Cas12f1 protein or mutant thereof, gene editing system containing Cas12f1 protein or mutant thereof and application

Abstract

The invention belongs to the technical field of gene editing, and particularly relates to a CRISPR / Cas12f1 gene editing system and application thereof. The gene editing system is a complex formed by wild type or mutant type PspCas12f1 protein and sgRNA, and can accurately locate a target DNA sequence and cut the target DNA sequence, so that the target DNA sequence is subjected to double-strand breakage damage; the gene editing may be performed in a cell or in vitro. The wild type or mutant type PspCas12f1 protein has a relatively small number of amino acids and has relatively high editing efficiency, and the recognized PAM sequence is very simple. On the basis, the CRISPR / Cas12f1 gene editing system disclosed by the invention has a wide application prospect in the field of gene editing.

Description

Technical Field

[0001] This invention relates to the field of gene editing technology, specifically to the Cas12f1 protein or its mutants, gene editing systems containing the Cas12f1 protein or its mutants, and their applications. Background Technology

[0002] The CRISPR / Cas system is an acquired immune system evolved by bacteria and archaea to defend against invasion by exogenous viruses or plasmids. The CRISPR / Cas12f1 system contains tracrRNA (trans-activating crRNA) and crRNA (CRISPR-derived RNA), which together with Cas12f1 form a complex to function. tracrRNA and crRNA can be fused together via a linker sequence to form single-stranded guide RNA (sgRNA). When DNA breaks occur, repair is handled by two main DNA damage repair mechanisms within the cell: non-homologous end-joining (NHEJ) and homologous recombination (HR). NHEJ repair results in base deletions or insertions, which can be used for gene knockout; HR repair, when a homologous template is provided, can be used for site-specific gene insertion and precise base substitution.

[0003] Beyond basic research, the CRISPR / Cas12f1 gene editing system also holds broad clinical application prospects. The CRISPR / Cas12f1 gene editing system can be used for gene therapy, which involves introducing the Cas enzyme and single-stranded guide RNA into the body. Currently, the most effective expression vector used in gene therapy is adeno-associated virus (AAV). However, AAV viral packaging has requirements on DNA length, generally not exceeding 4.5 kb. Therefore, although SpCas9 has been widely used in basic research due to its simple PAM sequence (recognizing NGG) and high activity, the 1368 amino acids in the SpCas9 protein, coupled with the sgRNA and promoter required for gene editing systems, make it difficult to effectively package into AAV viruses, limiting its clinical application. To overcome this problem, several small-molecule Cas9 proteins have been developed, such as SaCas9 (PAM sequence NNGRRT), St1Cas9 (PAM sequence NNAGAW), NmCas9 (PAM sequence NNNNGATT), Nme2Cas9 (PAM sequence NNNNCC), and CjCas9 (PAM sequence NNNNRYAC). However, these Cas9 proteins are either prone to off-target cleavage (i.e., non-target site cleavage), have complex PAM sequences, or have low editing activity, making them difficult to widely apply.

[0004] Therefore, finding a small CRISPR / Cas system with high editing activity, high specificity, and simple PAM sequences is the hope for solving the above problems. Summary of the Invention

[0005] To address the aforementioned problems, the inventors, through repeated research, discovered a Cas12f1 protein (PspCas12f1, derived from...) Phascolarctobacterium sp. Furthermore, the corresponding mutants were obtained and a corresponding single-stranded guide RNA was designed. The wild-type PspCas12f1 protein or its mutants and the above-mentioned single-stranded guide RNA can constitute an effective CRISPA / Cas12 gene editing system, thereby completing the present invention.

[0006] Therefore, in a first aspect, the present invention provides a PspCas12f1 mutant protein, wherein the PspCas12f1 mutant protein contains mutations at Q100 and / or E293 relative to the wild-type PspCas12f1 protein with the amino acid sequence as shown in SEQ ID NO: 1.

[0007] In a second aspect, the present invention provides a conjugate comprising: a) Cas12f1 protein or its homologs, wherein: The Cas12f1 protein is the wild-type PspCas12f1 protein with the amino acid sequence shown in SEQ ID NO: 1 or the PspCas12f1 mutant protein of the first aspect of the present invention. The homolog is a homolog whose amino acid sequence has at least 80% sequence identity with the amino acid sequence of the Cas12f1 protein and retains the biological activity of the Cas12f1 protein; b) Modified parts; and c) An optional adapter for connecting the Cas12f1 protein or its homolog to the modified portion.

[0008] In a third aspect, the present invention provides a fusion protein comprising: a) Cas12f1 protein or its homologs, wherein: The Cas12 protein is the wild-type PspCas12f1 protein with the amino acid sequence shown in SEQ ID NO: 1 or the PspCas12f1 mutant protein of the first aspect of the present invention. The homolog is a homolog whose amino acid sequence has at least 80% sequence identity with the amino acid sequence of the Cas12f1 protein and retains the biological activity of the Cas12f1 protein; b) Other proteins or peptides; and c) Optional adapters for connecting the Cas12f1 protein or its homologs to the other protein or polypeptide.

[0009] In a fourth aspect, the present invention provides a single-stranded guide RNA comprising a CRISPR scaffold sequence having: a) The nucleic acid sequence shown in SEQ ID NO: 5; b) A nucleic acid sequence that shares at least 90% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 5 and retains the biological activity of SEQ ID NO: 5; or c) A nucleic acid sequence that retains the biological activity of SEQ ID NO: 5, obtained by modifying the nucleic acid sequence described in SEQ ID NO: 5.

[0010] In a fifth aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a PspCas12f1 mutant protein or a homolog thereof of the first aspect of the present invention, a conjugate of the second aspect of the present invention, or a fusion protein of the third aspect of the present invention.

[0011] In a sixth aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a single-stranded guide RNA of the fourth aspect of the present invention.

[0012] In a seventh aspect, the present invention provides a vector comprising a nucleic acid sequence encoding a PspCas12f1 mutant protein or a homolog thereof of the first aspect of the present invention, a conjugate of the second aspect of the present invention, or a fusion protein of the third aspect of the present invention.

[0013] In an eighth aspect, the present invention provides a vector comprising a nucleic acid sequence encoding a single-stranded guide RNA of the fourth aspect of the present invention.

[0014] In a ninth aspect, the present invention provides a CRISPR / Cas12f1 gene editing system comprising: 1) Protein components, comprising Cas12f1 protein or its homologs, conjugates, or fusion proteins, wherein: The Cas12f1 protein is the wild-type PspCas12f1 protein with the amino acid sequence shown in SEQ ID NO: 1 or the PspCas12f1 mutant protein of the first aspect of the present invention. The homolog is a homolog whose amino acid sequence has at least 80% sequence identity with the amino acid sequence of the Cas12f1 protein and retains the biological activity of the Cas12f1 protein; The conjugate comprises: the Cas12f1 protein or its homolog, a modified moiety, and an optional linker; The fusion protein comprises: the Cas12f1 protein or its homologs, additional proteins or peptides, and optional linkers; The optional connector is used to connect the Cas12f1 protein or its homolog to the modified moiety or the other protein or polypeptide; and 2) Nucleic acid components, which include: The fourth aspect of this invention involves a single-stranded guide RNA; Furthermore, the protein component and the nucleic acid component combine with each other to form a complex.

[0015] In a tenth aspect, the present invention provides a cell comprising: isolated nucleic acid molecules of the fifth and / or sixth aspects of the present invention, or a vector of the seventh and / or eighth aspects of the present invention.

[0016] In an eleventh aspect, the present invention provides a method for gene editing of a target sequence in an intracellular or in vitro environment, the method comprising contacting any one of the following (1) to (4) with the target sequence in the intracellular or in vitro environment: (1) Cas12f1 protein or its homologs, conjugates or fusion proteins, and the single-stranded guide RNA of the fourth aspect of the present invention, wherein: The Cas12f1 protein is the wild-type PspCas12f1 protein with the amino acid sequence shown in SEQ ID NO: 1 or the PspCas12f1 mutant protein of the first aspect of the present invention. The homolog is a homolog whose amino acid sequence has at least 80% sequence identity with the amino acid sequence of the Cas12f1 protein and retains the biological activity of the Cas12f1 protein; The conjugate comprises: the Cas12f1 protein or its homolog, a modified moiety, and an optional linker; The fusion protein comprises: the Cas12f1 protein or its homologs, additional proteins or peptides, and optional linkers; The optional connector is used to connect the Cas12f1 protein or its homolog to the modified portion or the other protein or polypeptide; (2) The seventh aspect of the present invention does not contain a vector encoding a nucleic acid sequence of a single-stranded guide RNA of the fourth aspect of the present invention or a vector containing a nucleic acid sequence encoding a wild-type PspCas12f1 protein or its homolog as shown in SEQ ID NO: 1, and the eighth aspect of the present invention does not contain a vector encoding a nucleic acid sequence encoding a wild-type PspCas12f1 protein or its homolog as shown in SEQ ID NO: 1. (3) A vector comprising, in the seventh aspect of the present invention, a nucleic acid sequence encoding the PspCas12f1 mutant protein or its homolog, the conjugate of the second aspect of the present invention, or the fusion protein of the third aspect of the present invention, and a nucleic acid sequence encoding the single-stranded guide RNA of the fourth aspect of the present invention; or a vector comprising, in the eighth aspect of the present invention, a nucleic acid sequence encoding the single-stranded guide RNA of the fourth aspect of the present invention, and a nucleic acid sequence encoding the wild-type PspCas12f1 protein or its homolog as shown in SEQ ID NO: 1; or (4) The CRISPR / Cas12f1 gene editing system of the ninth aspect of the present invention; Upon contact with the target sequence, the Cas12f1 protein or its homologs, conjugates, or fusion proteins recognize the PAM sequence 5'-TTC located at the 5' end of the target sequence.

[0017] In a twelfth aspect, the present invention provides a kit for gene editing of target sequences in intracellular or in vitro environments, comprising: i. Choose any one of the following (1) to (6): (1) Cas12f1 protein or its homologs, conjugates or fusion proteins, and the single-stranded guide RNA of the fourth aspect of the present invention, wherein: The Cas12f1 protein is the wild-type PspCas12f1 protein with the amino acid sequence shown in SEQ ID NO: 1 or the PspCas12f1 mutant protein of the first aspect of the present invention; The homolog is a homolog whose amino acid sequence has at least 80% sequence identity with the amino acid sequence of the Cas12f1 protein and retains the biological activity of the Cas12f1 protein. The conjugate comprises: the Cas12f1 protein or its homolog, a modified moiety, and an optional linker; The fusion protein comprises: the Cas12f1 protein or its homologs, additional proteins or peptides, and optional linkers; The optional connector is used to connect the Cas12f1 protein or its homolog to the modified portion or the other protein or polypeptide; (2) The fifth aspect of the present invention does not contain an isolated nucleic acid molecule encoding a nucleic acid sequence of a single-stranded guide RNA of the fourth aspect of the present invention or an isolated nucleic acid molecule containing a nucleic acid sequence encoding a wild-type PspCas12f1 protein or its homolog as shown in SEQ ID NO: 1, and the sixth aspect of the present invention does not contain an isolated nucleic acid molecule encoding a nucleic acid sequence encoding a wild-type PspCas12f1 protein or its homolog as shown in SEQ ID NO: 1; (3) The fifth aspect of the present invention comprises a nucleic acid molecule encoding the PspCas12f1 mutant protein or its homolog, the conjugate of the second aspect of the present invention or the fusion protein of the third aspect of the present invention and an isolated nucleic acid molecule encoding the single-stranded guide RNA of the fourth aspect of the present invention; or the sixth aspect of the present invention comprises a nucleic acid molecule encoding the single-stranded guide RNA of the fourth aspect of the present invention and an isolated nucleic acid molecule encoding the wild-type PspCas12f1 protein or its homolog as shown in SEQ ID NO: 1. (4) The seventh aspect of the present invention does not contain a vector encoding a nucleic acid sequence of a single-stranded guide RNA of the fourth aspect of the present invention or a vector containing a nucleic acid sequence encoding a wild-type PspCas12f1 protein or its homolog as shown in SEQ ID NO: 1, and the eighth aspect of the present invention does not contain a vector encoding a nucleic acid sequence encoding a wild-type PspCas12f1 protein or its homolog as shown in SEQ ID NO: 1. (5) The seventh aspect of the present invention comprises a nucleic acid sequence encoding the PspCas12f1 mutant protein or its homolog, the conjugate of the second aspect of the present invention or the fusion protein of the third aspect of the present invention and a nucleic acid sequence encoding the single-stranded guide RNA of the fourth aspect of the present invention; or the eighth aspect of the present invention comprises a nucleic acid sequence encoding the single-stranded guide RNA of the fourth aspect of the present invention and a nucleic acid sequence encoding the wild-type PspCas12f1 protein or its homolog as shown in SEQ ID NO: 1; (6) The CRISPR / Cas12f1 gene editing system of the ninth aspect of the present invention; and ii. Instructions on how to perform gene editing on target sequences in intracellular or in vitro environments.

[0018] The inventors have developed a CRISPR / PspCas12f1 editing tool for efficient gene editing in a eukaryotic environment. This PspCas12f1 protein has a relatively small number of amino acids, particularly a small number currently available for eukaryotic gene editors, thus allowing for efficient packaging into expression vectors such as adeno-associated virus vectors. Furthermore, this protein exhibits high specificity, simple PAM (Physical Amino Acid) structure, and its small molecular weight allows for easy packaging into vectors such as adeno-associated viruses, making it highly suitable for future development as a gene therapy tool.

[0019] Furthermore, the PAM of the PspCas12f1 protein is TTC, a very simple sequence, thus allowing for a wide range of editing capabilities with the CRISPR / PspCas12f1 editing system. Moreover, our experiments have demonstrated that the PspCas12f1 mutant protein of this invention exhibits a significant advantage in editing efficiency at random sites compared to the wild-type PspCas12f1 protein, demonstrating strong gene editing capabilities in a eukaryotic environment. Compared to its sibling Cas12f1 protein, the mutant protein enPspCas12f1 possesses extremely significant editing advantages, making it more suitable for the development and application of gene editing research.

[0020] The PspCas12f1 protein of this invention has high activity and high specificity, and has a relatively simple PAM sequence, which expands the field of PspCas12f1 protein and increases its application range. Attached Figure Description

[0021] Figure 1 The images show cell photographs of GFP reporter cell lines after treatment with the CRISPR / PspCas12f1 gene editing system, with the left image being a fluorescence image and the right image being a conventional micrograph. Figure 2A schematic diagram illustrating the CRISPR / PspCas12f1 gene editing system's recognition of PAM sequences is shown. Figure 3 A schematic diagram showing the efficiency results of gene editing at a single target site using the CRISPR / PspCas12f1 gene editing system and nine modified CRISPR / PspCas12f1-mutant gene editing systems; Figure 4 This diagram illustrates the editing efficiency of the CRISPR / PspCas12f1 gene editing system after gene editing at six target sites. Figure 5 A schematic diagram showing the editing efficiency results of the CRISPR / enPspCas12f1 gene editing system after gene editing at six target sites; Figure 6 This diagram illustrates the results of the CRISPR / PspCas12f1 gene editing system's specific detection of single and double base mismatches at a single target site. Figure 7 This diagram illustrates the results of the CRISPR / enPspCas12f1 gene editing system's specific detection of single and double base mismatches at a single target site. Figure 8 This diagram illustrates a comparison of the editing efficiency of the CRISPR / enPspCas12f1, CRISPR / SpaCas12f1, and CRISPR / enOsCas12f1 gene editing systems after gene editing at six target sites. Detailed Implementation

[0022] Unless otherwise stated, the scientific and technical terms used in this application have the meanings commonly understood by those skilled in the art. Definitions and explanations of relevant terms are provided below for a better understanding of the invention.

[0023] The terms "Cas12f1 protein," "Cas12f1," and "Cas" used herein are interchangeable and refer to RNA-guided nucleases, including the Cas12f1 protein or its functionally active fragments. Therefore, in this document, the Cas12f1 protein may refer to the wild-type PspCas12f1 protein with the amino acid sequence shown in SEQ ID NO: 1, a mutant of the wild-type PspCas12f1 protein, or both, as the case may be. The Cas12f1 protein is a protein component of the CRISPR / Cas12f1 genome editing system that, guided by single-stranded guide RNA (sgRNA), targets and cleaves DNA target sequences, forming DNA double-strand breaks (DSBs). DNA double-strand breaks can activate the cell's inherent repair mechanisms non-homologous end-joining (NHEJ) and homologous recombination (HR), thereby repairing DNA damage in the cell. During the repair process, the specific DNA sequence is edited at specific sites.

[0024] The terms “single-stranded guide RNA” and “sgRNA” as used herein are interchangeable and have the meanings commonly understood by those skilled in the art. Generally, a single-stranded guide RNA may comprise a scaffold sequence and a guide sequence, also referred to herein as guide RNA (or gRNA). In the context of an endogenous CRISPR system, the guide sequence is also referred to as a spacer sequence. In some cases, the guide sequence is any polynucleotide sequence that is sufficiently similar to a target sequence to hybridize with said target sequence and guide the specific binding of the CRISPR / Cas12f1 complex to said target sequence. In some embodiments, when optimally aligned, the complementarity between the guide sequence and its corresponding target sequence is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%. Determining optimal alignment is within the capabilities of those skilled in the art. For example, there are publicly available and commercially available comparison algorithms and programs, such as, but not limited to, ClustalW, the Smith-Waterman algorithm in MATLAB, Bowtie, Geneious, Biopython, and SeqMan.

[0025] As used herein, the term "CRISPR / Cas12f1 complex" refers to a complex formed by the binding of a single-stranded guide RNA or mature crRNA:tracrRNA hybrid to a Cas12f1 protein (e.g., wild-type PspCas12f1 protein or a mutant thereof), which contains a guide sequence that hybridizes to a target sequence and thereby enables the Cas12f1 protein to bind to said target sequence. This complex is capable of recognizing and cleaving polynucleotides that hybridize with the single-stranded guide RNA or mature crRNA.

[0026] Therefore, in the formation of the CRISPR / Cas12f1 complex, the "target sequence" refers to a polynucleotide targeted by a guide sequence designed to be targeted, such as a sequence complementary to the guide sequence, where hybridization between the target sequence and the guide sequence will promote the activity of Cas12f1, such as cleavage of the target sequence. Perfect complementarity is not required, as long as sufficient complementarity exists to induce hybridization and promote the activity of Cas12f1. The target sequence can include any polynucleotide, such as DNA or RNA. In some cases, the target sequence is located in the cell nucleus or cytoplasm. In other cases, the target sequence may be located in an organelle of a eukaryotic cell, such as a mitochondrion or chloroplast.

[0027] As used herein, the term "target sequence" or "target polynucleotide" can refer to any endogenous or exogenous polynucleotide for a cell (e.g., a eukaryotic cell). For example, the target polynucleotide can be a polynucleotide present in the nucleus of a eukaryotic cell. The target polynucleotide can be a sequence encoding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or useless DNA). In some cases, the target sequence should be associated with a protospacer adjacent motif (PAM). The precise sequence and length requirements for the PAM vary depending on the Cas protein used, but the PAM is typically a 2-5 base sequence adjacent to the protospacer sequence (target sequence). Those skilled in the art can identify the PAM sequence used with a given Cas protein.

[0028] The terms “polynucleotide,” “nucleic acid sequence,” “nucleotide sequence,” or “nucleic acid fragment” used herein are used interchangeably and are single-stranded or double-stranded RNA or DNA polymers, optionally containing synthetic, non-natural, or modified nucleotide bases. Nucleotides are designated by their single-letter names as follows: “A” for adenosine or deoxyadenosine (corresponding to RNA or DNA, respectively), “C” for cytidine or deoxycytidine, “G” for guanosine or deoxyguanosine, “U” for uridine, “T” for deoxythymidine, “R” for purine (A or G), “Y” for pyrimidine (C or T), “K” for G or T, “H” for A, C, or T, “I” for inosine, and “N” for any nucleotide.

[0029] The terms “polypeptide,” “peptide,” and “protein” as used herein are used interchangeably 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, and 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 linkage, sulfation, γ-carboxylation, hydroxylation, and ADP-ribosylation of glutamate residues.

[0030] The terms “sequence identity” or “homology” as used herein have their generally accepted meaning in the art, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using publicly available techniques. Sequence identity can be measured along the full length of a polynucleotide or polypeptide or along a region of that molecule. (See, e.g., Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; SequenceAnalysis in Molecular Biology, von Heinje, G., Academic Press, 1987; andSequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). While many methods exist for measuring the identity between two polynucleotides or peptides, the term "identity" is known to those skilled in the art to refer to conserved amino acid substitutions in peptides or proteins that can generally be performed without altering the biological activity of the resulting molecule. Typically, those skilled in the art recognize that a single amino acid substitution in a non-essential region of a peptide does not substantially alter its biological activity (see, for example, Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. co., p. 224).

[0031] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. A vector is called an expression vector when it enables the expression of a protein encoded by the inserted polynucleotide, or when it enables transcription of the inserted polynucleotide (e.g., to generate mRNA or functional RNA). Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to, plasmid vectors and viral vectors. Vectors may also contain various regulatory sequences that regulate expression. The terms "regulatory sequence" and "regulatory element" are used interchangeably herein, referring to a nucleotide sequence located upstream (5' non-coding sequence), midway, or downstream (3' non-coding sequence) of a coding sequence that affects transcription, RNA processing, stability, or translation of the relevant coding sequence. Regulatory sequences may include, but are not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. These regulatory sequences may originate from different sources or from the same source but arranged in a manner different from what is typically found naturally. Additionally, vectors may contain a replication initiation site.

[0032] As used herein, the term "promoter" refers to a nucleic acid fragment capable of controlling the transcription of another nucleic acid fragment. In some embodiments of the invention, a promoter is a promoter capable of controlling gene transcription in a cell, regardless of whether it originates from the cell. A promoter can be a constitutive promoter, a tissue-specific promoter, a developmental regulatory promoter, or an inducible promoter.

[0033] As used in this article, the term "constitutive promoter" refers to a promoter that generally causes gene expression in most cell types and under most conditions. "Tissue-specific promoter" and "tissue-preferred promoter" are used interchangeably and refer to promoters that are primarily, but not necessarily, expressed specifically in one tissue or organ, and may also be expressed in a specific cell type. "Developmental regulatory promoter" refers to a promoter whose activity is determined by developmental events. "Inducible promoter" selectively expresses a manipulated DNA sequence in response to endogenous or exogenous stimuli (environment, hormones, chemical signals, etc.).

[0034] "Introducing" nucleic acid molecules (such as plasmids, linear nucleic acid fragments, RNA, etc.) or proteins into an organism refers to transforming the cells of an organism with the nucleic acid or protein, enabling the nucleic acid or protein to function within the cell. The term "transformation" as used in this invention includes both stable transformation and transient transformation.

[0035] As used in this article, the term "stable transformation" refers to the introduction of a foreign nucleotide sequence into the genome, resulting in the stable inheritance of the foreign gene. Once stable transformation occurs, the foreign nucleic acid sequence is stably integrated into the genome of the organism and its subsequent generations.

[0036] The term "transient transformation" as used in this article refers to the introduction of nucleic acid molecules or proteins into cells to perform their functions without the stable inheritance of the foreign gene. In transient transformation, the foreign nucleic acid sequence does not integrate into the genome.

[0037] The terms “identity,” “consistency,” or “homology” used in this article have the generally accepted meanings in the art, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using publicly available 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). While many methods exist for measuring the identity between two polynucleotides or peptides, the term "identity" is known to those skilled in the art to refer to conserved amino acid substitutions in peptides or proteins that can generally be performed without altering the biological activity of the resulting molecule. Typically, those skilled in the art recognize that a single amino acid substitution in a non-essential region of a peptide does not substantially alter its biological activity (see, for example, Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. co., p. 224).

[0038] As used herein, the term "complementarity" refers to the ability of one nucleic acid sequence to form one or more hydrogen bonds with another nucleic acid sequence via conventional Watson-Crick or other non-conventional types. The complementarity percentage indicates the percentage of residues in one nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with another nucleic acid sequence (e.g., 50%, 60%, 70%, 80%, 90%, and 100% complementarity out of 10). "Complete complementarity" means that all consecutive residues in one nucleic acid sequence form hydrogen bonds with the same number of consecutive residues in another nucleic acid sequence. As used herein, “substantially complementary” refers to a complementarity of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% in a region having 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more nucleotides, or to two nucleic acids hybridizing under stringent conditions.

[0039] As used in this paper, the term "strict condition" in relation to hybridization refers to conditions under which a nucleic acid complementary to a target sequence hybridizes primarily with that target sequence and substantially does not hybridize to non-target sequences. Strict conditions are typically sequence-dependent and depend on many factors. Generally, the longer the sequence, the higher the temperature at which it specifically hybridizes to its target sequence. A non-limiting example of a strict condition is described in Tijssen (1993), *Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization With Nucleic Acid Probes*, Part I, Chapter 2, "Overview of principles of hybridization and the strategy of nucleic acid probe assay", Elsevier, New York.

[0040] As used herein, the term "hybridization" refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized by hydrogen bonds between the bases of these nucleotide residues. Hydrogen bonds can occur via Watson-Crick base pairing, Hoogstein binding, or any other sequence-specific mechanism. The complex can consist of two strands forming a duplex, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination thereof. Hybridization can be a step in a broader process, such as the initiation of PCR or the cleavage of a polynucleotide by an enzyme. A sequence capable of hybridizing with a given sequence is called the "complement" of that given sequence.

[0041] Proteins, their conjugates and fusion proteins As described above, the inventors unexpectedly discovered that wild-type and mutant PspCas12f1 proteins, when combined with sgRNA, can effectively form a CRISPR / PspCas12f1 gene editing system. The PspCas12f1 protein has a relatively small number of amino acids, high editing efficiency, and recognizes very simple PAM sequences (TTC). Therefore, the CRISPR / PspCas12f1 gene editing system of this invention has a wide editing range and can be effectively used for site-specific editing of target sequences. This completes the present invention.

[0042] Therefore, in a first aspect, the present invention provides a PspCas12f1 mutant protein, wherein the PspCas12f1 mutant protein contains mutations at Q100 and / or E293 relative to the wild-type PspCas12f1 protein with the amino acid sequence as shown in SEQ ID NO: 1.

[0043] In one implementation, the mutation includes Q100R, E293R, or Q100R and E293R.

[0044] Experiments have demonstrated that the PspCas12f1 mutant protein of this invention exhibits a significant advantage in editing efficiency at random sites compared to the wild-type PspCas12f1 protein. Furthermore, compared to other Cas12f1 proteins in the same series, the double-point mutant proteins enPspCas12f1 (Q100R and E293R) possess extremely significant editing advantages, making them more suitable for the development and application of gene editing technologies.

[0045] Furthermore, the Cas12f1 protein can be derivatized, for example, by linking it to other molecules (e.g., other proteins or peptides, detectable markers). Typically, protein derivatization (e.g., labeling) does not adversely affect the protein's desired activity (e.g., activity binding to single-stranded guide RNA, endonuclease activity, activity to bind to and cleave a target sequence at a specific site guided by the guide RNA). Therefore, in this invention, the Cas12f1 protein can be functionally linked (through chemical coupling, gene fusion, non-covalent linkage, or other means) to one or more other molecular moieties, such as other proteins or peptides, detectable markers, pharmaceutical reagents, etc.

[0046] Specifically, the Cas12f1 protein can be linked to other functional units. For example, it can be linked to a nuclear localization signal (NLS) sequence to enhance the protein's ability to enter the cell nucleus. For example, it can be linked to a targeting moiety to make the Cas12f1 protein targeted. For example, it can be linked to a detectable tag to facilitate the detection of the Cas12f1 protein. For example, it can be linked to an epitope tag to facilitate the expression, detection, tracing, and / or purification of the Cas12f1 protein.

[0047] Therefore, in a second aspect, the present invention provides a conjugate comprising: a) Cas12f1 protein or its homologs, wherein: The Cas12f1 protein is the wild-type PspCas12f1 protein with the amino acid sequence shown in SEQ ID NO: 1 or the PspCas12f1 mutant protein of the first aspect of the present invention. The homolog is a homolog whose amino acid sequence has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.99%, or at least 99.999% sequence identity with the amino acid sequence of the Cas12f1 protein and retains the biological activity of the Cas12f1 protein. b) Modified parts; and c) An optional adapter for connecting the Cas12f1 protein or its homolog to the modified portion.

[0048] In this invention, the so-called "biological activity" of the Cas12f1 protein refers to its activity in binding to single-stranded guide RNA, its endonuclease activity (including single-stranded cleavage activity and double-stranded cleavage activity), and / or its activity in binding to and cleaving a specific site of a target sequence under the guidance of guide RNA (gRNA), but is not limited thereto.

[0049] It is understandable that, in addition to the Cas12f1 protein itself, the Cas12f1 protein can also be combined with other substances, such as other proteins or tagged objects, to give it other functions.

[0050] Therefore, in one embodiment, the modified portion may be another protein or peptide, a detectable marker, or a combination thereof.

[0051] In a further embodiment, the additional protein or polypeptide is selected from one or more of the following: epitope tags, reporter proteins or nuclear localization signal (NLS) sequences, cytosine deaminase (CBE), adenine deaminase (ABE), reverse transcriptase, cytosine methyltransferases DNMT3A and MQ1, cytosine demethylase Tet1, transcription activators VP64, p65 and RTA, transcription repressor KRAB, histone acetyltransferase p300, histone deacetyltransferase LSD1, and endonuclease FokI.

[0052] Epitope tags are well known to those skilled in the art, and examples include, but are not limited to, His, V5, FLAG, HA, Myc, VSV-G, Trx, etc., and those skilled in the art know how to select an appropriate epitope tag according to the desired purpose (e.g., purification, detection, or tracing).

[0053] Reporter proteins are well known to those skilled in the art, and examples include, but are not limited to, GST, HRP, CAT, GFP, HcRed, DsRed, CFP, YFP, and BFP.

[0054] Detectable markers are well known to those skilled in the art, and examples include fluorescent dyes such as fluorescein isothiocyanate (FITC) or DAPI.

[0055] The Cas12f1 protein of the present invention can be coupled, conjugated, or fused to the modified portion via a linker, or it can be directly linked to the modified portion without a linker. Linkers are well known in the art, and examples of them may include, but are not limited to, linkers containing 1-50 amino acids (such as Glu or Ser) or amino acid derivatives (such as Ahx, β-Ala, GABA, or Ava), or PEG, etc.

[0056] In a third aspect, the present invention provides a fusion protein comprising: a) Cas12f1 protein or its homologs, wherein: The Cas12 protein is the wild-type PspCas12f1 protein with the amino acid sequence shown in SEQ ID NO: 1 or the PspCas12f1 mutant protein of the first aspect of the present invention. The homolog is a homolog whose amino acid sequence has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.99%, or at least 99.999% sequence identity with the amino acid sequence of the Cas12f1 protein and retains the biological activity of the Cas12f1 protein; b) Other proteins or peptides; and c) Optional adapters for connecting the Cas12f1 protein or its homologs to the other protein or polypeptide.

[0057] In one embodiment, the additional protein or polypeptide is selected from one or more of the following: epitope tags, reporter proteins or nuclear localization signal (NLS) sequences, cytosine deaminase (CBE), adenine deaminase (ABE), reverse transcriptase, cytosine methyltransferases DNMT3A and MQ1, cytosine demethylase Tet1, transcription activators VP64, p65 and RTA, transcription repressor KRAB, histone acetyltransferase p300, histone deacetyltransferase LSD1, and endonuclease FokI.

[0058] Single-stranded guide RNA In a fourth aspect, the present invention provides a single-stranded guide RNA comprising a CRISPR scaffold sequence having: a) The nucleic acid sequence shown in SEQ ID NO: 5; b) A nucleic acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9% sequence identical to the nucleic acid sequence shown in SEQ ID NO: 5 and retains the biological activity of SEQ ID NO: 5; or c) A nucleic acid sequence obtained by modifying the nucleic acid sequence described in SEQ ID NO: 5 and retaining the biological activity of SEQ ID NO: 5.

[0059] In one embodiment, the modification may be one or more of the following: base phosphorylation, base sulfidation, base methylation, base hydroxylation, sequence shortening, and sequence lengthening.

[0060] In a further embodiment, the shortening of the sequence and the lengthening of the sequence include the deletion or addition of one, two, three, four, five, six, seven, eight, nine, or ten bases relative to the base sequence.

[0061] In yet another embodiment, the single-stranded guide RNA may further include a CRISPR spacer sequence at the 3' end of the CRISPR scaffold sequence, the CRISPR spacer sequence being a sequence of 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 nucleotides in length and capable of complementary pairing with the target sequence.

[0062] In a preferred embodiment, the CRISPR spacer sequence is a 20-nucleotide sequence that is complementary to the target sequence.

[0063] In a further embodiment, the single-stranded guide RNA further includes a terminator at the 3' end of the spacer sequence. As an example, the terminator may be a plurality of terminators, such as at least six (e.g., seven or eight) U.

[0064] The single-stranded guide RNA can bind to the Cas12f1 protein, conjugate, or fusion protein mentioned above to form a complex. This complex can recognize the corresponding PAM and thereby bind to the target sequence, thus achieving the cleavage of the target sequence or gene editing.

[0065] Nucleic acid encoding and vectors In a fifth aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a PspCas12f1 mutant protein or a homolog thereof of the first aspect of the present invention, a conjugate of the second aspect of the present invention, or a fusion protein of the third aspect of the present invention, wherein: The homolog is a homolog whose amino acid sequence has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.99%, or at least 99.999% sequence identity with the amino acid sequence of the PspCas12f1 mutant protein and retains the biological activity of the PspCas12f1 mutant protein.

[0066] In one embodiment, the isolated nucleic acid molecule comprises the nucleic acid sequence or degenerate sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 9 or SEQ ID NO: 10.

[0067] In a further embodiment, the isolated nucleic acid molecule also comprises a nucleic acid sequence encoding a single-stranded guide RNA of the fourth aspect of the present invention.

[0068] As an example, the isolated nucleic acid molecule contains the nucleic acid sequence or degenerate sequence shown in SEQ ID NO: 4, SEQ ID NO: 9 or SEQ ID NO: 10, and the isolated nucleic acid molecule also contains the nucleic acid sequence shown in SEQ ID NO: 6.

[0069] In a sixth aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a single-stranded guide RNA of the fourth aspect of the present invention.

[0070] In one embodiment, the isolated nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO: 6.

[0071] In a preferred embodiment, the isolated nucleic acid molecule further comprises a nucleic acid sequence encoding a CRISPR spacer sequence.

[0072] In one embodiment, the isolated nucleic acid molecule further comprises a nucleic acid sequence encoding the wild-type PspCas12f1 protein or a homolog thereof as shown in SEQ ID NO: 1, wherein: The homolog is a homolog whose amino acid sequence has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.99%, or at least 99.999% sequence identity with the wild-type PspCas12f1 protein and retains the biological activity of the wild-type PspCas12f1 protein.

[0073] In a preferred embodiment, the isolated nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO: 6 and the nucleic acid sequence shown in SEQ ID NO: 3 or a degenerate sequence thereof.

[0074] After the isolated nucleic acid molecules of the present invention are transfected into corresponding cells using certain tools known in the art, such as expression vectors, the isolated nucleic acid molecules of the present invention can express the PspCas12f1 mutant protein or its homologs described above, the conjugates of the second aspect of the present invention or the fusion protein of the third aspect of the present invention, encoding the wild-type PspCas12f1 protein or its homologs as shown in SEQ ID NO: 1, and / or the single-stranded guide RNA described above, and perform corresponding functions, such as gene editing.

[0075] In addition, the isolated nucleic acid molecules of the present invention can express PspCas12f1 mutant protein or its homologs, conjugates of the second aspect of the present invention or fusion proteins of the third aspect of the present invention, and single-stranded guide RNA individually or separately, or can express the above expression products in one go. The choice of expression method depends on the specific circumstances.

[0076] Furthermore, the aforementioned expressive products have the corresponding effects and / or functions described above, which will not be repeated here for the sake of brevity.

[0077] In a seventh aspect, the present invention provides a vector comprising a nucleic acid sequence encoding a PspCas12f1 mutant protein or a homolog thereof of the first aspect of the present invention, a conjugate of the second aspect of the present invention, or a fusion protein of the third aspect of the present invention, wherein: The homolog is a homolog whose amino acid sequence has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.99%, or at least 99.999% sequence identity with the amino acid sequence of the PspCas12f1 mutant protein and retains the biological activity of the PspCas12f1 mutant protein.

[0078] In one embodiment, the vector comprises a nucleic acid sequence or a degenerate sequence thereof shown in any one of SEQ ID NO: 4, SEQ ID NO: 9 or SEQ ID NO: 10.

[0079] In one embodiment, the vector may be an expression vector, such as a plasmid vector like pUC19 vector, an applicator vector, a pAAV2_ITR vector, a retroviral vector, a lentiviral vector, an adenovirus vector, or an adeno-associated virus vector.

[0080] In yet another embodiment, the vector further comprises a nucleic acid sequence encoding a single-stranded guide RNA of the fourth aspect of the present invention.

[0081] As an example, the vector contains the nucleic acid sequence or degenerate sequence shown in SEQ ID NO: 4, SEQ ID NO: 9 or SEQ ID NO: 10, and the vector also contains the nucleic acid sequence shown in SEQ ID NO: 6.

[0082] In an eighth aspect, the present invention provides a vector comprising a nucleic acid molecule encoding a single-stranded guide RNA of the fourth aspect of the present invention.

[0083] In one embodiment, the vector comprises the nucleic acid sequence shown in SEQ ID NO: 6 or a degenerate sequence thereof.

[0084] In a preferred embodiment, the vector further comprises a nucleic acid sequence encoding a CRISPR spacer sequence.

[0085] In one embodiment, the vector further comprises a nucleic acid sequence encoding the wild-type PspCas12f1 protein or a homolog thereof as shown in SEQ ID NO: 1, wherein: The homolog is a homolog whose amino acid sequence has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.99%, or at least 99.999% sequence identity with the wild-type PspCas12f1 protein and retains the biological activity of the wild-type PspCas12f1 protein.

[0086] In a preferred embodiment, the vector comprises the nucleic acid sequence shown in SEQ ID NO: 6 and the nucleic acid sequence shown in SEQ ID NO: 3 or a degenerate sequence thereof.

[0087] As described above, after the vector of the present invention is transfected into cells, the nucleic acid sequence cloned in the vector can be expressed as a PspCas12f1 mutant protein or its homolog, a conjugate of the second aspect of the present invention or a fusion protein of the third aspect of the present invention, a wild-type PspCas12f1 protein or its homolog as shown in SEQ ID NO: 1, and / or the single-stranded guide RNA described above, and perform corresponding functions, such as gene editing.

[0088] Alternatively, multiple vectors, such as two vectors, can be transfected into cells. One vector expresses the PspCas12f1 mutant protein or its homologs, the conjugate of the second aspect of the present invention, or the fusion protein of the third aspect of the present invention, or the wild-type PspCas12f1 protein or its homologs as shown in SEQ ID NO: 1, while the other vector expresses single-stranded guide RNA. Subsequently, the expressed PspCas12f1 mutant protein or its homologs, the conjugate of the second aspect of the present invention, or the fusion protein of the third aspect of the present invention, or the wild-type PspCas12f1 protein or its homologs as shown in SEQ ID NO: 1, complexes with the expressed single-stranded guide RNA to form a complex, which performs the corresponding function, such as gene editing.

[0089] Alternatively, the nucleic acid sequence encoding the PspCas12f1 mutant protein or its homolog, the conjugate of the second aspect of the present invention, or the fusion protein of the third aspect of the present invention, or the wild-type PspCas12f1 protein or its homolog as shown in SEQ ID NO: 1, and the nucleic acid sequence encoding the single-stranded guide RNA can be cloned into a vector. After the vector is transfected into cells, it expresses both the PspCas12f1 mutant protein or its homolog, the conjugate of the second aspect of the present invention, the fusion protein of the third aspect of the present invention, or the wild-type PspCas12f1 protein or its homolog as shown in SEQ ID NO: 1, and the single-stranded guide RNA, and performs the corresponding functions, such as gene editing.

[0090] CRISPR / Cas12f1 gene editing system In a ninth aspect, the present invention provides a CRISPR / Cas12f1 gene editing system comprising: 1) Protein components, comprising Cas12f1 protein or its homologs, conjugates, or fusion proteins, wherein: The Cas12f1 protein is the wild-type PspCas12f1 protein with the amino acid sequence shown in SEQ ID NO: 1 or the PspCas12f1 mutant protein of the first aspect of the present invention; The homolog is a homolog whose amino acid sequence has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.99%, or at least 99.999% sequence identity with the amino acid sequence shown by the Cas12f1 protein and retains the biological activity of the Cas12f1 protein; The conjugate comprises: the Cas12f1 protein or its homolog, a modified moiety, and an optional linker; The fusion protein comprises: the Cas12f1 protein or its homologs, additional proteins or peptides, and optional linkers; The optional connector is used to connect the Cas12f1 protein or its homolog to the modified moiety or the other protein or polypeptide; and 2) Nucleic acid components, comprising: single-stranded guide RNA of the fourth aspect of the present invention; Furthermore, the protein component and the nucleic acid component combine with each other to form a complex.

[0091] In one embodiment, the modified portion is selected from other proteins or peptides, detectable markers, or combinations thereof.

[0092] In one embodiment, the additional protein or polypeptide is selected from one or more of the following: epitope tags, reporter proteins or nuclear localization signal (NLS) sequences, cytosine deaminase (CBE), adenine deaminase (ABE), reverse transcriptase, cytosine methyltransferases DNMT3A and MQ1, cytosine demethylase Tet1, transcription activators VP64, p65 and RTA, transcription repressor KRAB, histone acetyltransferase p300, histone deacetyltransferase LSD1, and endonuclease FokI.

[0093] As an example, the protein component comprises: wild-type PspCas12f1 protein with an amino acid sequence as shown in SEQ ID NO: 1, or a PspCas12f1 mutant protein with an amino acid sequence as shown in any one of SEQ ID NO: 2, SEQ ID NO: 7 and SEQ ID NO: 8, and the nucleic acid component comprises the nucleic acid sequence shown in SEQ ID NO: 6.

[0094] The CRISPR / Cas12f1 gene editing system of the present invention can be directly constructed from the Cas12f1 protein or its homologs, conjugates or fusion proteins described herein and the single-stranded guide RNA described herein, or it can be constructed from the expression product obtained by the vector described herein.

[0095] The CRISPR / Cas12f1 gene editing system of the present invention achieves the recognition, localization, cutting and gene editing of target sequences through the combined action of the Cas12f1 protein or its homologs, conjugates or fusion proteins contained therein and single-stranded guide RNA.

[0096] The CRISPR / Cas12f1 gene editing system of this invention can precisely locate the target sequence. "Precisely located" has two meanings: first, the CRISPR / Cas12f1 gene editing system itself can recognize and bind to the target sequence; second, the CRISPR / Cas12f1 gene editing system can bring other proteins fused with the Cas12 protein or proteins that specifically recognize the sgRNA to the location of the target sequence.

[0097] The CRISPR / Cas12f1 gene editing system of the present invention has low tolerance for non-target sequences. In this document, "low tolerance" means that the CRISPR / Cas12f1 gene editing system of the present invention is substantially or completely unable to recognize and bind to non-target sequences, or substantially or completely unable to bring other proteins fused with the Cas12f1 protein or proteins that specifically recognize the sgRNA to the location of the non-target sequence.

[0098] The CRISPR / Cas12f1 gene editing system of the present invention can target more DNA sequences in the genome because the PAM sequence on the target sequence recognized by the Cas12f1 protein contained therein is simpler.

[0099] cell In a tenth aspect, the present invention provides a cell comprising: isolated nucleic acid molecules of the fifth and / or sixth aspects of the present invention, or a vector of the seventh and / or eighth aspects of the present invention.

[0100] As an example, the cell can be a prokaryotic cell or a eukaryotic cell. For the eukaryotic cell, as an example, it can be a plant cell or an animal cell. For the animal cell, as an example, it can be a mammalian cell, such as a human cell.

[0101] method In an eleventh aspect, the present invention provides a method for gene editing of a target sequence in an intracellular or in vitro environment, the method comprising contacting any one of the following (1) to (4) with the target sequence in the intracellular or in vitro environment: (1) Cas12f1 protein or its homologs, conjugates or fusion proteins, and the single-stranded guide RNA of the fourth aspect of the present invention, wherein: The Cas12f1 protein is the wild-type PspCas12f1 protein with the amino acid sequence shown in SEQ ID NO: 1 or the PspCas12f1 mutant protein of the first aspect of the present invention. The homolog is a homolog whose amino acid sequence has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.99%, or at least 99.999% sequence identity with the amino acid sequence of the Cas12f1 protein and retains the biological activity of the Cas12f1 protein. The conjugate comprises: the Cas12f1 protein or its homolog, a modified moiety, and an optional linker; The fusion protein comprises: the Cas12f1 protein or its homologs, additional proteins or peptides, and optional linkers; The optional connector is used to connect the Cas12f1 protein or its homolog to the modified portion or the other protein or polypeptide; (2) The seventh aspect of the present invention does not contain a vector encoding a nucleic acid sequence of a single-stranded guide RNA of the fourth aspect of the present invention or a vector containing a nucleic acid sequence encoding a wild-type PspCas12f1 protein or its homolog as shown in SEQ ID NO: 1, and the eighth aspect of the present invention does not contain a vector encoding a nucleic acid sequence encoding a wild-type PspCas12f1 protein or its homolog as shown in SEQ ID NO: 1. (3) A vector comprising, in the seventh aspect of the present invention, a nucleic acid sequence encoding the PspCas12f1 mutant protein or its homolog, the conjugate of the second aspect of the present invention, or the fusion protein of the third aspect of the present invention, and a nucleic acid sequence encoding the single-stranded guide RNA of the fourth aspect of the present invention; or a vector comprising, in the eighth aspect of the present invention, a nucleic acid sequence encoding the single-stranded guide RNA of the fourth aspect of the present invention, and a nucleic acid sequence encoding the wild-type PspCas12f1 protein or its homolog as shown in SEQ ID NO: 1; or (4) The CRISPR / Cas12f1 gene editing system of the ninth aspect of the present invention; Upon contact with the target sequence, the Cas12f1 protein or its homologs, conjugates, or fusion proteins recognize the PAM sequence 5'-TTC located at the 5' end of the target sequence.

[0102] In one embodiment, the modified portion is selected from other proteins or peptides, detectable markers, or combinations thereof.

[0103] In one embodiment, the additional protein or polypeptide is selected from one or more of the following: epitope tags, reporter proteins or nuclear localization signal (NLS) sequences, cytosine deaminase (CBE), adenine deaminase (ABE), reverse transcriptase, cytosine methyltransferases DNMT3A and MQ1, cytosine demethylase Tet1, transcription activators VP64, p65 and RTA, transcription repressor KRAB, histone acetyltransferase p300, histone deacetyltransferase LSD1, and endonuclease FokI.

[0104] As an example, for item (1) above, it can be: wild-type PspCas12f1 protein with an amino acid sequence as shown in SEQ ID NO: 1, or PspCas12f1 mutant protein with an amino acid sequence as shown in any one of SEQ ID NO: 2, SEQ ID NO: 7 and SEQ ID NO: 8, and a single-stranded guide RNA containing the CRISPR scaffold sequence shown in SEQ ID NO: 5.

[0105] As an example, for item (2) above, it can be: a vector containing the nucleic acid sequence or its degenerate sequence shown in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:9 or SEQ ID NO:10, and a vector containing the nucleic acid sequence shown in SEQ ID NO:6.

[0106] In one embodiment, the cell is a prokaryotic cell or a eukaryotic cell, the eukaryotic cell being, for example, a plant cell or an animal cell, the animal cell being, for example, a mammalian cell such as a human cell.

[0107] In one embodiment, the gene editing includes one or more of the following: gene knockout of a target sequence, site-specific base alteration, site-specific insertion, regulation of gene transcription, regulation of DNA methylation, DNA acetylation modification, histone acetylation modification, single base conversion, and chromatin imaging tracking.

[0108] Furthermore, in one embodiment, the single base conversion includes adenine to guanine, cytosine to thymine, or cytosine to uracil.

[0109] In one embodiment, in the method, the CRISPR spacer sequence of the single-stranded guide RNA forms a fully complementary base pairing structure with the target sequence, and an incompletely complementary base pairing structure with a non-target sequence.

[0110] In this document, the incomplete base pairing structure refers to a structure that includes a portion of base pairing and a portion of non-base pairing, wherein the non-base pairing includes, for example, base mismatch and / or base bulge.

[0111] In one embodiment, the incomplete base complementary pairing structure includes one or more, for example, two or more base mismatches.

[0112] Therefore, the Cas12f1 protein or its homologs, conjugates, or fusion proteins of the present invention can cleave target sites on the target sequence, and the cleavage action of the Cas12f1 protein or its homologs, conjugates, or fusion proteins causes double-strand breaks in the target sequence. Furthermore, when the method is performed intracellularly, the cleaved target sequence can be repaired through intracellular non-homologous end joining repair or homologous recombination repair pathways, thereby achieving gene editing of the target sequence.

[0113] The CRISPR / Cas12f1 gene editing system and gene editing method using this invention have been experimentally shown to have editing efficiencies of 23%-76% (for wild-type PspCas12f1 protein) and 63%-98% (for mutant PspCas12f1 protein). Furthermore, the mismatch rate of the first 18 bp guide RNA in the CRISPR / PspCas12f1 gene editing system is extremely low. Therefore, this gene editing system can edit target genes with high specificity, exhibiting high editing efficiency and low off-target rate, and can be widely applied to gene editing in cells or in vitro environments.

[0114] Reagent test kit In a twelfth aspect, the present invention provides a kit for gene editing of target sequences in intracellular or in vitro environments, comprising: i. Choose any one of the following (1) to (6): (1) Cas12f1 protein or its homologs, conjugates or fusion proteins, and the single-stranded guide RNA of the fourth aspect of the present invention, wherein: The Cas12f1 protein is the wild-type PspCas12f1 protein with the amino acid sequence shown in SEQ ID NO: 1 or the PspCas12f1 mutant protein of the first aspect of the present invention; The homolog is a homolog whose amino acid sequence has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.99%, or at least 99.999% sequence identity with the amino acid sequence of the Cas12f1 protein and retains the biological activity of the Cas12f1 protein; The conjugate comprises: the Cas12f1 protein or its homolog, a modified moiety, and an optional linker; The fusion protein comprises: the Cas12f1 protein or its homologs, additional proteins or peptides, and optional linkers; The optional connector is used to connect the Cas12f1 protein or its homolog to the modified portion or the other protein or polypeptide; (2) The fifth aspect of the present invention does not contain an isolated nucleic acid molecule encoding a nucleic acid sequence of a single-stranded guide RNA of the fourth aspect of the present invention or an isolated nucleic acid molecule containing a nucleic acid sequence encoding a wild-type PspCas12f1 protein or its homolog as shown in SEQ ID NO: 1, and the sixth aspect of the present invention does not contain an isolated nucleic acid molecule encoding a nucleic acid sequence encoding a wild-type PspCas12f1 protein or its homolog as shown in SEQ ID NO: 1; (3) The fifth aspect of the present invention comprises a nucleic acid sequence encoding the PspCas12f1 mutant protein or its homolog, the conjugate of the second aspect of the present invention or the fusion protein of the third aspect of the present invention and a nucleic acid sequence encoding the single-stranded guide RNA of the fourth aspect of the present invention; or the sixth aspect of the present invention comprises a nucleic acid sequence encoding the single-stranded guide RNA of the fourth aspect of the present invention and a nucleic acid sequence encoding the wild-type PspCas12f1 protein or its homolog as shown in SEQ ID NO: 1; (4) The seventh aspect of the present invention does not contain a vector encoding a nucleic acid sequence of a single-stranded guide RNA of the fourth aspect of the present invention or a vector containing a nucleic acid sequence encoding a wild-type PspCas12f1 protein or its homolog as shown in SEQ ID NO: 1, and the eighth aspect of the present invention does not contain a vector encoding a nucleic acid sequence encoding a wild-type PspCas12f1 protein or its homolog as shown in SEQ ID NO: 1. (5) The seventh aspect of the present invention comprises a nucleic acid sequence encoding the PspCas12f1 mutant protein or its homolog, the conjugate of the second aspect of the present invention or the fusion protein of the third aspect of the present invention and a nucleic acid sequence encoding the single-stranded guide RNA of the fourth aspect of the present invention; or the eighth aspect of the present invention comprises a nucleic acid sequence encoding the single-stranded guide RNA of the fourth aspect of the present invention and a nucleic acid sequence encoding the wild-type PspCas12f1 protein or its homolog as shown in SEQ ID NO: 1; (6) The CRISPR / Cas12f1 gene editing system of the ninth aspect of the present invention; and ii. Instructions on how to perform gene editing on target sequences in intracellular or in vitro environments.

[0115] In one embodiment, the modified portion is selected from other proteins or peptides, detectable markers, or combinations thereof.

[0116] In one embodiment, the additional protein or polypeptide is selected from one or more of the following: epitope tags, reporter proteins or nuclear localization signal (NLS) sequences, cytosine deaminase (CBE), adenine deaminase (ABE), reverse transcriptase, cytosine methyltransferases DNMT3A and MQ1, cytosine demethylase Tet1, transcription activators VP64, p65 and RTA, transcription repressor KRAB, histone acetyltransferase p300, histone deacetyltransferase LSD1, and endonuclease FokI.

[0117] As an example, for item (1) above, it can be: wild-type PspCas12f1 protein with an amino acid sequence as shown in SEQ ID NO: 1, or PspCas12f1 mutant protein with an amino acid sequence as shown in any one of SEQ ID NO: 2, SEQ ID NO: 7 and SEQ ID NO: 8, and a single-stranded guide RNA containing the CRISPR scaffold sequence shown in SEQ ID NO: 5.

[0118] As an example, for item (2) above, it can be: an isolated nucleic acid molecule containing the nucleic acid sequence or its degenerate sequence shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 9 or SEQ ID NO: 10, and an isolated nucleic acid molecule containing the nucleic acid sequence shown in SEQ ID NO: 6.

[0119] As an example, for item (4) above, it can be: a vector containing the nucleic acid sequence or its degenerate sequence shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 9 or SEQ ID NO: 10, and a vector containing the nucleic acid sequence shown in SEQ ID NO: 6.

[0120] Of course, those skilled in the art will understand that the kit of the present invention may also contain other reagents that facilitate gene editing.

[0121] A brief description of the sequence involved in this invention. SEQ ID NO: 1: PspCas12f1 protein sequence SEQ ID NO: 2: enPspCas12f1 protein sequence SEQ ID NO: 3: Coding sequence of PspCas12f1 protein SEQ ID NO: 4: The coding sequence of the enPspCas12f1 protein SEQ ID NO: 5: Sequence of a single-stranded guide RNA scaffold used in conjunction with PspCas12f1 or enPspCas12f1 protein. SEQ ID NO: 6: DNA sequence of a single-stranded guide RNA scaffold sequence used in conjunction with PspCas12f1 protein or enPspCas12f1 protein. SEQ ID NO: 7: PspCas12f1-Q100R mutant protein sequence SEQ ID NO: 8: PspCas12f1-E293R mutant protein sequence SEQ ID NO: 9: Coding sequence of the PspCas12f1-Q100R mutant protein SEQ ID NO: 10: Coding sequence of the PspCas12f1-E293R mutant protein SEQ ID NO: 11: SpaCas12f1 protein sequence SEQ ID NO: 12: enOsCas12f1 protein sequence SEQ ID NO: 13: Coding sequence of SpaCas12f1 protein SEQ ID NO: 14: Coding sequence of enOsCas12f1 protein SEQ ID NO: 15: Sequence of a single-stranded guide RNA scaffold used in conjunction with the SpaCas12f1 protein SEQ ID NO: 16: Sequence of a single-stranded guide RNA scaffold used in conjunction with the enOsCas12f1 protein SEQ ID NO: 17: DNA sequence of a single-stranded guide RNA scaffold sequence used in conjunction with SpaCas12f1 protein. SEQ ID NO: 18: DNA sequence of a single-stranded guide RNA scaffold sequence used in conjunction with enOsCas12f1 protein. Example The invention will now be described with reference to the following embodiments, which are intended to be illustrative and not limiting. Those skilled in the art will understand that the embodiments provided herein are for the purpose of describing the invention in detail only and are not intended to limit the scope of protection claimed by the invention.

[0122] Unless otherwise specified, the experiments and methods described in the examples were generally performed according to conventional methods well known in the art and described in the various references. Furthermore, for conditions not specifically specified in the examples, conventional conditions or conditions recommended by the manufacturer were followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0123] Example 1 (1) Constructing plasmid pAAV2_PspCas12f1_ITR Download the amino acid sequence of the PspCas12f1 protein from NCBI (SEQ ID NO: 1, its gene search number is QAMT01000002).

[0124] The nucleic acid sequence encoding the PspCas12f1 protein was codon-optimized to obtain the gene sequence of the PspCas12f1 protein that is highly expressed in human cells, as shown in SEQ ID NO: 3.

[0125] Genes were synthesized from the gene sequence shown in SEQ ID NO: 3 obtained above and constructed onto the pSpCas9(BB)-2A-Puro (PX459) backbone plasmid V2.0 (Addgene platform, catalog#62988) to obtain plasmid pAAV2_PspCas12f1_ITR.

[0126] (2) Construction of plasmid PspCas12f1-PSK-hU6-sgRNA-scaffold The pSKB plasmid (available commercially from the Addgene platform, catalog #62540) was digested with ClaI and XhoI restriction endonucleases. The digestion system consisted of 1 μg pSKB plasmid, 5 μL 10×rCutSmart buffer (from NEB), 1 μL ClaI and 1 μL XhoI restriction endonucleases (from NEB), and water to a final volume of 50 μL. The digestion was incubated at 37°C for 1 hour.

[0127] Then, the enzyme digestion products were electrophoresed on a 1% agarose gel at 120V for 30 min.

[0128] The target size DNA fragment was cut from the agarose gel and recovered using a gel recovery kit (Tiangen Biotech (Beijing) Co., Ltd., DP209) according to the manufacturer's instructions. Finally, it was eluted with ultrapure water.

[0129] Genes were synthesized based on the DNA sequence of the sgRNA scaffold (SEQ ID NO: 6) and constructed on a linearized pSKB backbone to obtain the plasmid PspCas12f1-PSK-hU6-sgRNA-scaffold.

[0130] (3) Construction of plasmid pAAV2_PspCas12f1-hU6-sgRNA-scaffold_ITR vector The pAAV2_PspCas12f1_ITR plasmid expressing Cas12f1 protein in (1) and the PspCas12f1-PSK-hU6-sgRNA-scaffold plasmid expressing sgRNA in (2) were linearized using PCR.

[0131] For the pAAV2_PspCas12f1_ITR plasmid, the primer sequences are as follows: ccgcaggaacccctagtgatTTTTTGCGGCCGCAGGAA; and ggacgcaaagccgccctgtaGGTGTTTCGTCCTTCCACA; For the PspCas12f1-PSK-hU6-sgRNA-scaffold plasmid, the primer sequences are as follows: gtacagggcggctttgcgt; and atcactaggggttcctgcg.

[0132] The PCR reaction system is as follows: The PCR procedure is as follows: The obtained PCR products were electrophoresed on a 1% agarose gel at 120V for 30 min. The target DNA fragment was purified using a gel extraction kit according to the manufacturer's instructions. The DNA concentration was measured using a NanoDrop™ Lite spectrophotometer (ThermoScientific) for later use or for long-term storage at -20℃.

[0133] The linearized pAAV2_PspCas12f1_ITR fragment (linearized backbone) and the linearized PspCas12f1-PSK-hU6-sgRNA-scaffold fragment (insertion fragment) were homologously recombinated according to the ratio specified in the instructions. The homologous recombinase used was NEBuilder® High Fidelity DNA Assembly Premix (NEB). The reaction system is as follows: The reaction conditions are as follows: The obtained ligation product was added to Escherichia coli DH5α competent cells (purchased from Shanghai Weidi Biotechnology Co., Ltd.), incubated on ice for 30 min, heat-shocked at 42℃ for 1 min, incubated on ice for 2 min, and then 900 μL of LB medium was added and cultured at 37℃ for 1 hour to activate and revive Escherichia coli DH5α competent cells.

[0134] The revived Escherichia coli DH5α competent cells were plated on LB solid plates containing ampicillin resistance, incubated upside down in a 37°C incubator, and the obtained Escherichia coli DH5α monoclonal cells were verified by Sanger sequencing.

[0135] The correctly ligated E. coli DH5α clone was subjected to culture by shaking, and then the plasmid was extracted to obtain the plasmid pAAV2_PspCas12f1-hU6-sgRNA-scaffold_ITR, which was then used for later use.

[0136] (4) Preparation of linearized plasmid pAAV2_PspCas12f1-hU6-sgRNA-scaffold_ITR The plasmid pAAV2_PspCas12f1-hU6-sgRNA-scaffold_ITR prepared in (3) was digested with BbsI restriction endonuclease. The digestion system consisted of 1 μg plasmid pAAV2_PspCas12f1-hU6-sgRNA-scaffold_ITR, 5 μL 10×CutSmart buffer (purchased from NEB), 1 μL BbsI restriction endonuclease (purchased from NEB), and water to a final volume of 50 μL. The digestion system was incubated at 37°C for 1 hour.

[0137] Then, the obtained enzyme digestion products were electrophoresed on a 1% agarose gel at 120V for 30 min.

[0138] DNA fragments were excised from agarose gels and recovered using a gel extraction kit (Tiangen Biotech (Beijing) Co., Ltd., DP209) according to the manufacturer's instructions. The fragments were then eluted with ultrapure water. The resulting DNA fragment was the linearized plasmid pAAV2_PspCas12f1-hU6-sgRNA-scaffold_ITR, containing the encoding genes for the PspCas12f1 protein and sgRNA scaffold, with a size of 6562 bp.

[0139] The DNA concentration of the recovered linearized plasmid pAAV2_PspCas12f1-hU6-sgRNA-scaffold_ITR was determined using a NanoDrop™ Lite spectrophotometer (Thermo Scientific) and used for later use or stored at -20°C for long-term preservation.

[0140] (5) Preparation of plasmid pAAV2_PspCas12f1-hU6-sgRNA_ITR We designed a gRNA sequence and added sticky end sequences (indicated by uppercase letters) to both the sense and antisense strands of the linearized plasmid pAAV2_PspCas12f1-hU6-sgRNA-scaffold_ITR. We then synthesized these two oligonucleotide single-stranded DNA sequences, as shown below: gRNA: ggatatgttgaagaacacca Oligo-F:AAGAggatatgttgaagaacacca Oligo-R: AAAAtggtgttcttcaacatatcc Oligonucleotide single-stranded DNA was annealed to obtain double-stranded DNA. The annealing reaction system consisted of 3 μL 10 μM oligo-F, 3 μL 10 μM oligo-R, and 4 μL water. After vortexing and mixing the annealing system, it was placed in a PCR instrument and the annealing program was run as follows: 95℃_5 min, 85℃_1 min, 75℃_1 min, 65℃_1 min, 55℃_1 min, 45℃_1 min, 35℃_1 min, 25℃_1 min, and stored at 4℃ with a cooling rate of 0.3℃ / s. After annealing, the obtained product was ligated into the linearized pAAV2_PspCas12f1-hU6-sgRNA-scaffold_ITR plasmid obtained in step (4) using DNA ligase (purchased from NEB).

[0141] 1 μL of the obtained ligation product was added to Escherichia coli DH5α competent cells (purchased from Shanghai Weidi Biotechnology Co., Ltd.), incubated on ice for 30 min, heat-shocked at 42℃ for 1 min, incubated on ice for 2 min, and then 900 μL of LB medium was added and cultured at 37℃ for 1 hour to activate and reactivate Escherichia coli DH5α competent cells.

[0142] The revived Escherichia coli DH5α competent cells were plated on LB solid plates containing the corresponding antibiotics and incubated upside down at 37°C to obtain Escherichia coli DH5α monoclonal cells, which were then verified by Sanger sequencing.

[0143] The correctly ligated E. coli DH5α clone was subjected to culture in a shaking process, and then the plasmid was extracted to obtain the plasmid pAAV2_PspCas12f1-hU6-sgRNA-scaffold_ITR containing the target sgRNA sequence, which was then used for later use.

[0144] (6) Transfection of a GFP reporter system HEK293T cell line library containing the target sequence by plasmid pAAV2_PspCas12f1-hU6-sgRNA_ITR expressing PspCas12f1 protein and sgRNA. The HEK293T cell line library containing the target sequence of the GFP reporter system was obtained as follows: a 5 bp random sequence (as the PAM sequence) and a 24 bp protospacer (as the target sequence) were inserted between the start codon ATG and the GFP coding sequence, resulting in a frameshift mutation that prevents GFP expression. This GFP gene containing the inserted fragment was started using the CMV promoter and constructed into a lentiviral expression vector. This sequence was then randomly inserted into the genome of HEK293T cells via lentiviral mediation, creating a stable GFP reporter cell line library. When the target sequence was cut using the gene editing system, the cells' self-repair system caused some cells to recover the GFP reading frame, producing green fluorescence. Flow cytometry analysis was used to statistically analyze the percentage of GFP-positive cells, which allowed for the assessment of the editing capability and specificity of the gene editing system.

[0145] The transfection process includes the following steps: On day 0, the HEK293T cell line library containing the target sequence of the GFP reporter system was plated in 10 cm dishes as required for transfection, with the cell density controlled at 30%.

[0146] The HEK293T cell line library containing the target sequence of the GFP reporter system contains the nucleotide sequence CMV-ATG-PAM-targetsite-GFP, where the PAM sequence is a 5 bp random sequence and the target site sequence is GGATATGTTGAAGAACACCATGAC.

[0147] Day 1, transfection was performed. The transfection process is as follows: Take 2 μg of the plasmid pAAV2_PspCas12f1-hU6-sgRNA_ITR to be transfected and add it to 100 μL of Opti-MEM medium (purchased from Gibco). Gently pipette to mix.

[0148] Gently mix Lipofectamine® 2000 (purchased from Invitrogen) or PEI (purchased from Polysciences), then add 5 μL of Lipofectamine® 2000 or PEI to 100 μL of Opti-MEM medium, mix gently, and let stand at room temperature for 5 min.

[0149] The diluted plasmid and diluted transfection reagent were mixed and gently pipetted to mix. The resulting mixture was allowed to stand at room temperature for 20 min, and then added to the culture medium of the HEK293T cell line library containing the target sequence of the GFP reporter system. The mixture was then placed in a 37°C, 5% CO2 incubator for further culture.

[0150] Five days after culture, the editing of target genes in the HEK293T cell line library by the CRISPR / PspCas12f1 system was observed under a fluorescence microscope. Under the microscope, cells transfected with the CRISPR / PspCas12f1 system produced green fluorescence, as shown in the image. Figure 1 As shown, this indicates that the CRISPR / PspCas12f1 system successfully edited the target gene in the cell. Subsequently, cells expressing GFP fluorescence were sorted using flow cytometry and further enriched for growth.

[0151] (7) Preparation of next-generation sequencing libraries The enriched HEK293T cell library was collected, and genomic DNA was extracted using a DNA kit (Tiangen Biotech (Beijing) Co., Ltd., DP304) according to the instructions provided with the DNA kit.

[0152] The first round of PCR for library preparation was performed using a 2×Q5 Mastermix. The PCR primers are shown below: F1 primer: ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNcagcacaaaaggaaactcacc; R1 primer: ACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNtggtgcagatgaacttcagg.

[0153] The PCR reaction system is as follows: The PCR procedure is as follows: The second round of PCR for sequencing library preparation was performed using a 2×Q5 Mastermix. The PCR primers are shown below: F2 primer: AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGAC; R2 primer: CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTGACTGGAGTTCAGACGTGTG.

[0154] The PCR reaction system is as follows: The PCR procedure is as follows: The second-round PCR products were purified into DNA fragments of the target size using a gel extraction kit following the manufacturer's instructions, and the next-generation sequencing library was prepared.

[0155] (8) Analysis of second-generation sequencing results The prepared next-generation sequencing library was subjected to paired-end sequencing on the high-throughput sequencer HiseqXTen (illumina).

[0156] Based on the results obtained from next-generation sequencing, the PAM sequences of the HEK293T cell library were analyzed, and a PAM-logo diagram was plotted, as shown below. Figure 2 As shown, the PspCas12f1 protein specifically recognizes the 5'-TTC-3' PAM sequence, which is simple and universal, significantly expanding the targetable genome range. PspCas12f1 demonstrates promising application potential in mammalian cell gene editing.

[0157] Example 2 (1) Construct plasmid pAAV2_PspCas12f1_ITR and 9 modified pAAV2_PspCas12f1-mutant_ITR plasmids. Genes were synthesized from the gene sequences of SEQ ID NO: 3 and 9 modified gene sequences (Q97R, Q100R, N108R, S157L, P191R, I194R, P211R, E293R, and enPspCas12f1 (Q100R & E293R)) and constructed onto the pSpCas9(BB)-2A-Puro (PX459) backbone plasmid V2.0 (Addgene platform, catalog#62988) to obtain plasmid pAAV2_PspCas12f1_ITR and 9 modified pAAV2_PspCas12f1-mutant_ITR plasmids.

[0158] (2) Constructing plasmid Cas12f1-PSK-hU6-sgRNA-scaffold The plasmid Cas12f1-PSK-hU6-sgRNA-scaffold was prepared using the same method as in Example 1 (2).

[0159] (3) Construction of plasmid pAAV2_PspCas12f1-hU6-sgRNA-scaffold_ITR and 9 modified pAAV2_PspCas12f1-mutant-hU6-sgRNA-scaffold_ITR vectors Using a method similar to that in Example 1 (3), the following primer sequences were used to prepare plasmid pAAV2_PspCas12f1-hU6-sgRNA-scaffold_ITR and 9 modified pAAV2_PspCas12f1-mutant-hU6-sgRNA-scaffold_ITR for later use.

[0160] For the pAAV2_PspCas12f1_ITR plasmid and 9 modified pAAV2_PspCas12f1-mutant_ITR plasmids, the primer sequences are as follows: ccgcaggaacccctagtgatTTTTTGCGGCCGCAGGAA; and ggacgcaaagccgccctgtaGGTGTTTCGTCCTTCCACA; For the Cas12f1-PSK-hU6-sgRNA-scaffold plasmid, the primer sequences are as follows: gtacagggcggctttgcgt; and atcactaggggttcctgcg.

[0161] (4) Preparation of linearized plasmid pAAV2_PspCas12f1-hU6-sgRNA-scaffold_ITR and 9 modified pAAV2_PspCas12f1-mutant-hU6-sgRNA-scaffold_ITR Using a method similar to that in Example 1 (4), linearized plasmid pAAV2_PspCas12f1-hU6-sgRNA-scaffold_ITR and 9 modified pAAV2_PspCas12f1-mutant-hU6-sgRNA-scaffold_ITR were prepared, and the DNA concentration was determined using a NanoDrop™ Lite spectrophotometer (Thermo Scientific). The DNA was then stored for later use or at -20°C for long-term preservation.

[0162] (5) Preparation of plasmid pAAV2_PspCas12f1-hU6-sgRNA_ITR and 9 modified pAAV2_PspCas12f1-mutant-hU6-sgRNA_ITR The gRNA sequence was designed, and the linearized plasmid pAAV2_PspCas12f1-hU6-sgRNA_ITR and nine modified pAAV2_PspCas12f1-mutant-hU6-sgRNA_ITR with corresponding sticky ends (indicated by uppercase letters) were added to the sense and antisense strands, respectively. These oligonucleotide single-stranded DNAs were then synthesized, and their specific sequences are shown below: gRNA: ttgctgtcagtccatggttc Oligo-F:AAGAttgctgtcagtccatggttc Oligo-R: AAAAgaaccatggactgacagcaa Oligonucleotide single-stranded DNA was annealed to obtain double-stranded DNA. The annealing reaction system consisted of 3 μL 10 μM oligo-F, 3 μL 10 μM oligo-R, and 4 μL water. After vortexing and mixing the annealing system, it was placed in a PCR instrument and the annealing program was run as follows: 95℃_5 min, 85℃_1 min, 75℃_1 min, 65℃_1 min, 55℃_1 min, 45℃_1 min, 35℃_1 min, 25℃_1 min, and stored at 4℃ with a cooling rate of 0.3℃ / s. After annealing, the obtained product was ligated to the linearized pAAV2_PspCas12f1-hU6-sgRNA_scaffold_ITR obtained in step (2) and 9 modified pAAV2_PspCas12f1-mutant-hU6-sgRNA_scaffold_ITR plasmids using DNA ligase (purchased from NEB).

[0163] 1 μL of the obtained ligation product was added to Escherichia coli DH5α competent cells (purchased from Shanghai Weidi Biotechnology Co., Ltd.), incubated on ice for 30 min, heat-shocked at 42℃ for 1 min, incubated on ice for 2 min, and then 900 μL of LB medium was added and cultured at 37℃ for 1 hour to activate and reactivate Escherichia coli DH5α competent cells.

[0164] The revived Escherichia coli DH5α competent cells were plated on LB solid plates containing the corresponding antibiotics and incubated upside down in a 37°C incubator. The obtained Escherichia coli DH5α monoclonal cells were then verified by Sanger sequencing.

[0165] The correctly ligated E. coli DH5α clone was subjected to culture and then plasmids were extracted, resulting in plasmid pAAV2_PspCas12f1-hU6-sgRNA_ITR containing the target sgRNA sequence and 9 modified pAAV2_PspCas12f1-mutant-hU6-sgRNA_ITR, which were then used for later use.

[0166] (6) Transfection of HEK293T cell line with plasmid pAAV2_PspCas12f1-hU6-gRNA_ITR expressing PspCas12f1 protein and sgRNA and plasmid pAAV2_PspCas12f1-mutant-hU6-sgRNA_ITR expressing modified protein and sgRNA. On day 0, HEK293T cells containing the target sequence were seeded in 6-well plates as needed for transfection, with a cell density of approximately 30%.

[0167] Day 1, transfection was performed. The transfection process is as follows: Add 2 μg of the plasmid pAAV2_PspCas12f1-hU6-sgRNA_ITR to be transfected or 9 modified pAAV2_PspCas12f1-mutant-hU6-sgRNA_ITR to 100 μL of Opti-MEM medium (purchased from Gibco) and gently pipette to mix.

[0168] Gently mix the transfection reagents Lipofectamine® 2000 (purchased from Invitrogen) or polyethyleneimine (hereinafter referred to as PEI, purchased from Polysciences). Add 5 μL of Lipofectamine® 2000 or PEI to 100 μL of Opti-MEM medium (purchased from Gibco), mix gently, and let stand at room temperature for 5 min.

[0169] Mix the diluted transfection reagent and diluted plasmid, gently pipette to mix, let stand at room temperature for 20 min, then add to the culture medium containing HEK293T cells to be transfected, and then place the cells in a 37℃, 5% CO2 incubator for 5 days.

[0170] (7) Preparation of next-generation sequencing libraries HEK293T cells were collected five days after editing, and genomic DNA was extracted using a DNA kit (Tiangen Biotech (Beijing) Co., Ltd., DP304) according to the instructions provided with the DNA kit.

[0171] The first round of PCR for library preparation was performed using a 2×Q5 Mastermix. The PCR primers are shown below: F1 primer: ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCCATCCAGCTTCCTTCGTGT R1 primer: ACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNtGGGAGTCTCTCTTCCCACCT The PCR reaction system is as follows: The PCR procedure is as follows: The second round of PCR for sequencing library preparation was performed using a 2×Q5 Mastermix. The PCR primers are shown below: F2 primer: AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGAC; R2 primer: CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTGACTGGAGTTCAGACGTGTG.

[0172] The PCR reaction system is as follows: The PCR procedure is as follows: The second-round PCR products were purified into DNA fragments of the target size using a gel extraction kit following the manufacturer's instructions, and the next-generation sequencing library was prepared.

[0173] (8) Analysis of second-generation sequencing results The prepared next-generation sequencing library was subjected to paired-end sequencing on the high-throughput sequencer HiseqXTen (illumina).

[0174] The editing efficiency of PspCas12f1 and nine modified PspCas12f1-mutants at the same endogenous site, as calculated by next-generation sequencing, is as follows: Figure 3 As shown, the X-axis represents the target site, and the Y-axis represents the editing efficiency (Indels%). From Figure 3As can be seen, compared with the unmodified PspCas12f1, the editing efficiency of mutants Q97R, N108R, S157L, P191R, I194R, and P211R is reduced to varying degrees. Conversely, mutants Q100R, E293R, and the double mutant enPspCas12f1 (E293R & Q100R) all show significantly improved editing efficiency. Among them, enPspCas12f1 has the highest efficiency, significantly better than wild-type and other single-point mutants. This indicates that in this invention, by rationally modifying the key amino acid sites of PspCas12f1, especially by introducing the combined mutation of E293R and Q100R, the resulting enPspCas12f1 can significantly improve gene editing performance, thus effectively serving as a gene editing agent for cells.

[0175] Example 3 (1) Construct plasmids pAAV2_PspCas12f1_ITR and pAAV2_enPspCas12f1_ITR Genes of SEQ ID NO: 3 and SEQ ID NO: 4 were synthesized and constructed into the pSpCas9(BB)-2A-Puro(PX459) backbone plasmid V2.0 (Addgene platform, catalog#62988) to obtain plasmids pAAV2_PspCas12f1_ITR and pAAV2_enPspCas12f1_ITR.

[0176] (2) Constructing plasmid Cas12f1-PSK-hU6-sgRNA-scaffold Using a method similar to that in Example 1 (2), the plasmid Cas12f1-PSK-hU6-sgRNA-scaffold was prepared using the DNA sequence of the sgRNA scaffold (SEQ ID NO: 6).

[0177] (3) Construction of plasmid pAAV2_PspCas12f1-hU6-sgRNA-scaffold_ITR and pAAV2_enPspCas12f1-hU6-sgRNA-scaffold_ITR vector Using a method similar to that in Example 1 (3), plasmids pAAV2_PspCas12f1-hU6-sgRNA-scaffold_ITR and pAAV2_enPspCas12f1-hU6-sgRNA-scaffold_ITR were prepared using the following primer sequences for later use.

[0178] For the pAAV2_PspCas12f1_ITR plasmid and the pAAV2_enPspCas12f1_ITR plasmid, the primer sequences are as follows: ccgcaggaacccctagtgatTTTTTGCGGCCGCAGGAA; and ggacgcaaagccgccctgtaGGTGTTTCGTCCTTCCACA; For the Cas12f1-PSK-hU6-sgRNA-scaffold plasmid, the primer sequences are as follows: gtacagggcggctttgcgt; and atcactaggggttcctgcg.

[0179] (4) Preparation of linearized plasmids pAAV2_PspCas12f1-hU6-sgRNA-scaffold_ITR and pAAV2_enPspCas12f1-hU6-sgRNA-scaffold_ITR Linearized plasmids pAAV2_PspCas12f1-hU6-sgRNA-scaffold_ITR and pAAV2_enPspCas12f1-hU6-sgRNA-scaffold_ITR were prepared using a method similar to that in Example 1 (4), and the DNA concentration was determined using a NanoDrop™ Lite spectrophotometer (Thermo Scientific). The plasmids were then stored for later use or at -20°C for long-term preservation.

[0180] (5) Preparation of plasmids pAAV2_PspCas12f1-hU6-sgRNA_ITR and pAAV2_enPspCas12f1-hU6-sgRNA_ITR Each gRNA was designed, and its sequence is shown in Table 1. Sticky end sequences (indicated by uppercase letters) corresponding to the linearized plasmids pAAV2_PspCas12f1-hU6-sgRNA_ITR and pAAV2_enPspCas12f1-hU6-sgRNA_ITR were added to the sense and antisense strands, respectively. These oligonucleotide single-stranded DNAs were then synthesized, and their specific sequences are also shown in Table 1 below.

[0181] Table 1. Sequences of gRNA and oligonucleotide single-stranded DNA Then, using a method similar to that in Example 1 (5), plasmids pAAV2_PspCas12f1-hU6-sgRNA_ITR and pAAV2_enPspCas12f1-hU6-sgRNA_ITR containing the target sgRNA sequence were prepared for later use.

[0182] (6) Transfection of HEK293T cell line with plasmids pAAV2_PspCas12f1-hU6-sgRNA_ITR expressing PspCas12f1 protein and sgRNA and pAAV2_enPspCas12f1-hU6-sgRNA_ITR expressing enPspCas12f1 protein and sgRNA. Using a method similar to that in Example 2 (6), the plasmids pAAV2_PspCas12f1-hU6-sgRNA_ITR expressing PspCas12f1 protein and sgRNA and pAAV2_enPspCas12f1-hU6-sgRNA_ITR expressing enPspCas12f1 protein and sgRNA were transfected into the HEK293T cell line.

[0183] (7) Preparation of next-generation sequencing libraries HEK293T cells were collected five days after editing, and genomic DNA was extracted using a DNA kit (Tiangen Biotech (Beijing) Co., Ltd., DP304) according to the instructions provided with the DNA kit.

[0184] The first round of PCR for library preparation was performed using a 2×Q5 Mastermix. The PCR primers are shown in Table 2. Table 2. List of primers for the first round of PCR in next-generation sequencing The PCR reaction system is as follows: The PCR procedure is as follows: The second round of PCR for sequencing library preparation was performed using a 2xQ5 Mastermix. The PCR primers are shown below: F2 primer: AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGAC; R2 primer: CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTGACTGGAGTTCAGACGTGTG.

[0185] The PCR reaction system is as follows: The PCR procedure is as follows: The second-round PCR products were purified into DNA fragments of the target size using a gel extraction kit following the manufacturer's instructions, and the next-generation sequencing library was prepared.

[0186] (8) Analysis of second-generation sequencing results The prepared next-generation sequencing library was subjected to paired-end sequencing on the high-throughput sequencer HiseqXTen (illumina).

[0187] The editing efficiency of PspCas12f1 on six target sites, calculated by next-generation sequencing, is as follows: Figure 4 As shown, the X-axis represents the target site, and the Y-axis represents the editing efficiency (Indels%). From Figure 4 As can be seen, the unengineered PspCas12f1 protein exhibited editing activity at all six target sites, with editing efficiencies ranging from 23% to 76%. This result preliminarily confirms that PspCas12f1 possesses the basic ability to perform gene editing in mammalian cells. Furthermore, protein engineering optimization of PspCas12f1 was performed, resulting in the enhanced mutant enPspCas12f1. Figure 5 As shown, enPspCas12f1 exhibited significantly improved editing efficiency compared to wild-type PspCas12f1 protein at all tested sites, with a minimum editing efficiency of 63% and a maximum of 98%, representing an average improvement of more than two times. These results demonstrate that both the CRISPR / PspCas12f1 gene editing system and the CRISPR / enPspCas12f1 gene editing system of this invention can be effectively used for cellular gene editing, with the CRISPR / enPspCas12f1 gene editing system showing superior gene editing performance.

[0188] Example 4 (1)-(4) Linearized plasmids pAAV2_PspCas12f1-hU6-sgRNA-scaffold_ITR and pAAV2_enPspCas12f1-hU6-sgRNA-scaffold_ITR were prepared using the same method as in Examples 3(1)-(4).

[0189] (5) Preparation of plasmids pAAV2_PspCas12f1-hU6-on target sgRNA_ITR, pAAV2_PspCas12f1-hU6-mismatch-sgRNA_ITR, pAAV2_enPspCas12f1-hU6-on target sgRNA_ITR, and pAAV2_enPspCas12f1-hU6-mismatch-sgRNA_ITR The sequences of each on-target gRNA and mismatch gRNA were designed, and their corresponding oligonucleotide single-stranded DNA are shown in Table 3. The mismatch bases are shown as underlined uppercase bold bases in the sequence listing.

[0190] Table 3. Oligonucleotide single-stranded DNA corresponding to on target gRNA and mismatch gRNA The oligonucleotide single-stranded DNA corresponding to the obtained on-target gRNA and the oligonucleotide single-stranded DNA corresponding to different mismatch gRNAs were annealed separately. The annealing reaction system was: 3 μL 10 μM oligo-F, 3 μL 10 μM oligo-R, and 4 μL water. After vortexing and mixing the annealing system, it was placed in a PCR instrument and the annealing program was run as follows: 95℃_5 min, 85℃_1 min, 75℃_1 min, 65℃_1 min, 55℃_1 min, 45℃_1 min, 35℃_1 min, 25℃_1 min, stored at 4℃, and the cooling rate was 0.3℃ / s. After annealing, the resulting products were ligated into the linearized pAAV2_PspCas12f1-hU6-sgRNA_scaffold_ITR plasmid and pAAV2_enPspCas12f1-hU6-sgRNA_scaffold_ITR plasmid respectively using DNA ligase (purchased from NEB).

[0191] 1 μL of the obtained ligation product was added to Escherichia coli DH5α competent cells (purchased from Shanghai Weidi Biotechnology Co., Ltd.), incubated on ice for 30 min, heat-shocked at 42℃ for 1 min, incubated on ice for 2 min, and then 900 μL of LB medium was added. The cells were cultured at 37℃ for 1 h to activate and reactivate Escherichia coli DH5α competent cells.

[0192] The revived Escherichia coli DH5α competent cells were plated on LB solid plates containing the corresponding antibiotics and incubated upside down in a 37°C incubator. The obtained Escherichia coli DH5α monoclonal cells were then verified by Sanger sequencing.

[0193] The correctly ligated *E. coli* DH5α clones were subjected to culture in a shaking process, and then plasmids were extracted to obtain plasmids pAAV2_PspCas12f1-hU6-on target-sgRNA_ITR and pAAV2_enPspCas12f1-hU6-on target-sgRNA_ITR expressing the above-mentioned on target gRNA sequences, and plasmids pAAV2_PspCas12f1-hU6-mismatch-sgRNA_ITR and pAAV2_enPspCas12f1-hU6-mismatch-sgRNA_ITR expressing the above-mentioned different mismatch gRNA sequences, which were then used for future purposes.

[0194] (6) Transfection of HEK293T cell line with plasmids pAAV2_PspCas12f1-hU6-ontarget-sgRNA_ITR and pAAV2_enPspCas12f1-hU6-on target-sgRNA_ITR expressing Cas12f1 protein and on-target-sgRNA, as well as plasmids pAAV2_PspCas12f1-hU6-mismatch-sgRNA_ITR and pAAV2_enPspCas12f1-hU6-mismatch-sgRNA_ITR expressing Cas12f1 protein and mismatch-sgRNA. Using a method similar to that in Example 2 (6), the obtained plasmids pAAV2_PspCas12f1-hU6-on target-sgRNA_ITR and pAAV2_enPspCas12f1-hU6-ontarget-sgRNA_ITR expressing Cas12f1 protein and on target gRNA sequence, and plasmids pAAV2_PspCas12f1-hU6-mismatch-sgRNA_ITR and pAAV2_enPspCas12f1-hU6-mismatch-sgRNA_ITR expressing Cas12f1 protein and mismatch gRNA sequence, were transfected into the HEK293T cell line containing the target sequence of the GFP reporter system.

[0195] (7) Preparation of next-generation sequencing libraries HEK293T cells were collected five days after editing, and genomic DNA was extracted using a DNA kit (Tiangen Biotech (Beijing) Co., Ltd., DP304) according to the instructions provided with the DNA kit.

[0196] The first round of PCR for library preparation was performed using a 2×Q5 Mastermix. The PCR primers are shown below: F1 primer: CTGTAAAGTGGAGTTAATGACAGAC R1 primer: TCCTTTCATTCGTGTGGCTCT The PCR reaction system is as follows: The PCR procedure is as follows: The second round of PCR for sequencing library preparation was performed using a 2×Q5 Mastermix. The PCR primers are shown below: F2 primer: AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGAC; R2 primer: CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTGACTGGAGTTCAGACGTGTG.

[0197] The PCR reaction system is as follows: The PCR procedure is as follows: The second-round PCR products were purified into DNA fragments of the target size using a gel extraction kit following the manufacturer's instructions, and the next-generation sequencing library was prepared.

[0198] (8) Analysis of second-generation sequencing results The prepared next-generation sequencing library was subjected to paired-end sequencing on the high-throughput sequencer HiseqXTen (illumina).

[0199] Next-generation sequencing calculations yielded the specificity of PspCas12f1 at a single target site, such as... Figure 6 As shown, the X-axis represents the base mismatch positions, and the Y-axis represents the editing efficiency (Indels%). From Figure 6It can be seen that PspCas12f1 only exhibits detectable off-target activity when a single or double base mismatch occurs at position 19 or 20 of the gRNA sequence. No significant editing activity was observed when mismatches occurred at other positions, indicating that PspCas12f1 has high specificity. Furthermore, the specificity of enPspCas12f1 at the same target site was also analyzed using next-generation sequencing, and the results are presented below. Figure 7 .from Figure 7 It can be seen that when a single base mismatch occurs in the sgRNA sequence of enPspCas12f1 at positions 8 to 20, it can induce a certain degree of off-target editing activity; however, under the condition of a double base mismatch, a detectable off-target effect is only observed when the mismatch is located at positions 17 to 20. Therefore, compared with PspCas12f1, the specificity of enPspCas12f1 is somewhat reduced, but its overall specificity remains at a high level.

[0200] Example 5 (1) Construct plasmids pAAV2_enPspCas12f1_ITR, pAAV2_SpaCas12f1_ITR and pAAV2_enOsCas12f1_ITR Genes of the enPspCas12f1 gene sequence shown in SEQ ID NO: 3 and the known SpaCas12f1 (amino acid sequence shown in SEQ ID NO: 11, nucleic acid sequence shown in SEQ ID NO: 13) and enOsCas12f1 (amino acid sequence shown in SEQ ID NO: 12, nucleic acid sequence shown in SEQ ID NO: 14) were synthesized and constructed onto the pSpCas9(BB)-2A-Puro(PX459) backbone plasmid V2.0 (Addgene platform, catalog#62988) to obtain plasmids pAAV2_enPspCas12f1_ITR, pAAV2_SpaCas12f1_ITR and pAAV2_enOsCas12f1_ITR.

[0201] (2) Construct plasmids enPspCas12f1-PSK-hU6-sgRNA-scaffold, SpaCas12f1-PSK-hU6-sgRNA-scaffold and enOsCas12f1-PSK-hU6-sgRNA-scaffold Using a method similar to that in Example 1 (2), the DNA sequences of the enPspCas12f1-sgRNA scaffold (SEQ ID NO: 6) and the known DNA sequences of the SpaCas12f1-sgRNA scaffold (SEQ ID NO: 15) (SEQ ID NO: 17) and the enOsCas12f1-sgRNA scaffold (SEQ ID NO: 16) (SEQ ID NO: 18) were used to prepare plasmids enPspCas12f1-PSK-hU6-sgRNA-scaffold, SpaCas12f1-PSK-hU6-sgRNA-scaffold, and enOsCas12f1-PSK-hU6-sgRNA-scaffold.

[0202] (3) Construction of plasmids pAAV2_enPspCas12f1-hU6-sgRNA-scaffold_ITR, pAAV2_SpaCas12f1-hU6-sgRNA-scaffold_ITR and pAAV2_enOsCas12f1-hU6-sgRNA-scaffold_ITR vectors Using a method similar to that in Example 1 (3), plasmids pAAV2_enPspCas12f1-hU6-sgRNA-scaffold_ITR, pAAV2_SpaCas12f1-hU6-sgRNA-scaffold_ITR, and pAAV2_enOsCas12f1-hU6-sgRNA-scaffold_ITR were prepared using the following primer sequences and are ready for use.

[0203] For the pAAV2_enPspCas12f1_ITR plasmid, the primer sequences are as follows: ccgcaggaacccctagtgatTTTTTGCGGCCGCAGGAA; and ggacgcaaagccgccctgtaGGTGTTTCGTCCTTCCACA; For the enPspCas12f1-PSK-hU6-sgRNA-scaffold plasmid, the primer sequences are as follows: gtacagggcggctttgcgt; and atcactaggggttcctgcg.

[0204] For the pAAV2_SpaCas12f1_ITR plasmid, the primer sequences are as follows: gtttcaatccacgcgcttacTTTTTGCGGCCGCAGGAACC; and actgccctggcgcgcgaaacGGTGTTTCGTCCTTTCCA; For the SpaCas12f1-PSK-hU6-sgRNA-scaffold plasmid, the primer sequences are as follows: gtttcgcgcgccagggcagt; and gtttcaatccacgcgcttac.

[0205] For the pAAV2_enOsCas12f1_ITR plasmid, the primer sequences are as follows: ccgcgaaagcggcttgaaggTTTTTTGCGGCCGCAGGAACC; and tgggccgggaagtcggccctGGTGTTTCGTCCTTCCACC; For the enOsCas12f1-PSK-hU6-sgRNA-scaffold plasmid, the primer sequences are: agggccgacttcccggccca; and ccttcaagccgctttcgcgg.

[0206] (4) Preparation of linearized plasmids pAAV2_enPspCas12f1-hU6-sgRNA-scaffold_ITR, pAAV2_SpaCas12f1-hU6-sgRNA-scaffold_ITR and pAAV2_enOsCas12f1-hU6-sgRNA-scaffold_ITR Linearized plasmids pAAV2_enPspCas12f1-hU6-sgRNA-scaffold_ITR, pAAV2_SpaCas12f1-hU6-sgRNA-scaffold_ITR, and pAAV2_enOsCas12f1-hU6-sgRNA-scaffold_ITR were prepared using a method similar to that in Example 1 (4). The DNA concentration was measured using a NanoDrop™ Lite spectrophotometer (ThermoScientific) for later use or for long-term storage at -20°C.

[0207] (5) Preparation of plasmids pAAV2_enPspCas12f1-hU6-sgRNA_ITR, pAAV2_SpaCas12f1-hU6-sgRNA_ITR and pAAV2_enOsCas12f1-hU6-sgRNA_ITR Each gRNA was designed, and its sequence is shown in Table 4. Sticky end sequences (indicated by uppercase letters) corresponding to the linearized plasmids pAAV2_enPspCas12f1-hU6-sgRNA_ITR, pAAV2_SpaCas12f1-hU6-sgRNA_ITR, and pAAV2_enOsCas12f1-hU6-sgRNA_ITR were added to the sense and antisense strands, respectively. These oligonucleotide single-stranded DNAs were then synthesized, and their specific sequences are also shown in Table 4 below.

[0208] Table 4. Sequences of gRNA and oligonucleotide single-stranded DNA Then, using a method similar to that in Example 1 (5), plasmids pAAV2_enPspCas12f1-hU6-sgRNA_ITR, pAAV2_SpaCas12f1-hU6-sgRNA_ITR, and pAAV2_enOsCas12f1-hU6-sgRNA_ITR containing the target sgRNA sequence were prepared for later use.

[0209] (6) Transfection of HEK293T cell line with plasmids pAAV2_enPspCas12f1-hU6-sgRNA_ITR expressing enPspCas12f1 protein and sgRNA, plasmid pAAV2_SpaCas12f1-hU6-sgRNA_ITR expressing SpaCas12f1 protein and sgRNA, and plasmid pAAV2_enOsCas12f1-hU6-sgRNA_ITR expressing enOsCas12f1 protein and sgRNA. Using a method similar to that in Example 2 (6), plasmids pAAV2_enPspCas12f1-hU6-sgRNA_ITR expressing enPspCas12f1 protein and sgRNA, pAAV2_SpaCas12f1-hU6-sgRNA_ITR expressing SpaCas12f1 protein and sgRNA, and pAAV2_enOsCas12f1-hU6-sgRNA_ITR expressing enOsCas12f1 protein and sgRNA were transfected into the HEK293T cell line.

[0210] (7) Preparation of next-generation sequencing libraries HEK293T cells were collected five days after editing, and genomic DNA was extracted using a DNA kit (Tiangen Biotech (Beijing) Co., Ltd., DP304) according to the instructions provided with the DNA kit.

[0211] The first round of PCR for library preparation was performed using a 2×Q5 Mastermix. The PCR primers are shown in Table 5. Table 5. List of primers for the first round of PCR in next-generation sequencing The PCR reaction system is as follows: The PCR procedure is as follows: The second round of PCR for sequencing library preparation was performed using a 2×Q5 Mastermix. The PCR primers are shown below: F2 primer: AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGAC; R2 primer: CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTGACTGGAGTTCAGACGTGTG.

[0212] The PCR reaction system is as follows: The PCR procedure is as follows: The second-round PCR products were purified into DNA fragments of the target size using a gel extraction kit following the manufacturer's instructions, and the next-generation sequencing library was prepared.

[0213] (8) Analysis of second-generation sequencing results The prepared next-generation sequencing library was subjected to paired-end sequencing on a high-throughput sequencer, HiseqXTen (Illumina). The editing efficiencies of enPspCas12f1, SpaCas12f1, and enOsCas12f1 at the six target sites were calculated using next-generation sequencing. Figure 8 As shown, the X-axis represents the target site, and the Y-axis represents the editing efficiency (Indels%). From Figure 8As can be seen, at six different endogenous sites, the editing efficiency of enPspCas12f1 is significantly higher than that of the existing SpaCas12f1 and enOsCas12f1. This result fully demonstrates that the CRISPR / enPspCas12f1 gene editing system constructed in this invention can be effectively used for cellular gene editing. Compared with existing CRISPR / Cas12f1 gene editing systems, enPspCas12f1, modified based on PspCas12f1, significantly improves editing performance while maintaining a small molecular weight for easy delivery, demonstrating its great potential in the field of gene therapy, especially in in vivo applications.

[0214] The sequences used in this invention: SEQ ID NO: 1: PspCas12f1 protein sequence MSIKAIRLEILKPYNEPDTATPVTWNELGQVLRDVRYACSKTENYAITKCYLWEQFKIEYKNNNGIFPNAKDFKEMTDLYSQLTAMFPDVAAAIVNQADQVATRKWNNEKKDVLSL RRSLTSFKLDVPIPIHNKSYKLRKISEDNKIIYVINVNLLSKKSERQTTYSMVLKVKDNSSKTILDRLINKELSPKGIQIIGSNKNRQKWFCLIPYDFTEKDTELNPDRIMGIDLG IAKAVYYAFSDSYKRGYIEGGEIEHFRKSVRARRIAIQNQGKYCGDGGRIGHGVKRRLQPVEALREKEKNFRNLTNHRYARRLVEVAVKNHCGVIQMEDLTSITKDNTFLKDWPYYD LQTKIAEKASEYGIVFKKINPYKTSQRCSRCGYIDSENRPEQSVFVCRECGYGNMYLCEDCNKEQNHAGKCDSCGGVTNLITVNADYNAAKNIATKDIEQIIKKTMGKDYNPPKKK SEQ ID NO: 2: enPspCas12f1 protein sequence MSIKAIRLEILKPYNEPDTATPVTWNELGQVLRDVRYACSKTENYAITKCYLWEQFKIEYKNNNGIFPNAKDFKEMTDLYSQLTAMFPDVAAAIVNQADRVATRKWNNEKKDVLSLRRSLTSFKLDVPIPIHNKSYKLRKISEDNKIIYVINVNLLSKKSERQTTYSMVLKVKDNSSKTILDRLINKELSPKGIQIIGSNKNRQKWFCLIPYDFTEKDTELNPDRIMGIDLGIAKAVYYAFSDSYKRGYIEGGEIEHFRKSVRARRIAIQNQGKYCGDGRIGHGVKRRLQPVRALREKEKNFRNLTNHRYARRLVEVAVKNHCGVIQMEDLTSITKDNTFLKDWPYYDLQTKIAEKASEYGIVFKKINPYKTSQRCSRCGYIDSENRPEQSVFVCRECGYGNMYLCEDCNKEQNHAGKCDSCGGVTNLITVNADYNAAKNIATKDIEQIIKKTMGKDYNPPKKK SEQ ID NO: 3: Coding sequence of PspCas12f1 protein SEQ ID NO: 4: The coding sequence of the enPspCas12f1 protein SEQ ID NO: 5: Sequence of a single-stranded guide RNA scaffold used in conjunction with PspCas12f1 or enPspCas12f1 protein. UACAGGGCGGCUUUGCGUCCUUAAAGCGAGAAGUGUCAGCUUUUAUGCUGAUAACGCUCGCUAAGUGAUUAUGUGCGAACAUAAGUCACACAUCUGCUUUUAGCAGACAAUUUACAGUUGCAGAAAGUAACAACUUAUGUGCAGCUGUGAAUUGAAGA SEQ ID NO: 6: DNA sequence of a single-stranded guide RNA scaffold sequence used in conjunction with PspCas12f1 protein or enPspCas12f1 protein. TACAGGGCGGCTTTGCGTCCTTAAAGCGAGAAGTGTCAGCTTTTATGCTGATAACGCTCGCTAAGTGATTATGTGCGAACATAAGTCACACATCTGCTTTTTAGCAGACAATTTACAGTTGCAGAAAGTAACAACTTATGTGCAGCTGTGAATTGAAGA SEQ ID NO: 7: PspCas12f1-Q100R mutant protein sequence MSIKAIRLEILKPYNEPDTATPVTWNELGQVLRDVRYACSKTENYAITKCYLWEQFKIEYKNNNGIFPNAKDFKEMTDLYSQLTAMFPDVAAAIVNQADRVATRKWNNEKKDVLSLRRSLTSFKLDVPIPIHNKSYKLRKISEDNKIIYVINVNLLSKKSERQTTYSMVLKVKDNSSKTILDRLINKELSPKGIQIIGSNKNRQKWFCLIPYDFTEKDTELNPDRIMGIDLGIAKAVYYAFSDSYKRGYIEGGEIEHFRKSVRARRIAIQNQGKYCGDGRIGHGVKRRLQPVEALREKEKNFRNLTNHRYARRLVEVAVKNHCGVIQMEDLTSITKDNTFLKDWPYYDLQTKIAEKASEYGIVFKKINPYKTSQRCSRCGYIDSENRPEQSVFVCRECGYGNMYLCEDCNKEQNHAGKCDSCGGVTNLITVNADYNAAKNIATKDIEQIIKKTMGKDYNPPKKK SEQ ID NO: 8: PspCas12f1-E293R mutant protein sequence MSIKAIRLEILKPYNEPDTATPVTWNELGQVLRDVRYACSKTENYAITKCYLWEQFKIEYKNNNGIFPNAKDFKEMTDLYSQLTAMFPDVAAAIVNQADQVATRKWNNEKKDVLSLRRSLTSFKLDVPIPIHNKSYKLRKISEDNKIIYVINVNLLSKKSERQTTYSMVLKVKDNSSKTILDRLINKELSPKGIQIIGSNKNRQKWFCLIPYDFTEKDTELNPDRIMGIDLGIAKAVYYAFSDSYKRGYIEGGEIEHFRKSVRARRIAIQNQGKYCGDGRIGHGVKRRLQPVRALREKEKNFRNLTNHRYARRLVEVAVKNHCGVIQMEDLTSITKDNTFLKDWPYYDLQTKIAEKASEYGIVFKKINPYKTSQRCSRCGYIDSENRPEQSVFVCRECGYGNMYLCEDCNKEQNHAGKCDSCGGVTNLITVNADYNAAKNIATKDIEQIIKKTMGKDYNPPKKK SEQ ID NO: 9: Coding sequence of the PspCas12f1-Q100R mutant protein SEQ ID NO: 10: Coding sequence of the PspCas12f1-E293R mutant protein SEQ ID NO: 11: SpaCas12f1 protein sequence MGESVKAIKLKILDMFLDPECTKQDDNWRKDLSTMSRFCAEAGNMCLRDLYNYFSMPKEDRISSKDLYNAMYHKTKLLHPELPGKVANQIVNHAKDVWKRNAKLIYRNQISMPTYKITTAPIRLQNNIYKLIKNKNKYIIDVQLYSKEYSKDSGKGTHRYFLVAVRDSSTRMIFDRIMSKDHIDSSKSYTQGQLQIKKDHQGKWYCIIPYTFPTHETVLDPDKVMGVDLGVAKAVYWAFNSSYKRGCIDGGEIEHFRKMIRARRVSIQNQIKHSGDARKGHGRKRALKPIETLSEKEKNFRDTINHRYANRIVEAAIKQGCGTIQIENLEGIADTTGSKFLKNWPYYDLQTKIVNKAKEHGITVVAINPQYTSQRCSMCGYIEKTNRSSQAVFECKQCGYGSRTICINCRHVQVSGDVCEECGGIVKKENVNADYNAAKNISTPYIDQIIMEKCLELGIPYRSITCKECGHIQASGNTCEVCGSTNILKPKKIRKAK SEQ ID NO: 12: enOsCas12f1 protein sequence MGKGVLAKVMKYELRYLDGCGDFSNMQEQVWALQRQTREILNRSIQIAFQWRCANSEHHRKTGEYLDLKTETGYKRLDGHIYNCLKGQYEDMATSNLNATIQKAWKKYNSSKKEILRGSMSIPSYKMNQPLRLDKNTVKLSEGERNPIVTLTLFSDKFKRAQGVSNVKFSMPLHDGTQRAIFANLMNGTYQLGECQLVYKRPKWFLFVTYKFPPVEHPLDPDKILGVDMGEACALYASTFGEHGYLKIDGGEITKYAKKMEARIRSMQKQAAHCGEGRIGHGTKTRVSVVYQAKDKVARFRDTINHRYSKALIDYALKNQCGTIQMEDLTGIKEDTGFPKFLRHWTYYDLQSKIEAKAAEHGIQVVKINPRHTSQRCSRCGHIDKANRTSQADFCCTKCGFSANADFNASQNISIRNIDKIIAKAIGANRKQT SEQ ID NO: 13: Coding sequence of SpaCas12f1 protein SEQ ID NO: 14: Coding sequence of enOsCas12f1 protein SEQ ID NO: 15: Sequence of a single-stranded guide RNA scaffold used in conjunction with the SpaCas12f1 protein GUUUCGCGCCCAGGGCAGUUAGGUGCCCUAAAAGAGCGAAGUGGCCGAAAGGAAAGGCUAACGCUUCUCUAACGCUACGGCGACCUUGGCGAAAUGCCAUCAAUACCACGCGGCCCGAAAGGGUUCGCGCGAAACAAGGUAAGCGCGUGGAUUGAAAC SEQ ID NO: 16: Sequence of a single-stranded guide RNA scaffold used in conjunction with the enOsCas12f1 protein AGGGCCGACUUCCCGGCCCAAAAUCGAGACAGUAGCCGUAAAACGUUGAGUUUCAGCGUGGGCGACACACUCGAAAAGGUUAAGAUAUGCACAUAGUAAUCCGUGCAUGAGCCGCGAAAGCGGCUUGAAGG SEQ ID NO: 17: DNA sequence of a single-stranded guide RNA scaffold sequence used in conjunction with SpaCas12f1 protein. GTTTCGCGCGCCAGGGCAGTTAGGTGCCCTAAAAGAGCGAAGTGGCCGAAAGGAAAGGCTAACGCTTCTCTAACGCTACGGCCGACCTTGGCGAAATGCCATCAATACCACGCGGCCCGAAAGGGTTCGCGCGAAACAAGGTAAGCGCGTGGATTGAAAC SEQ ID NO: 18: DNA sequence of a single-stranded guide RNA scaffold sequence used in conjunction with enOsCas12f1 protein. AGGGCCGACTTCCCGGCCCAAAATCGAGACAGTAGCCGTAAAACGTTGAGTTTCAGCGTGGGCGACACACTCGAAAAGGTTAAGATATGCACATAGTAATCCGTGCATGAGCCGCGAAAGCGGCTTGAAGG

Claims

1. A PspCas12fl mutant protein comprising a mutation at Q100 and / or E293 relative to a wild-type PspCas12fl protein having an amino acid sequence set forth in SEQ ID NO:

1. Preferably, the mutation comprises Q100R, E293R, or Q100R and E293R.

2. A conjugate comprising: a) a Cas12fl protein or a homolog thereof, wherein: the Cas12fl protein is a wild-type PspCas12fl protein having an amino acid sequence set forth in SEQ ID NO: 1 or a PspCas12fl mutant protein of claim 1, the homolog is a homolog having a sequence identity of at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.99%, or at least 99.999% to the amino acid sequence of the Cas12fl protein and retains a biological activity of the Cas12fl protein; b) a modification moiety; for example, the modification moiety is selected from the group consisting of another protein or polypeptide, a detectable label, or a combination thereof; for example, the another protein or polypeptide is selected from the group consisting of one or more of an epitope tag, a reporter protein, or a nuclear localization signal (NLS) sequence, a cytosine deaminase (CBE), an adenine deaminase (ABE), a reverse transcriptase, a cytosine methylase DNMT3A and MQ1, a cytosine demethylase Tetl, a transcriptional activator protein VP64, p65 and RTA, a transcriptional repressor protein KRAB, a histone acetylase p300, a histone deacetylase LSDl, and an endonuclease Fokl; and c) an optional linker for connecting the Cas12fl protein or the homolog thereof and the modification moiety.

3. A fusion protein comprising: a) a Cas12fl protein or a homolog thereof, wherein: the Cas12fl protein is a wild-type PspCas12fl protein having an amino acid sequence set forth in SEQ ID NO: 1 or a PspCas12fl mutant protein of claim 1, the homolog is a homolog having a sequence identity of at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.99%, or at least 99.999% to the amino acid sequence of the Cas12fl protein and retains a biological activity of the Cas12fl protein. the homolog has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.99%, or at least 99.999% sequence identity to the amino acid sequence of the Cas12f1 protein and retains the biological activity of the Cas12f1 protein; b) a further protein or polypeptide; For example, the further protein or polypeptide is selected from one or more of an epitope tag, a reporter protein, or a nuclear localization signal (NLS) sequence, a cytosine deaminase (CBE), an adenine deaminase (ABE), a reverse transcriptase, a cytosine methylase DNMT3A and MQ1, a cytosine demethylase Tet1, a transcriptional activator VP64, p65 and RTA, a transcriptional repressor KRAB, a histone acetylase p300, a histone deacetylase LSD1, and an endonuclease Fokl; and c) an optional linker for connecting the Cas12f1 protein or the homolog thereof to the further protein or polypeptide.

4. A single guide RNA comprising a CRISPR scaffold sequence, the CRISPR scaffold sequence having: a) the nucleic acid sequence set forth in SEQ ID NO: 5; b) a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 5 and retaining the biological activity of SEQ ID NO: 5; or c) a nucleic acid sequence engineered based on the nucleic acid sequence set forth in SEQ ID NO: 5 and retaining the biological activity of SEQ ID NO: 5, For example, the engineering is one or more of base phosphorylation, base sulfuration, base methylation, base hydroxylation, sequence shortening, and sequence lengthening. For example, the sequence shortening and the sequence lengthening include deletion or addition of one, two, three, four, five, six, seven, eight, nine, or ten bases relative to the base sequence.

5. The single guide RNA of claim 4, wherein, The single guide RNA further comprises a CRISPR spacer sequence at the 3’ end of the CRISPR scaffold sequence, the CRISPR spacer sequence being a sequence of 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 nucleotides in length (preferably 20 nucleotides in length) and capable of complementary pairing to a target sequence.

6. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the PspCas12fl mutant protein of claim 1 or a homolog thereof, the conjugate of claim 2, or the fusion protein of claim 3, wherein: the homolog is a homolog having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.99%, or at least 99.999% sequence identity to the amino acid sequence of the PspCas12fl mutant protein and retains the biological activity of the PspCas12fl mutant protein; for example, the isolated nucleic acid molecule comprises the nucleic acid sequence set forth in any one of SEQ ID NO: 4, SEQ ID NO: 9, or SEQ ID NO: 10, or a degenerate sequence thereof.

7. The isolated nucleic acid molecule of claim 6, wherein the isolated nucleic acid molecule further comprises a nucleic acid sequence encoding the single guide RNA of any one of claims 4-5. for example, the isolated nucleic acid molecule comprises the nucleic acid sequence set forth in any one of SEQ ID NO: 4, SEQ ID NO: 9, or SEQ ID NO: 10, or a degenerate sequence thereof, and the isolated nucleic acid molecule further comprises the nucleic acid sequence set forth in SEQ ID NO:

6.

8. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the single guide RNA of any one of claims 4-5. for example, the isolated nucleic acid molecule comprises the nucleic acid sequence set forth in SEQ ID NO: 6, and preferably further comprises a nucleic acid sequence encoding a CRISPR spacer sequence.

9. The isolated nucleic acid molecule of claim 8, wherein the isolated nucleic acid molecule further comprises a nucleic acid sequence encoding the wild-type PspCas12fl protein set forth in SEQ ID NO: 1 or a homolog thereof, wherein: the homolog has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.99%, or at least 99.999% sequence identity to the amino acid sequence of the wild-type PspCas12fl protein and retains the biological activity of the wild-type PspCas12fl protein; For example, the isolated nucleic acid molecule comprises the nucleic acid sequence set forth in SEQ ID NO: 6 and the nucleic acid sequence set forth in SEQ ID NO: 3 or a degenerate sequence thereof.

10. A vector comprising a nucleic acid sequence encoding the PspCas12fl mutant protein of claim 1 or a homolog thereof, the conjugate of claim 2, or the fusion protein of claim 3, wherein: the homolog has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.99%, or at least 99.999% sequence identity to the amino acid sequence of the PspCas12fl mutant protein and retains the biological activity of the PspCas12fl mutant protein; For example, the vector comprises the nucleic acid sequence set forth in any one of SEQ ID NO: 4, SEQ ID NO: 9, or SEQ ID NO: 10 or a degenerate sequence thereof. For example, the vector is a plasmid vector, such as a pUC19 vector, an episomal vector, a pAAV2_ITR vector, a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector.

11. The vector of claim 10, wherein, the vector further comprises a nucleic acid sequence encoding the single guide RNA of any one of claims 4-5; For example, the vector comprises the nucleic acid sequence set forth in any one of SEQ ID NO: 4, SEQ ID NO: 9, or SEQ ID NO: 10 or a degenerate sequence thereof, and the vector further comprises the nucleic acid sequence set forth in SEQ ID NO:

6.

12. A vector comprising a nucleic acid sequence encoding the single guide RNA of any one of claims 4 to 5; For example, the vector comprises the nucleic acid sequence set forth in SEQ ID NO: 6, and preferably further comprises a nucleic acid sequence encoding a CRISPR spacer sequence.

13. The vector of claim 12, wherein the vector further comprises a nucleic acid sequence encoding a wild-type PspCas12fl protein as set forth in SEQ ID NO: 1 or a homolog thereof, wherein: the homolog is a homolog having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.99%, or at least 99.999% sequence identity to the amino acid sequence of the wild-type PspCas12fl protein and retains the biological activity of the wild-type PspCas12fl protein; For example, the vector comprises the nucleic acid sequence set forth in SEQ ID NO: 6 and the nucleic acid sequence set forth in SEQ ID NO: 3 or a degenerate sequence thereof.

14. A CRISPR / Cas12fl gene editing system comprising: 1) a protein component comprising a Cas12fl protein or a homolog thereof, a conjugate, or a fusion protein, wherein: the Cas12fl protein is a wild-type PspCas12fl protein having an amino acid sequence set forth in SEQ ID NO: 1 or a PspCas12fl mutant protein of claim 1; the homolog is a homolog having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.99%, or at least 99.999% sequence identity to the amino acid sequence set forth in the Cas12fl protein and retains the biological activity of the Cas12fl protein; the conjugate comprises: the Cas12fl protein or a homolog thereof, a modification moiety, and optionally a linker; the fusion protein comprises: the Cas12f1 protein or homolog thereof, the additional protein or polypeptide, and optionally a linker; the optional linker is used to connect the Cas12f1 protein or homolog thereof and the modification moiety or the additional protein or polypeptide; for example, the modification moiety is selected from the additional protein or polypeptide, the detectable label, or a combination thereof; for example, the additional protein or polypeptide is selected from one or more of an epitope tag, a reporter protein, or a nuclear localization signal (NLS) sequence, a cytosine deaminase (CBE), an adenine deaminase (ABE), a reverse transcriptase, a cytosine methylase DNMT3A and MQ1, a cytosine demethylase Tet1, a transcriptional activator VP64, p65 and RTA, a transcriptional repressor KRAB, a histone acetylase p300, a histone deacetylase LSD1, and an endonuclease Fokl; and 2) a nucleic acid component comprising: the single guide RNA of any one of claims 4-5; and the protein component and the nucleic acid component combine with each other to form a complex; for example, the protein component comprises a wild-type PspCas12f1 protein having an amino acid sequence as set forth in SEQ ID NO: 1, or a PspCas12f1 mutant protein having an amino acid sequence as set forth in any one of SEQ ID NO: 2, SEQ ID NO: 7, and SEQ ID NO: 8, and the nucleic acid component comprises a nucleic acid sequence as set forth in SEQ ID NO:

5.

15. A cell comprising: the isolated nucleic acid molecule of any one of claims 6-9, or the vector of any one of claims 10-13. for example, the cell is a prokaryotic cell or a eukaryotic cell, such as a plant cell or an animal cell, such as a mammalian cell, such as a human cell.

16. A method of genetically editing a target sequence in a cell or in an in vitro environment, the method comprising contacting any one of (1) to (4) below with a target sequence in a cell or in an in vitro environment: (1) a Cas12f1 protein or homolog thereof, a conjugate or a fusion protein, and a single guide RNA according to any one of claims 4-5, wherein: the Cas12f1 protein is a wild-type PspCas12f1 protein having an amino acid sequence as set forth in SEQ ID NO: 1 or a PspCas12f1 mutant protein of claim 1, the homolog has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.99%, or at least 99.999% sequence identity to the amino acid sequence of the Cas12f1 protein and retains the biological activity of the Cas12f1 protein; the conjugate comprises the Cas12f1 protein or the homolog thereof, a modification moiety, and optionally a linker; the fusion protein comprises the Cas12f1 protein or the homolog thereof, another protein or polypeptide, and optionally a linker; the optional linker is used to connect the Cas12f1 protein or the homolog thereof to the modification moiety or the another protein or polypeptide; for example, the modification moiety is selected from the group consisting of another protein or polypeptide, a detectable label, or a combination thereof; for example, the another protein or polypeptide is selected from one or more of an epitope tag, a reporter protein, or a nuclear localization signal (NLS) sequence, a cytosine deaminase (CBE), an adenine deaminase (ABE), a reverse transcriptase, a cytosine methylase DNMT3A and MQ1, a cytosine demethylase Tet1, a transcriptional activator VP64, p65 and RTA, a transcriptional repressor KRAB, a histone acetylase p300, a histone deacetylase LSD1, and an endonuclease Fokl; for example, a wild-type PspCas12f1 protein having an amino acid sequence as set forth in SEQ ID NO: 1, or a PspCas12f1 mutant protein having an amino acid sequence as set forth in any one of SEQ ID NO: 2, SEQ ID NO: 7, and SEQ ID NO: 8, and a single guide RNA comprising a CRISPR scaffold sequence as set forth in SEQ ID NO: 5; (2) the vector of claim 10 or a vector comprising a nucleic acid sequence encoding a wild-type PspCas12f1 protein as set forth in SEQ ID NO: 1 or a homolog thereof, and the vector of claim 12, wherein the homolog is a homolog having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.99%, or at least 99.999% sequence identity to the amino acid sequence of the wild-type PspCas12f1 protein and retains the biological activity of the wild-type PspCas12f1 protein; for example, a vector comprising the nucleic acid sequence set forth in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 9, or SEQ ID NO: 10, or a degenerate sequence thereof, and a vector comprising the nucleic acid sequence set forth in SEQ ID NO: 6; (3) the vector according to claim 11 or 13; or (4) the CRISPR / Cas12f1 gene editing system according to claim 14; wherein the Cas12f1 protein or the homolog, conjugate, or fusion protein thereof recognizes a PAM sequence 5’-TTC located 5’ to the target sequence upon contact with the target sequence; for example, the cell is a prokaryotic cell or a eukaryotic cell, the eukaryotic cell is, for example, a plant cell or an animal cell, the animal cell is, for example, a mammalian cell such as a human cell; for example, the gene editing comprises one or more of gene knockout, site-directed base alteration, site-directed insertion, transcription level regulation of a gene, DNA methylation regulation, DNA acetylation modification, histone acetylation modification, single base conversion, and chromatin imaging tracking, for example, the single base conversion comprises conversion of a base adenine to guanine, conversion of cytosine to thymine, or conversion of cytosine to uracil.

17. The method of claim 16, wherein, the CRISPR spacer sequence of the single guide RNA forms a perfect base complementary pairing structure with the target sequence, and forms an imperfect base complementary pairing structure with a non-target sequence; for example, the imperfect base complementary pairing structure comprises one or more, for example, two or more, base mismatches.

18. A kit for gene editing a target sequence in a cell or in an in vitro environment, comprising: i. any one selected from the following (1) to (6): (1) a Cas12f1 protein or a homolog, conjugate, or fusion protein thereof, and a single guide RNA according to any one of claims 4 to 5, wherein: (2) a vector according to any one of claims 1 to 3, and a single guide RNA according to any one of claims 4 to 5, wherein: the Cas12f1 protein is a wild-type PspCas12f1 protein having an amino acid sequence as set forth in SEQ ID NO: 1 or a PspCas12f1 mutant protein as claimed in claim 1; the homolog has an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.99%, or at least 99.999% sequence identity to the amino acid sequence of the Cas12f1 protein and retains the biological activity of the Cas12f1 protein; the conjugate comprises the Cas12f1 protein or the homolog thereof, a modification moiety, and optionally a linker; the fusion protein comprises the Cas12f1 protein or the homolog thereof, another protein or polypeptide, and optionally a linker; the optional linker is used to link the Cas12f1 protein or the homolog thereof to the modification moiety or the another protein or polypeptide; for example, the modification moiety is selected from the group consisting of another protein or polypeptide, a detectable label, or a combination thereof; for example, the another protein or polypeptide is selected from one or more of an epitope tag, a reporter protein or a nuclear localization signal (NLS) sequence, a cytosine deaminase (CBE), an adenine deaminase (ABE), a reverse transcriptase, a cytosine methylase DNMT3A and MQ1, a cytosine demethylase Tet1, a transcriptional activator VP64, p65 and RTA, a transcriptional repressor KRAB, a histone acetylase p300, a histone deacetylase LSD1, and an endonuclease FokI; for example, the wild-type PspCas12f1 protein having an amino acid sequence as set forth in SEQ ID NO: 1, or the PspCas12f1 mutant protein having an amino acid sequence as set forth in any one of SEQ ID NO: 2, SEQ ID NO: 7 and SEQ ID NO: 8, and a single guide RNA comprising a CRISPR scaffold sequence as set forth in SEQ ID NO: 5; (2) the isolated nucleic acid molecule of claim 6 or 8, or an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a wild-type PspCas12f1 protein as set forth in SEQ ID NO: 1 or a homolog thereof, and (2) the isolated nucleic acid molecule of claim 6 or 8, or an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a wild-type PspCas12f1 protein as set forth in SEQ ID NO: 1 or a homolog thereof, and wherein the homolog has an amino acid sequence that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.99%, or at least 99.999% sequence identity to the amino acid sequence of the wild-type PspCas12fl protein and retains the biological activity of the wild-type PspCas12fl protein; for example, an isolated nucleic acid molecule comprising the nucleic acid sequence set forth in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 9, or SEQ ID NO: 10, or a degenerate sequence thereof, and an isolated nucleic acid molecule comprising the nucleic acid sequence set forth in SEQ ID NO: 6; (3) the isolated nucleic acid molecule according to claim 7 or 9; (4) the vector according to claim 10 or a vector comprising a nucleic acid sequence encoding a wild-type PspCas12fl protein as set forth in SEQ ID NO: 1 or a homolog thereof, and the vector according to claim 12, wherein the homolog has an amino acid sequence that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%, at least 99.99%, or at least 99.999% sequence identity to the amino acid sequence of the wild-type PspCas12fl protein and retains the biological activity of the wild-type PspCas12fl protein; for example, a vector comprising the nucleic acid sequence set forth in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 9, or SEQ ID NO: 10, or a degenerate sequence thereof, and a vector comprising the nucleic acid sequence set forth in SEQ ID NO: 6; (5) the vector according to claim 11 or 13; or (6) the CRISPR / Cas12fl gene editing system according to claim 14; and ii. instructions for how to genetically edit a target sequence in a cell or in an in vitro environment. iii. instructions for how to genetically edit a target sequence in a cell or in an in vitro environment. iv. instructions for how to

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