Modular gene editing tool and use thereof

Abstract

The present invention belongs to the field of genetic engineering. Provided are a modular gene editing tool and the use thereof. Specifically, provided are a modular gene editing tool protein that can precisely edit a genomic nucleotide sequence of an organism, a gene editing system comprising the protein, and a method for gene editing by using the system. The modular gene editing tool can realize efficient, precise and universal gene editing in cells of various eukaryotes, including animals, plants, etc. The present invention enriches the types of existing editing tools, and has wide application prospects.

Description

A modular gene editing tool and its application

[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”. The entire contents of this application, including the appendix, are incorporated herein 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 performing 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 organisms and has become a disruptive technology in the field of life sciences. It is applied in many areas such as biomedicine development, genetic engineering therapy, crop genetic improvement, and ecological and environmental protection.

[0005] Early gene editing technologies, exemplified by zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), target genes through protein-nucleic acid interactions. In 2012, gene editing technology based on clustered regularly interspaced short palindromic repeats (CRISPR) sequences was developed. Since then, this system has been rapidly adopted by researchers in the field due to its ease of use and high editing efficiency. The principle is that, guided by a small RNA sequence called a sgRNA, the endonuclease Cas protein localizes to the target DNA site, cuts it, and triggers a double-strand break (DSB), thereby activating DNA repair pathways such as non-homologous end joining (NHEJ). The NHEJ pathway performs DNA repair inaccurately, potentially introducing insertions or deletions (indels) of bases into the repaired DNA. This can cause frameshift mutations in the target gene's expression frame, leading to gene knockout. Because NHEJ indels are random, this type of gene editing system is both random and imprecise.

[0006] To achieve more precise gene editing, base editing and base editing systems that do not rely on DSBs were developed in 2016. Base editing is a type of gene editing technology that refers to the process of replacing nucleotides at specific DNA sites through genetic engineering. The base editor system is a fusion protein containing Cas protein and deaminase. When the specific Cas protein recognizes and opens the DNA double strand, 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. They can only achieve C-to-T and A-to-G base conversions, and cannot achieve other types of base replacements, which limits their application areas and scenarios.

[0007] To achieve more types of precise base substitutions, a prime editor (PE) was developed in 2019. PE introduces target mutations into target DNA through reverse transcription of template RNA by reverse transcriptase, enabling the replacement of all 12 types of bases and the insertion or deletion of any short nucleotide segment in a site-specific manner. Notably, PE editing can achieve a lower rate of unexpected indels. Therefore, PE can achieve more precise targeted insertions, 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 less research on the structural optimization and modular improvement of editing tools, and their application scenarios and fields are limited.

[0009] Summary of the Invention

[0010] Problems to be solved by the invention

[0011] In order to enrich the application scenarios of gene editing and provide a variety of gene editing tool options in this field, the inventors of this application, after a lot of experiments and exploration, artificially synthesized a modular gene editing tool with nucleotide replacement, insertion or deletion functions through modular assembly.

[0012] Solutions for solving problems

[0013] The first aspect of the present invention provides a modular protein, wherein the modular protein comprises the following functional domains:

[0014] (a) a functional domain comprising the amino acid sequence of SEQ ID NO: 2, 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: 2;

[0015] (b) a functional domain comprising the amino acid sequence of 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 of SEQ ID NO: 3;

[0016] (c) a functional domain comprising the amino acid sequence 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 set forth in SEQ ID NO:4;

[0017] (d) a functional domain comprising the amino acid sequence of 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 of SEQ ID NO: 5;

[0018] (e) a functional 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

[0019] (f) a domain comprising the amino acid sequence of 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 of SEQ ID NO: 7;

[0020] The modular protein also includes:

[0021] (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.

[0022] In some embodiments, the DNA polymerase is an RNA-dependent DNA polymerase or a DNA-dependent DNA polymerase, preferably a reverse transcriptase.

[0023] In some specific embodiments, the DNA polymerase domain sequence comprises the amino acid sequence 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. In some embodiments, the DNA polymerase domain comprises (a) a mutation selected from any one of F156Y, F156V, F157Y, D525N, and D201C, the amino acid positions 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).

[0024] In some embodiments, the modular protein further comprises:

[0025] (h) a functional domain comprising the amino acid sequence of 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 of SEQ ID NO: 8.

[0026] In some specific embodiments, the modular protein comprises one or more of the following sequences:

[0027] (i) the amino acid sequence shown in any one of SEQ ID NOs: 9-35, 56-58;

[0028] (ii) an amino acid sequence that has at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 9-35, 56-58;

[0029] (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

[0030] (iv) an amino acid sequence encoded by a nucleotide sequence that hybridizes to a polynucleotide sequence encoding the amino acid sequence 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.

[0031] In some embodiments, the DNA polymerase domain is embedded in the functional domain (f)

[0032] 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.

[0033] In some specific embodiments, the modular protein comprises one or more of the following sequences:

[0034] (i) the amino acid sequence shown in any one of SEQ ID NOs: 29-31, 56-58, and 13-15;

[0035] (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;

[0036] (iii) an amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence of any one of SEQ ID NOs: 29-31, 56-58 and 13-15; or

[0037] (iv) an amino acid sequence encoded by a nucleotide sequence that hybridizes to a polynucleotide sequence encoding the amino acid sequence shown in any one of SEQ ID NOs: 29 to 31, 56 to 58, and 13 to 15 under stringent conditions, which are medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions.

[0038] In some preferred embodiments, the modular protein comprises one or more of the following sequences:

[0039] (i) the amino acid sequence shown in any one of SEQ ID NOs: 29-31, 56-58;

[0040] (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;

[0041] (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

[0042] (iv) an amino acid sequence encoded by a nucleotide sequence that hybridizes to 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.

[0043] 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.

[0044] In some specific embodiments, the modular protein comprises one or more of the following sequences:

[0045] (i) the amino acid sequences shown in SEQ ID NOs: 11, 16-19, and 34-35;

[0046] (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;

[0047] (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

[0048] (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, which are medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions.

[0049] In some preferred embodiments, the modular protein comprises one or more of the following sequences:

[0050] (i) the amino acid sequence shown in SEQ ID NO: 11;

[0051] (ii) an amino acid sequence that has at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 11;

[0052] (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

[0053] (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 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.

[0054] In some embodiments, the terminal fusion or insertion comprises direct fusion and / or connection through a linker.

[0055] 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 an (XP)n motif, or a combination thereof, wherein n is independently an integer from 1 to 32, and wherein X is any amino acid.

[0056] A second aspect of the present invention provides a base editing system, comprising:

[0057] (i) a modular protein according to the first aspect of the present invention, or an expression construct of a nucleotide sequence encoding the modular protein; and

[0058] (ii) a modular protein guide RNA (mgRNA), or an expression construct of a nucleotide sequence encoding mgRNA.

[0059] 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.

[0060] In some embodiments, the RT sequence is an exogenous donor DNA or RNA for inserting an exogenous nucleotide sequence.

[0061] 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;

[0062] Preferably, the PBS sequence is complementary to at least a portion of the 3' free single strand caused by the nick in the DNA chain where the target sequence of the nucleic acid molecule is located.

[0063] 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.

[0064] In some embodiments, the base editing system further comprises: (iii) a nuclear localization sequence (NLS).

[0065] The third aspect of the present invention provides a method for producing at least one genetically modified cell, which comprises introducing 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 into at least one cell, thereby causing substitution, insertion or deletion of the nucleic acid sequence within the target nucleic acid region in the at least one cell.

[0066] 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)-mediated, gene gun method, PEG-mediated protoplast transformation and / or Agrobacterium-mediated transformation.

[0067] 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.

[0068] In some embodiments, the vertebrate is a mammal such as a human, mouse, rat, monkey, dog, pig, sheep, cow, cat; and 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.

[0069] The fourth aspect of the present invention provides a polynucleotide encoding 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.

[0070] The fifth aspect of the present invention provides an expression construct, which comprises the polynucleotide according to the fourth aspect of the present invention.

[0071] The sixth aspect of the present invention provides a pharmaceutical composition, comprising the modular protein according to the first aspect of the present invention, the polynucleotide according to the fourth aspect of the present invention, the expression construct according to the fifth aspect of the present invention, or the base editing system according to the second aspect of the present invention;

[0072] Optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0073] The seventh aspect of the present invention provides a kit 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.

[0074] The eighth aspect of the present invention provides a method for modifying a target nucleic acid, 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.

[0075] 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.

[0076] 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 drug for preventing and / or treating diseases related to or caused by gene mutations.

[0077] Effects of the Invention

[0078] 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

[0079] Figure 1. Schematic diagram of the assembly of a DNA polymerase domain (labeled as reverse transcriptase: RT in Figure 1) with a protein functional domain in an embedded and terminal fusion manner.

[0080] Figure 2. Schematic diagram of the structure of protein expression constructs based on modular gene editing tools.

[0081] Figure 3. Efficiency of base substitution by ME editor.

[0082] Figure 4. Efficiency of base fragment insertion by ME editor.

[0083] Figure 5. Efficiency of base segment deletion by ME editor.

[0084] Figure 6. Preferred ME editors and their outstanding effects on base substitution.

[0085] Figure 7. Preferred ME editors for indels in base substitution editing. DETAILED DESCRIPTION

[0086] 1. Definition

[0087] In the present invention, unless otherwise indicated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology 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 literature: 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, definitions and explanations of relevant terms are provided below.

[0088] As used herein, the term "and / or" encompasses all combinations of items connected by the term, and should be treated as if each combination had been individually listed 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."

[0089] When the word "comprising" is used herein to describe a sequence of a protein or nucleic acid, the protein or nucleic acid may be composed 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, and accordingly, its encoding nucleotide sequence may also contain a start codon; and vice versa.

[0090] "Gene" and "genome" as used herein encompass not only chromosomal DNA present in the cell nucleus, but also organelle DNA present in subcellular components of the cell (eg, mitochondria, plastids).

[0091] As used herein, "organism" includes any organism suitable for genome editing, such as prokaryotes and eukaryotes, preferably eukaryotes. Examples of organisms include, but are not limited to, mammals such as humans, mice, rats, monkeys, dogs, pigs, sheep, cattle, and cats; poultry such as chickens, ducks, and geese; and plants, including monocots and dicots, such as wheat, rice, corn, soybeans, sunflowers, sorghum, rapeseed, alfalfa, cotton, barley, millet, sugarcane, tomatoes, tobacco, cassava, and potatoes.

[0092] "Genetically modified organism" or "genetically modified cell" refers to an organism or cell that contains an exogenous polynucleotide or a modified gene or expression control sequence within its genome. For example, the exogenous polynucleotide is capable of stably integrating into the genome of the organism or cell and being inherited through successive generations. The exogenous polynucleotide can be integrated into the genome alone or as part of a recombinant DNA construct. A modified gene or expression control sequence is one that contains single or multiple deoxynucleotide substitutions, deletions, and additions within the genome of the organism or cell.

[0093] "Polynucleotide," "nucleic acid sequence," "nucleotide sequence," or "nucleic acid fragment" are used interchangeably and are single-stranded or double-stranded polymers of RNA or DNA that optionally 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" is cytidine or deoxycytidine, "G" is guanosine or deoxyguanosine, "U" is uridine, "T" is deoxythymidine, "R" is a purine (A or G), "Y" is a pyrimidine (C or T), "K" is G or T, "H" is A or C or T, "I" is inosine, and "N" is any nucleotide.

[0094] "Polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical analog of a corresponding naturally occurring amino acid, 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.

[0095] 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 entire 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). While there are many methods to measure the identity between two polynucleotides or polypeptides, the term "identity" is well known to those of skill in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48: 1073 (1988)).

[0096] In peptides or proteins, suitable conservative amino acid substitutions are known to those skilled in the art and can generally be made without altering 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 alter biological activity (see, e.g., Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub.co., p. 224).

[0097] The "RNA-dependent DNA polymerase" of the present invention refers to an enzyme that can synthesize a DNA chain using RNA as a template. It can recognize the base sequence on the RNA chain and synthesize a complementary DNA sequence on the DNA chain.

[0098] In the present invention, a "domain" refers to a portion of a protein with a relatively stable three-dimensional structure. Proteins are typically composed of one or more domains, each with its own spatial folding configuration. Domains can be continuous or discontinuous and typically consist of one or more segments of an amino acid sequence. The presence of domains allows for functional diversity in proteins, and new functions can be achieved through the recombination and combination of domains. An example is the DNA polymerase domain shown in SEQ ID NO:1.

[0099] 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 domain of the protein of the present invention includes the RuvC functional domain (SEQ ID NO: 2-4), which is responsible for the cutting of the non-complementary chain 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).

[0100] 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 new functions obtained by connecting two or more functional domains or structural domains with specific functions.

[0101] 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 can refer to the transcription of the nucleotide sequence (e.g., transcription to produce mRNA or functional RNA) and / or translation of RNA into a precursor or mature protein.

[0102] 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 an RNA (such as mRNA) that can be translated.

[0103] 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.

[0104] "Regulatory sequence" and "regulatory element" are used interchangeably to refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence and that influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include, but are not limited to, promoters, translation leader sequences, introns, and polyadenylation recognition sequences.

[0105] "Promoter" refers to a nucleic acid fragment that is capable of controlling the transcription of another nucleic acid fragment. In some embodiments of the present invention, a promoter is a promoter that is capable of controlling the transcription of a gene in a cell, whether or not it is derived from the cell. A promoter can be a constitutive promoter, a tissue-specific promoter, a developmentally regulated promoter, or an inducible promoter.

[0106] "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.).

[0107] 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 transcriptional regulatory element. Techniques for operably linking regulatory element regions to nucleic acid molecules are known in the art.

[0108] "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 herein includes stable transformation and transient transformation.

[0109] "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.

[0110] "Transient transformation" refers to the introduction of a nucleic acid molecule or protein into a cell where it functions without the exogenous nucleotide sequence being stably inherited. In transient transformation, the exogenous nucleic acid sequence does not integrate into the genome.

[0111] "Trait" refers to a physiological, morphological, biochemical, or physical characteristic of a cell or organism.

[0112] "Agronomic traits" specifically refer to measurable indicator parameters of crop plants, including but not limited to: leaf greenness, 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 tiller number, etc.

[0113] 2. Modular gene editing tool proteins

[0114] In one aspect, the present invention provides a modular protein comprising the following functional domains:

[0115] (a) a functional domain comprising the amino acid sequence of SEQ ID NO: 2, 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: 2;

[0116] (b) a functional domain comprising the amino acid sequence of 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 of SEQ ID NO: 3;

[0117] (c) a functional domain comprising the amino acid sequence 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 set forth in SEQ ID NO:4;

[0118] (d) a functional domain comprising the amino acid sequence of 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 of SEQ ID NO: 5;

[0119] (e) a functional 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

[0120] (f) a functional domain comprising the amino acid sequence of 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 of SEQ ID NO:7.

[0121] In some embodiments, the modular protein further includes a (g) 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.

[0122] 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.

[0123] 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.

[0124] "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.

[0125] In some embodiments, the DNA polymerase is an RNA-dependent DNA polymerase or a DNA-dependent DNA polymerase, preferably a reverse transcriptase.

[0126] In some embodiments, the DNA polymerase domain comprises the amino acid sequence 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.

[0127] In some embodiments, the DNA polymerase domain comprises (a) a mutation selected from any one of F156Y, F156V, F157Y, D525N, and D201C, with reference 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).

[0128] In some embodiments, the modular protein further comprises:

[0129] (h) a functional domain comprising the amino acid sequence of 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 of SEQ ID NO: 8.

[0130] In some embodiments, the terminal fusion or insertion comprises direct fusion and / or connection through a linker.

[0131] 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 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 an (XP)n motif, or a combination thereof, wherein n is independently an integer from 1-32, and wherein X is any amino acid.

[0132] In some embodiments, the modular protein comprises one or more of the following sequences:

[0133] (i) the amino acid sequence shown in any one of SEQ ID NOs: 9-35, 56-58;

[0134] (ii) an amino acid sequence that has 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;

[0135] (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

[0136] (iv) an amino acid sequence encoded by a nucleotide sequence that hybridizes to a polynucleotide sequence encoding the amino acid sequence 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.

[0137] As used herein, "moderately stringent conditions," "medium-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. Both aqueous and nonaqueous methods are described in this document, and either method 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 (the wash temperature can be increased to 55°C for low stringency conditions); (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.

[0138] In some preferred embodiments, the modular protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 11, 13-19, 29-31, 56-58, and 33-35.

[0139] 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.

[0140] In some further preferred embodiments, the modular protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 11, 29-31, and 56-58.

[0141] In some specific embodiments, the DNA polymerase domain is embedded in the functional domain (f) (SEQ ID NO: 7).

[0142] 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 with 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).

[0143] In other specific embodiments, the DNA polymerase domain is connected to the C-terminus of functional domain (b) (SEQ ID NO: 3) and the N-terminus of 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 NO: 11, 16-19 and 34-35 (i.e., ME3, 8-11, 26-27).

[0144] In another aspect, the present invention also provides a polynucleotide encoding the above modular protein.

[0145] In another aspect, the present invention also provides an expression construct comprising the above-mentioned polynucleotide.

[0146] 3. Base editing system containing modular gene editing tool proteins

[0147] In another aspect, the present invention provides a base editing system comprising:

[0148] (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

[0149] (ii) a modular protein guide RNA (mgRNA), or an expression construct of a nucleotide sequence encoding mgRNA.

[0150] On the other hand, the present invention also provides a polynucleotide encoding the above-mentioned base editing system.

[0151] In another aspect, the present invention also provides an expression construct comprising the above-mentioned polynucleotide.

[0152] 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 a cut 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, writing 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.

[0153] In some embodiments, the mgRNA comprises additional modifications that 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, by modifying the base pairs in its hairpin structure; 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.

[0154] In some embodiments, the RT sequence is present episomal or contained within the mgRNA.

[0155] In some embodiments, the RT sequence is an exogenous donor DNA or RNA for inserting an exogenous nucleotide sequence.

[0156] In some embodiments, the PBS sequence is configured to be complementary to at least a portion of the target nucleic acid 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 chain where the target nucleic acid sequence is located (i.e., the target DNA chain).

[0157] 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.

[0158] In some embodiments, the base editing system further includes: (iii) a nuclear localization sequence (NLS). Generally speaking, the 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. Generally speaking, the strength of nuclear localization activity is determined by the number and position of the NLSs in the modular protein, the one or more specific NLSs used, or a combination of these factors.

[0159] In some embodiments, the NLS of the modular protein of the present invention can 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 can be located between each functional domain or structural 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 more than one NLS is present, each can be selected to be independent of the other NLSs.

[0160] Generally speaking, NLS consists of one or more short sequences of positively charged lysine or arginine amino acids 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).

[0161] 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.

[0162] IV. Methods for Producing Genetically Modified Cells

[0163] 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.

[0164] 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.

[0165] In some embodiments, the methods of the present invention are performed in vitro. For example, the cells are isolated cells, or cells in isolated tissues or organs.

[0166] In another aspect, the present invention also provides a genetically modified organism comprising a genetically modified cell or progeny thereof produced by the method of the present invention. Preferably, the genetically modified cell or progeny thereof has a desired one or more nucleotide substitutions.

[0167] In the present invention, the target nucleic acid region to be modified can be located at any position in the genome, for example, in a functional gene such as a protein-coding gene, or, for example, in a gene expression regulatory region such as a promoter region or an enhancer region, thereby achieving modification of the gene function or modification of gene expression. In some embodiments, the desired nucleotide substitution results in a desired gene function modification or gene expression modification.

[0168] 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 an albumen. In some embodiments, the function-related motif or domain of the target nucleic acid region encoding protein. In some preferred embodiments, one or more nucleotide substitutions in the target nucleic acid region cause an amino acid substitution in the amino acid sequence of the albumen. In some embodiments, one or more nucleotide substitutions cause a change in the function of the albumen.

[0169] 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.

[0170] 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.

[0171] In some embodiments, organisms that can be genomic modified by the methods of the present invention include any organism suitable for base editing, preferably a eukaryotic organism. Examples of organisms include, but are not limited to, mammals such as humans, mice, rats, monkeys, dogs, pigs, sheep, cattle, and cats; poultry such as chickens, ducks, and geese; and plants, including monocots and dicots. For example, the plant is a crop plant, including but not limited to wheat, rice, corn, soybeans, sunflowers, sorghum, rapeseed, alfalfa, cotton, barley, millet, sugarcane, tomatoes, tobacco, cassava, and potatoes.

[0172] 5. Test kit

[0173] 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.

[0174] In some specific embodiments, the kit can be a disease treatment kit.In other specific embodiments, the kit can be a kit for target nucleic acid modification or generating genetically modified cells.

[0175] VI. Pharmaceutical Composition

[0176] The present disclosure provides a pharmaceutical composition comprising 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.

[0177] In some optional embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0178] In some embodiments, the pharmaceutically acceptable carrier can be a delivery carrier, such as a lipid, a cationic lipid, or other polymers with drug delivery function.

[0179] VII. Methods and Uses of Modifying Target Nucleic Acids

[0180] The present invention provides a method for modifying a target nucleic acid, 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.

[0181] 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.

[0182] 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.

[0183] 8. Methods and uses of treating diseases

[0184] The present invention provides a method for preventing and / or treating diseases associated with or caused by gene mutations, 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. Genetic mutations can be corrected, for example, by modular proteins or base editing systems provided herein.

[0185] 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.

[0186] 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.

[0187] Modular proteins or base editing systems provided herein produce nucleotide changes that correct disease-associated genes. Exemplary, disease-associated 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, pachyonychia congenita, phenylketonuria, severe combined immunodeficiency, sickle cell disease, Stevens-Lewis-Owen syndrome, and Tay-Sachs disease. In other embodiments, disease-associated genes may be associated with a polygenic disorder selected from the group consisting of heart disease, hypertension, Alzheimer's disease, arthritis, diabetes, cancer, and obesity.

[0188] Example

[0189] Example 1: Acquisition of new modular gene editing tools

[0190] The inventors conducted a modular analysis of the functional domain / domain SEQ ID NO: 1-8 through bioinformatics, and rearranged and fused the DNA polymerase domain (shown as reverse transcriptase: RT in Figure 1) with multiple different protein functional domains in various ways such as terminal fusion (B in Figure 1) or embedding (A in Figure 1), 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 Figure 2. Figure 1 A and Figure 1 B respectively show the way of assembling the DNA polymerase domain with the protein functional domain in an embedded and terminal fusion manner, and provide a scheme that optionally contains the HNH functional domain. It should be noted that the schematic diagram of Figure 1 is only an exemplary display of the combination scheme between the functional domains, and the arrangement order and fusion method of the functional domains are not limited to the scheme shown in the schematic diagram.

[0191] Example 2: Multiple editing functions on the human cell genome

[0192] In this example, the 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 a base editor plasmid 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 Figure 2. Different sites are targeted for different types of editing. The specific targets are described in detail in the following experiments. After 72 hours of transfection, DNA was extracted; the Mock group was used as a blank control without transfection of the base editor plasmid, and the other treatment methods were the same as above. The base editing efficiency of the target was detected using second-generation sequencing technology,

[0193] Experiment (1) Base substitution

[0194] ME editors can achieve effective base substitutions. This experiment (1) demonstrates the editing effects of different ME editors on base substitutions at the APP1 site. The APP1 site sequence (SEQ ID NO: 38) is: The underlined 20 bp are 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):

[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 perform single-base substitutions. As shown in Figure 3, the editing abilities of ME3, ME21, ME22, and ME23 are better than those of ME editors in terms of single-base substitution editing.

[0197] Table 1

[0198] In the above table, Null means that no editing efficiency was detected.

[0199] Experiment (2) Base fragment deletion

[0200] The ME editor with high single-base editing ability in experiment (1) was experimentally verified by base segment deletion. This experiment demonstrates the editing effect of base deletion at the RUNX1 site, deleting a 15bp segment of bases from positions +1 to +15. The RUNX1 site sequence (SEQ ID NO: 39) is as follows: The underlined 20 bp are the target sequences, and the bold italic sequences are the fragments that need to be deleted through ME editing.

[0201] The mgRNA used was (SEQ ID NO: 53):

[0202] The underlined part is the guide sequence.

[0203] The results are shown in Figure 4. 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.

[0204] Experiment (3) Base fragment insertion

[0205] This experiment demonstrates the editing effects of different ME editors on base fragment insertion at the RNF2 site, starting at position +1 and inserting a 24bp long DNA sequence. RNF2 site sequence (SEQ ID NO:40):

[0206] 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.

[0207] The mgRNA used was (SEQ ID NO: 54):

[0208] The underlined part is the guide sequence.

[0209] The results are shown in Figure 5. 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 embedded between amino acids 143 / 144, 144 / 145, and 145 / 146 of SEQ ID NO: 7, respectively) have higher efficiency and better effect in base fragment insertion editing.

[0210] In summary, after screening, this example obtained a batch of ME editing tools that can perform various base editing functions such as base substitution, base fragment deletion, and base fragment insertion. Among them, 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.

[0211] Example 3: Preferred ME editors have outstanding effects on base substitution

[0212] This example further validates the ME editor selected in Example 2. The inventors selected HEK293T cells as a validation model, targeting the HEK3 site, 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.

[0213] The mgRNA sequence used in the experiment is as follows (SEQ ID NO: 36):

[0214] The underlined part is the guide sequence.

[0215] 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 the reverse transcription editing results of the reverse transcription template at the DNA target site were then analyzed using second-generation sequencing (NGS).

[0216] Figure 6 shows that each modular gene editing tool protein achieved efficient and precise editing, with the highest editing efficiency reaching 25.3%. Referring to the reported PE editors (James W. Nelson et al., 2022; Omer Habib et al., 2022), the four preferred ME editing tools of the present invention have significantly improved editing efficiency compared to PE1; compared with PE2, the editing efficiency of ME3, ME21, and ME22 editors is comparable to PE2, but the ME23 editor still has a significant improvement over PE2. This shows that the modular gene editing tool of the present invention still has significantly improved gene editing capabilities compared to the relatively mature existing technologies.

[0217] Example 4: Preferred ME editors with significantly reduced indels

[0218] Also at the HEK3 locus, edit-replacement editing was performed as in Example 3. This example analyzed the frequency of random insertion / deletion (indel) byproducts generated during editing. Indels are random base insertions or deletions outside of the target site, and a high number of indels can compromise the accuracy of gene editing.

[0219] As shown in Figure 7, ME3 and ME21-23 achieved efficient base substitutions at the HEK3 locus while inducing significantly fewer indel byproducts than the PE2 system. The ME3, ME21, and ME22 editors significantly reduced indel production while maintaining similar editing efficiency to PE2. The ME23 editor significantly reduced indel production while improving editing efficiency. This result demonstrates that the ME editors of the present invention have made significant progress in precise gene editing, further ensuring the safety of gene editing.

[0220] Example 5: Replacement and optimization of DNA polymerase domain

[0221] 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 to obtain ME28-ME30 editors (SEQ ID NO: 56-58) 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 level of the fusion protein is increased by point mutation of the reverse transcriptase or deletion of its redundant domain.

[0222] The base substitution efficiency was verified using 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.

[0223] Table 2

[0224] Although the present invention has been described with reference to specific embodiments thereof, it will be understood 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 invention. In addition, many modifications may be made to adapt specific circumstances, materials, compositions of matter, processes, process steps or 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.

[0225] In the above SEQ ID NOs: 9-35, the underlined portion is the sequence of the DNA polymerase domain.

[0226] 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 also includes: (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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