) with terminal fusion ( Figure 1 B) or embedded in ( Figure 1 A) in the figure is rearranged and fused with multiple different protein functional domains in various ways to obtain a series of modular gene editing tool proteins ME1-ME27 (SEQ ID NO: 9-35). The overall schematic diagram of the expression construct is shown in FIG. Figure 2 shown. Figure 1A in Figure 1 B in the figure shows how to assemble the DNA polymerase domain with the protein domain by embedding and terminal fusion, and provides a scheme that can selectively contain the HNH domain. It should be noted that Figure 1 The schematic diagram is only an exemplary display of the combination scheme between the functional domains, and the arrangement order and integration method of the functional domains are not limited to the scheme shown in the schematic diagram.

[0190] Example 2: Multiple editing functions on the human cell genome

[0191] In this example, various functions of a series of modular gene editing tool proteins obtained by the design method of Example 1 were tested. In this example, the above-mentioned mgRNA and base editor plasmids based on a series of modular gene editing tool proteins were transfected into HEK293T cells. The overall schematic diagram of the expression construct is shown in FIG. Figure 2 As shown, different types of editing are performed on different sites. The specific targets are described in detail in the following experiments. DNA was extracted 72 hours after transfection; the Mock group was used as a blank control without transfection of the base editor plasmid, and the other treatments were the same as above. The base editing efficiency of the target was detected using the second-generation sequencing technology.

[0192] Experiment (1) Base substitution

[0193] ME editors can achieve effective base substitution. This experiment (1) demonstrates the editing effects of different ME editors on base substitution at the APP1 site. The APP1 site sequence (SEQ ID NO: 38): The underlined 20 bp is the target sequence, and the bold italic A is the base to be edited. Through ME editing, the bold italic A base in the sequence is replaced with G. The mgRNA used is (SEQ ID NO: 52):

[0194] GGAGATCTCTGAAGTGAAGA GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCGGAATTCTGTATCCATCTTCACTTCAGAGATAATAAGAC

[0195] The underlined part is the guide sequence.

[0196] The results are shown in Table 1. ME3, ME5-11, ME21-23 and ME25-27 all have the ability to make single base substitutions. Figure 3 As shown, the editing abilities of ME3, ME21, ME22, and ME23 are better in single-base substitution editing among a series of ME editors.

[0197] Table 1

[0198]

[0199] Among them, Null in the above table means: no editing efficiency was detected.

[0200] Experiment (2) Base fragment deletion

[0201] The ME editor with higher single-base editing ability in experiment (1) was experimentally verified for base fragment deletion. This experiment demonstrates the editing effect of base deletion at the RUNX1 site, deleting a 15bp segment of bases from +1 to +15. The RUNX1 site sequence (SEQ ID NO: 39) is: The underlined 20 bp are the target sequences, and the bold italic sequences are the fragments that need to be deleted through ME editing.

[0202] The mgRNA used was (SEQ ID NO: 53):

[0203] GCATTTTCAGGAGGAAGCGA GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCGCACTGTGGGTACGAAGGAAATGACTCAAATATGCCTTCCTCCTGAAAATAACTCTCT

[0204] The underlined part is the guide sequence.

[0205] The results are as follows Figure 4 As shown, compared with the blank control group, ME3, ME9-11 and ME21-23 all have the ability to delete bases, among which ME3 and ME21-23 have higher efficiency and better effect in base deletion editing.

[0206] Experiment (3) Base fragment insertion

[0207] This experiment shows the editing effect of different ME editors on base fragment insertion at the RNF2 site, starting from position +1, inserting a 24bp long DNA sequence. RNF2 site sequence (SEQ ID NO: 40): The underlined 20 bp is the target sequence, and the bold italic sequence is a 24 bp DNA fragment that needs to be inserted through ME editing.

[0208] The mgRNA used was (SEQ ID NO: 54):

[0209] GTCATCTTAGTCATTACCTG GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTGAGTTACAACGAACACCTCAGCTTATCGTCGTCATCCTTGTAATCGTAATGACTAAGATGTCATCTCT

[0210] The underlined part is the guide sequence.

[0211] The results are as follows Figure 5 As shown, compared with the blank control group, ME3, ME9-11 and ME21-23 all have the ability to insert base fragments, among which ME3 and ME21-23 (i.e., the modules of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 3, SEQ ID NO: 8, SEQ ID NO: 4, and SEQ ID NO: 7 are connected sequentially, and the sequence of the DNA polymerase domain is respectively embedded between the 143 / 144, 144 / 145, and 145 / 146 amino acids of SEQ ID NO: 7) have higher efficiency and better effect in base fragment insertion editing.

[0212] In summary, after screening, this embodiment obtained a batch of ME editing tools that can complete various base editing functions such as base replacement, base fragment deletion, base fragment insertion, etc., among which the editing capabilities of ME3, ME21, ME22, and ME23 editing tools all showed high efficiency in the above three gene editing methods, indicating that the modular gene editing tool of the present invention is an effective gene editing tool with the potential for precise gene editing.

[0213] Example 3: Preferred ME editors have outstanding effects in base substitution

[0214] This example further verifies the ME editor selected in Example 2. The inventors selected HEK293T cells as a verification model, targeting the HEK3 site, and the HEK3 site sequence (SEQ ID NO: 37): The underlined 20 bp are the target sequence, and the bold italic T is the base that needs to be edited. Through ME editing, the bold italic T base in the sequence is replaced with A.

[0215] The mgRNA sequence used in the experiment is as follows (SEQ ID NO: 36):

[0216] GGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTCCTCTGCCATCTCGTGCTCAGTCTGTCTCTCTTGACGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAAA

[0217] The underlined part is the guide sequence.

[0218] HEK293T cells were co-transfected with modular gene editing proteins indicated by ME1-ME3 and mgRNA targeting the HEK3 target. DNA was extracted 72 hours after transfection, and then the reverse transcription editing results of the reverse transcription template at the DNA target site were analyzed using next-generation sequencing (NGS).

[0219] Figure 6 It shows that each modular gene editing tool protein has achieved efficient and precise editing, with the highest editing efficiency reaching 25.3%. Referring to the reported PE editor (James W. Nelson et al., 2022; Omer Habib et al., 2022), the editing efficiency of the four preferred ME editing tools of the present invention is greatly improved compared with PE1; compared with PE2, the editing efficiency of ME3, ME21, and ME22 editors is comparable to PE2, but ME23 editor is still significantly improved compared with PE2. It shows that the modular gene editing tool of the present invention still has significantly improved gene editing capabilities compared with the relatively mature prior art.

[0220] Example 4: Preferred ME editor indels are significantly reduced

[0221] Also at the HEK3 site, edit-replacement editing was performed as in Example 3, and this example analyzed the frequency of random insertion / deletion (indel) byproducts generated during editing. Indel refers to random base insertion or deletion outside of the non-targeted site, and high indel levels will destroy the accuracy of gene editing.

[0222] like Figure 7 As shown, ME3 and ME21-23 achieve effective base substitution at the HEK3 site and induce significantly lower indel byproducts than the PE2 system. ME3, ME21, and ME22 editors significantly reduce indels while ensuring that the editing efficiency is similar to that of PE2; the ME23 editor can significantly reduce indels while improving the editing efficiency. This result shows that the ME editor of the present invention has made great progress in the precise editing of genes, further ensuring the safety of gene editing.

[0223] Example 5: Replacement and optimization of DNA polymerase domain

[0224] In this example, a functional variant of the DNA polymerase domain (M-MLV) (SEQ ID NO: 55) was used to replace the M-MLV domain in the aforementioned preferred ME21-ME23 editors, and ME28-ME30 editors (SEQ ID NO: 56-58) were obtained to analyze the effect of the selection of the DNA polymerase domain on the ME editor. The M-MLV variant used refers to the M-MLV protein modified by the inventor's prior patent WO2023030534A1, and the stability or expression amount of the fusion protein is increased by point mutation of the reverse transcriptase or deletion of its redundant domain.

[0225] The base substitution efficiency was verified by the same method as in Experiment (1) of Example 2. The results are shown in Table 2. ME28-ME30 also has a single base substitution editing effect.

[0226] Table 2

[0227] Editing Protein Editing efficiency (%) ME28 3.70 ME29 2.94 ME30 3.08

[0228] Although the present invention has been described with reference to its specific embodiments, it will be appreciated by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the present invention. In addition, many modifications may be made to adapt specific circumstances, materials, compositions of matter, processes, process steps or multiple steps to the purpose, spirit and scope of the present invention. All such modifications are intended to be within the scope of the appended claims.

[0229] Sequence Listing:

[0230] >SEQ ID NO: 1DNA polymerase domain (M-MLV, Figure 1 Reverse transcriptase (RT)

[0231] STLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP

[0232] >SEQ ID NO:2RuvC I

[0233] DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAE

[0234] >SEQ ID NO:3RuvC II

[0235] SLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHK

[0236] >SEQ ID NO:4RuvC III

[0237] ELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTE

[0238] >SEQ ID NO:5BH

[0239] ATRLKRTARRRRYTRRKNRICYLQEIFSNEMAKVD

[0240] >SEQ ID NO:6REC

[0241] DSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKLVDSTDKADLLRIYLALAMHIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP NFKSNFDLAEDAKLQLSKDTYDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFD NGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVK QLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRIEMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGD

[0242] >SEQ ID NO:7PI

[0243] VQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD

[0244] >SEQ ID NO:8HNH

[0245] PENIVIEMARENQTTQKGQKNSRERMKRIEGIGELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLS

[0246] >SEQ ID NO:9ME1

[0247] MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYSGGSSGGSSGSETPGTSESATPESSGGSSGGS STLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIIPLKATSTPVSIKQYPMSQEAR LGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYT VLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYV DDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQ LREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQG YAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRW LSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSS LLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGW LTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP

[0248] >SEQ ID NO:10 ME2

[0249] MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETEATRLKRTARRRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKLVDSTDKADLRIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEEN PINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDL LRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILK STLNIEDEYRLHETSKEPDVSLGSTWLSDFP QAWAETGGMGLAVRQAPLIIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGT NDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQ LTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKK AQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLF NWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCL RMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPL PEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAE LIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGH QKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSPSGGSSGGSSGSETPGTSESATPESSGGSSGGSNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD*

[0250] >SEQ ID NO:11 ME3

[0251] MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRNASLGTYHDLLKIIK DKDFLDNEENEDILEDIVLTLTLFEDROMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVEN STLNIEDEYRLHETSKEPDVSLGST WLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPE MGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGY RASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLT KPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAA GWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNP ATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGT SAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSI IHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP SGGSSGGSSGSETPGTSESATPESSSGGSSGGSLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPK RNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILDANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD*

[0252] >SEQ ID NO:12 ME4

[0253] STLNIEDEYR LHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQG ILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLH PTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQ QGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLF IPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWR RPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLL DTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVT TETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP EKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD*

[0254] >SEQ ID NO:13 ME5

[0255] STLNIEDEY RLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQ GILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRL HPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDC QQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRL FIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPW RRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALL LDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAV TTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDE ILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP KLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD*

[0256] >SEQ ID NO:14 ME6

[0257] STLNIEDE YRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLD QGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELD CQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCR LFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGP WRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQAL LLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAA VTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKD EILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP LKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD*

[0258] >SEQ ID NO:15 ME7

[0259] STLNIED EYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLL DQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCL RLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSEL DCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFC RLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLG PWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQA LLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGA AVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNK DEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP KGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILDANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD*

[0260] >SEQ ID NO:16 ME8

[0261] MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIK DKDFLDNEENEDILEDIVLTLTLFEDRAMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVEN STLNIEDEYRLHETSKEPDVSLGST WLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPE MGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGY RASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLT KPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAA GWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNP ATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGT SAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSI IHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP*

[0262] >SEQ ID NO:17 ME9

[0263] MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQ RTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKD KDFLDNEENEDILEDIVLTLTLFEDRAMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQ STLNIEDEYRLHETSKEPDVSLG STWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPL LPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRD PEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNL GYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYP LTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPV AAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVAL NPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPA GTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRL SIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP*

[0264] >SEQ ID NO:18 ME10

[0265] MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQR TFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKD FLDNEENEDILEDIVLTLTLFEDREMIEERLKTYALFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIGELGSQILKEHPVENTQLQ STLNIEDEYRLHETSKEPDVS LGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNT PLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEW RDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLG NLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPL YPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLD PVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVV ALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKAL PAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPK RLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP *

[0266] >SEQ ID NO:19 ME11

[0267] MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRT FDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDF LDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIGELGSQILKEHPVENTQLQN STLNIEDEYRLHETSKEPDV SLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWN TPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFE WRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTL GNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAP LYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKL DPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPV VALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKA LPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLP KRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP *

[0268] >SEQ ID NO:20 ME12

[0269] STL NIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHI QRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDA FFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAA TSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGK AGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLT QKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMT HYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQR KAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKE IKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP SGGSSGGSSGSETPGTSESATPESSGGSSGGSNGEIRKRPLIETNGETGEIVWDKGRDFATVRLKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD*

[0270] >SEQ ID NO:21 ME13

[0271] S TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKP HIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLK DAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLL AATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFL GKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGV LTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNAR MTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEG QRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEG KEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP SGGSSGGSSSGSETPGTSESATPESSSGGSSGGSEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLII KLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD*

[0272] >SEQ ID NO:22 ME14

[0273] STLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGI KPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLD LKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDL LLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLRE FLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAK GVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSN ARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQ EGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTS EGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP SGGSSGGSSGSETPGTSESATPESSGGSSGGSRKRPLIETNGETGEIVWDKGRDFATVRLQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD*

[0274] >SEQ ID NO:23 ME15

[0275] MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKLVDSTDKADLRIYLALAMHIKFRGHFLIEGDLNPDNSDV STLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGG MGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQD LREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAEWRDPEMGISGQLTWTRLPQ GFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASACKAQICQKQV KYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQK AYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAV LTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHN CLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQAL KMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEA RGNRMADQAARKAAITETPDTSTLLIENSSP

[0276] >SEQ ID NO:24 ME16

[0277] MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI STLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAW AETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDY RPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAEWRDPEMGISGQLTW TRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQI CQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWG PDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMV AAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEE GLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIA LTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKG HSAEARGNRMADQAARKAAITETPDTSTLLIENSSP

[0278] >SEQ ID NO:25 ME17

[0279] MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEEN STLNIEDEYRLHETSKEPDVS LGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNT PLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEW RDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLG NLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPL YPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLD PVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVV ALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKAL PAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPK RLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP

[0280] >SEQ ID NO:26 ME18

[0281] MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETEATRLKRTARRRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKLVDSTDKADLRIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFE ENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNR EDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKE STLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETG GMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQ DLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLP QGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQ VKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQ KAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIA VLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQH NCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQA LKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAE ARGNRMADQAARKAAITETPDTSTLLIENSSP SGGSSGGSSSGSETPGTSESATPESSSGGSSSGGSLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYKLESEFVYGDYKYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR KKDWDKPKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD*

[0282] >SEQ ID NO:27 ME19

[0283] MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEE LLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISG VEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDROMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKN STLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIP LKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHP TVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEAL HRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRW LTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAP ALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQP LVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRP DLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTD SRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKA AITETPDTSTLLIENSSP SGGSSGGSSSGSETPGTSESATPESSSGGSSSGGSLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYKLESEFVYGDYKYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR KKDWDKPKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD*

[0284] >SEQ ID NO:28 ME20

[0285] MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELL VKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVED RFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRAMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRER STLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAP LIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVE DIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLF NEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKE GQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQAL LTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLT MGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAH GTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLN VYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQA ARKAAITETPDTSTLLIENSSP SGGSSGGSSSGSETPGTSESATPESSSGGSSGGSGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYKLESEFVYGDYKYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIA RKKDWDKPKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD*

[0286] >SEQ ID NO:29 ME21

[0287] STLNIEDEYR LHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQG ILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLH PTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQ QGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLF IPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWR RPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLL DTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVT TETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEI LALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP SGGSSGGSSGSETPGTSESATPESSGGSSGGSEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD*

[0288] >SEQ ID NO:30 ME22

[0289] STLNIEDEY RLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQ GILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRL HPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDC QQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRL FIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPW RRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALL LDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAV TTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDE ILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP SGGSSGGSSGSETPGTSESATPESSGGSSGGSKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD*

[0290] >SEQ ID NO:31 ME23

[0291] STLNIEDE YRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLD QGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLR LHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELD CQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCR LFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGP WRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQAL LLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAA VTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKD EILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP SGGSSGGSSGSETPGTSESATPESSGGSSGGSLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD*

[0292] >SEQ ID NO:32 ME24

[0293] STLNIED EYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLL DQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCL RLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSEL DCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFC RLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLG PWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQA LLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGA AVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNK DEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP SGGSSGGSSGSETPGTSESATPESSGGSSGGSKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD*

[0294] >SEQ ID NO:33 ME25

[0295] MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYSGGSSGGSSGSETPGTSESATPESSGGSSGGS STLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEAR LGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYT VLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYV DDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQ LREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQG YAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRW LSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSS LLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGW LTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP

[0296] >SEQ ID NO:34 ME26

[0297] MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETEATRLKRTARRRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKLVDSTDKADLRIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEEPINASGVD AKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQI HLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKSGGSSGGSSGSETPGTSESATPESSGGSSGGSSG STLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGI KPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLD LKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDL LLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLRE FLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAK GVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSN ARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQ EGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTS EGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP SGGSSGGSSGSETPGTSESATPESSGGSSGGSNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD*

[0298] >SEQ ID NO:35ME27

[0299] MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLNLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLG ELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLF EDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIGELGSQILKEHPVENSGGSSGGSSGSETPGTSESATPESSGGSSGGS STLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQ EARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQ WYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILL QYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKT PRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDE KQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPP DRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTD GSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRR RGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSS PSGGSSGGSSGSETPGTSESATPESSGGSSGGSLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD*

[0300] Among the above SEQ ID NO:9 - 35, the underlined part is the sequence of the DNA polymerase domain.

[0301] >SEQ ID NO:36 mgRNA to HEK3(HEK293T)

[0302] GGCCCAGACTGAGCACGTGA GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCttcctctgccatctCGTGCTCAGTCTGTCTCTCTCTTGACGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAAA

[0303] >SEQ ID NO:52 mgRNA to APP1

[0304] GGAGATCTCTGAAGTGAAGA GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCGGAATTCTGTATCCATCTTCACTTCAGAGATAATAAGAC

[0305] >SEQ ID NO:53mgRNA to RUNX1

[0306] GCATTTTCAGGAGGAAGCGA GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCGCACTGTGGGTACGAAGGAAATGACTCAAATATGCCTTCCTCCTGAAAATAACTCTCT

[0307] >SEQ ID NO:54mgRNA to RNF2

[0308] GTCATCTTAGTCATTACCTG GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTGAGTTACAACGAACACCTCAGCTTATCGTCGTCATCCTTGTAATCGTAATGACTAAGATGTCATCTCT

[0309] >SEQ ID NO:37HEK3

[0310]

[0311] >SEQ ID NO:38APP1:

[0312]

[0313] >SEQ ID NO:39RUNX1:

[0314]

[0315] >SEQ ID NO:40RNF2:

[0316]

[0317] >SEQ ID NO:41

[0318] YGGGS

[0319] >SEQ ID NO:42

[0320] EAAAK

[0321] >SEQ ID NO:43

[0322] GGGS

[0323] >SEQ ID NO:44

[0324] SGGS

[0325] >SEQ ID NO:45

[0326] SGSETPGTSESATPES

[0327] >SEQ ID NO:46

[0328] SGGSSGGSSGSETPGTSESATPESSGGSSGGS

[0329] >SEQ ID NO:47PE1 amino acid sequence:

[0330]

[0331] >SEQ ID NO:48 PE2 amino acid sequence:

[0332]

[0333] >SEQ ID NO:49

[0334] PKKKRKV

[0335] >SEQ ID NO:50

[0336] KRPAATKKAGQAKKKK

[0337] >SEQ ID NO:51

[0338] KKRKV

[0339] >SEQ ID NO:55 DNA polymerase domain (M-MLV) variant

[0340] STLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTTLLIENSSP

[0341] >SEQ ID NO:56 ME28

[0342] STLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPM SQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPS HQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLI LLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTP KTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFV DEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQ PPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTTL LIENSSP SGGSSGGSSGSETPGTSESATPESSSGGSSGGSEKLKGSPEDNEQKQLFVEQHHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSKRTADGSEFEPKKKRKV*

[0343] >SEQ ID NO:57ME29

[0344] STLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYP MSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPP SHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDL ILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPT PKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELF VDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVK QPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTT LLIENSSP SGGSSGGSSGSETPGTSESATPESSGGSSGGSKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSKRTADGSEFEPKKKRKV*

[0345] >SEQ ID NO:58 ME30

[0346] STLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQY PMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLP PSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPD LILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQP TPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFEL FVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALV KQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHT TLLIENSSP SGGSSGGSSGSETPGTSESATPESSGGSSGGSLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSKRTADGSEFEPKKKRKV*

[0347] In the above SEQ ID NOs: 56-58, the underlined portion is the sequence of the DNA polymerase domain.

Claims

1. A modular protein comprising the following functional domains: (a) comprising an amino acid sequence as set forth in SEQ ID NO:2, or a functional domain of an amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:2; (b) a domain comprising the amino acid sequence as set forth in SEQ ID NO:3, or an amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:3; (c) a domain comprising the amino acid sequence as set forth in SEQ ID NO:4, or an amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:4; (d) a domain comprising the amino acid sequence as set forth in SEQ ID NO:5, or an amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:5; (e) a domain comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:6; and (f) a domain comprising the amino acid sequence as set forth in SEQ ID NO:7, or an amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:7; It is characterized in that The modular protein also includes: (g) a DNA polymerase domain, which is located in the middle region of the modular protein at the non-terminal end and is connected to at least one of the functional domains in (a) to (f) above by terminal fusion or embedding.

2. The modular protein according to claim 1, characterized in that The DNA polymerase is an RNA-dependent DNA polymerase or a DNA-dependent DNA polymerase, preferably a reverse transcriptase.

3. The modular protein according to claim 2, characterized in that The DNA polymerase domain sequence comprises an amino acid sequence as shown in SEQ ID NO: 1, or an amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, or a functional variant thereof.

4. The modular protein according to any one of claims 1 to 3, characterized in that The modular protein also includes: (h) a functional domain comprising the amino acid sequence as set forth in SEQ ID NO:8, or an amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:

8.

5. The modular protein according to any one of claims 1 to 4, characterized in that The modular protein comprises one or more of the following sequences: (i) the amino acid sequence shown in any one of SEQ ID NOs: 9-35, 56-58; (ii) an amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in any one of SEQ ID NOs: 9-35, 56-58; (iii) an amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in any one of SEQ ID NOs: 9-35, 56-58; or, (iv) an amino acid sequence encoded by a nucleotide sequence that hybridizes with a polynucleotide sequence encoding an amino acid sequence as shown in any one of SEQ ID NOs: 9-35, 56-58 under stringent conditions, wherein the stringent conditions are medium stringency conditions, medium-high stringency conditions, high stringency conditions or very high stringency conditions.

6. The modular protein according to any one of claims 1 to 5, characterized in that The DNA polymerase domain is embedded in the functional domain (f); preferably, the DNA polymerase domain is embedded in the functional domain (f) at any position between amino acids 143-146 relative to SEQ ID NO:

7.

7. The modular protein according to claim 6, characterized in that The modular protein comprises one or more of the following sequences: (i) an amino acid sequence as shown in any one of SEQ ID NOs: 29-31, 56-58 and 13-15; (ii) an amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 29-31, 56-58 and 13-15; (iii) an amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in any one of SEQ ID NOs: 29-31, 56-58 and 13-15; or, (iv) an amino acid sequence encoded by a nucleotide sequence that hybridizes to a polynucleotide sequence encoding an amino acid sequence as shown in any one of SEQ ID NOs: 29-31, 56-58 and 13-15 under stringent conditions, wherein the stringent conditions are medium stringency conditions, medium-high stringency conditions, high stringency conditions or very high stringency conditions; Preferably, the modular protein comprises one or more of the following sequences: (i) an amino acid sequence as shown in any one of SEQ ID NOs: 29-31, 56-58; (ii) an amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 29-31, 56-58; (iii) an amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in any one of SEQ ID NOs: 29-31, 56-58; or, (iv) an amino acid sequence encoded by a nucleotide sequence that hybridizes with a polynucleotide sequence encoding the amino acid sequence shown in any one of SEQ ID NOs: 29-31, 56-58 under stringent conditions, wherein the stringent conditions are medium stringency conditions, medium-high stringency conditions, high stringency conditions or very high stringency conditions.

8. The modular protein according to any one of claims 1 to 5, characterized in that The DNA polymerase domain is connected to the C-terminus of the functional domain (b) and the N-terminus of the functional domain (c) in a terminal fusion manner.

9. The modular protein according to claim 8, characterized in that The modular protein comprises one or more of the following sequences: (i) the amino acid sequences shown in SEQ ID NOs: 11, 16-19 and 34-35; (ii) an amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 11, 16-19 and 34-35; (iii) an amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in SEQ ID NOs: 11, 16-19 and 34-35; or, (iv) an amino acid sequence encoded by a nucleotide sequence that hybridizes to a polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NOs: 11, 16-19 and 34-35 under stringent conditions, wherein the stringent conditions are medium stringency conditions, medium-high stringency conditions, high stringency conditions or very high stringency conditions; Preferably, the modular protein comprises one or more of the following sequences: (i) the amino acid sequence shown in SEQ ID NO: 11; (ii) an amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 11; (iii) an amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in SEQ ID NO: 11; or, (iv) an amino acid sequence encoded by a nucleotide sequence that hybridizes with a polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 11 under stringent conditions, wherein the stringent conditions are medium stringency conditions, medium-high stringency conditions, high stringency conditions or very high stringency conditions.

10. The modular protein according to any one of claims 1 to 9, characterized in that The terminal fusion or insertion method includes direct fusion and / or connection through a linker.

11. The modular protein according to claim 10, characterized in that The linker comprises the amino acid sequence (GGGS)n (SEQ ID NO:43), (GGGGS)n (SEQ ID NO:41), (G)n, (EAAAK)n (SEQ ID NO:42), (GGS)n, (SGGS)n (SEQ ID NO:44), SGSETPGTSESATPES (SEQ ID NO:45), SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO:46) or (XP)n motif or a combination thereof, wherein n is independently an integer from 1-32, and wherein X is any amino acid.

12. A base editing system, characterized in that: The base editing system comprises: (i) a modular protein according to any one of claims 1 to 11, or an expression construct of a nucleotide sequence encoding the modular protein; and (ii) a modular protein guide RNA (mgRNA), or an expression construct of a nucleotide sequence encoding mgRNA.

13. The base editing system according to claim 12, characterized in that The mgRNA comprises a guide sequence, a scaffold sequence, a reverse transcription template (RT) sequence and a primer binding site (PBS) sequence from 5' to 3'.

14. The base editing system according to claim 13, characterized in that The RT sequence is an exogenous donor DNA or RNA, which is used to insert an exogenous nucleotide sequence.

15. The base editing system according to claim 13, characterized in that The PBS sequence is configured to be complementary to at least a portion of a target nucleic acid molecule sequence; Preferably, the PBS sequence is complementary to at least a portion of the 3' free single strand caused by the nick in the DNA strand where the target sequence of the nucleic acid molecule is located.

16. The base editing system according to any one of claims 12 to 15, characterized in that The modular protein binds to the nucleic acid molecule target sequence under the guidance of mgRNA, and replaces or inserts the RT sequence into the corresponding endogenous nucleotide sequence through DNA repair.

17. The base editing system according to any one of claims 12 to 16, characterized in that The base editing system further comprises: (iii) a nuclear localization sequence (NLS).

18. A method for producing at least one genetically modified cell, characterized in that The method comprises introducing a modular protein as described in any one of claims 1 to 11 or a base editing system as described in any one of claims 12 to 17 into at least one cell, thereby causing substitution, insertion or deletion of a nucleic acid sequence within a target nucleic acid region in the at least one cell.

19. The method according to claim 18, characterized in that The modular protein or base editing system is introduced into cells by a method selected from the following: calcium phosphate transfection, protoplast fusion, electroporation, liposome transfection, microinjection, viral infection (such as baculovirus, vaccinia virus, adenovirus, adeno-associated virus, lentivirus or other virus), N-acetylgalactosamine (GalNAc) mediation, gene gun method, PEG-mediated protoplast transformation and / or soil Agrobacterium-mediated transformation.

20. The method according to claim 18 or 19, characterized in that The cells are derived from prokaryotes such as bacteria, eukaryotes such as plants, fungi or vertebrates.

21. The method according to claim 20, characterized in that The vertebrate is a mammal such as a human, mouse, rat, monkey, dog, pig, sheep, cow, cat; the plant is a crop plant, such as wheat, rice, corn, soybean, sunflower, sorghum, rapeseed, alfalfa, cotton, barley, millet, sugarcane, tomato, tobacco, cassava or potato.

22. A polynucleotide, characterized in that The polynucleotide encodes a modular protein as described in any one of claims 1-11 or a base editing system as described in any one of claims 12-17.

23. An expression construct, characterized in that The expression construct comprises the polynucleotide of claim 22.

24. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises a modular protein as described in any one of claims 1 to 11, a polynucleotide as described in claim 22, an expression construct as described in claim 23, or a base editing system as described in any one of claims 12 to 17; Optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

25. A kit, characterized in that The kit comprises a modular protein as described in any one of claims 1-11, a polynucleotide as described in claim 22, an expression construct as described in claim 23, or a base editing system as described in any one of claims 12-17.

26. A method for modifying a target nucleic acid, characterized in that: The method comprises the step of contacting the target nucleic acid with a modular protein as described in any one of claims 1 to 11, a polynucleotide as described in claim 22, an expression construct as described in claim 23, a base editing system as described in any one of claims 12 to 17, a pharmaceutical composition as described in claim 24, or a kit as described in claim 25.

27. Use of the modular protein of any one of claims 1 to 11, the polynucleotide of claim 22, the expression construct of claim 23, or the base editing system of any one of claims 12 to 17 in the preparation of a reagent for modifying a target nucleic acid.

28. Use of the modular protein of any one of claims 1 to 11, the polynucleotide of claim 22, the expression construct of claim 23, or the base editing system of any one of claims 12 to 17 in the preparation of a medicament for preventing and / or treating diseases associated with or caused by gene mutations.

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