WO2023206872A1 - 一种工程优化的核酸酶、向导rna、编辑系统和应用 - Google Patents

一种工程优化的核酸酶、向导rna、编辑系统和应用 Download PDF

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WO2023206872A1
WO2023206872A1 PCT/CN2022/113357 CN2022113357W WO2023206872A1 WO 2023206872 A1 WO2023206872 A1 WO 2023206872A1 CN 2022113357 W CN2022113357 W CN 2022113357W WO 2023206872 A1 WO2023206872 A1 WO 2023206872A1
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sequence
nuclease
gene
ascas12f1
guide rna
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季泉江
吴兆韡
潘登
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上海科技大学
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    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]

Definitions

  • the invention belongs to the field of biotechnology, and specifically relates to an engineered optimized nuclease, guide RNA, editing system and application. In particular, it relates to an engineered optimized ultra-small CRISPR/AsCas12f1 novel genome editing system and its application and editing method. .
  • Genome editing technology refers to the use of gene editing machines, such as programmable nucleases (molecular scissors), to interrupt specific gene sequences and then introduce gene insertion, deletion or replacement to achieve the transformation of specific segments of the organism's genomic DNA, thereby A genetic engineering technology to edit target genes.
  • gene editing machines such as programmable nucleases (molecular scissors)
  • Cas nuclease can use guide RNA to locate specific target sites in the genome of various cells, cut it to generate DNA double-strand breaks, and then use the cell's endogenous or external DNA repair mechanisms, such as homologous recombination and non-homologous recombination. End-joining repair mechanisms enable editing. Depending on the activation of different DNA repair pathways, genome editing will lead to gene inactivation or mutation correction.
  • the CRISPR/Cas system can be further transformed into a base editing system and a lead editing system, which are widely used in biology, agricultural research, and disease treatment.
  • the currently widely used CRISPR/Cas genome editing systems mainly include CRISPR/Cas9 and CRISPR/Cas12a.
  • the CRISPR effector protein nucleases Cas9 and Cas12a are both large proteins containing more than 1,000 amino acids.
  • the gene editors developed with them as the core have large molecular sizes and are difficult to be secreted.
  • Commonly used vectors such as related viruses are efficiently packaged and delivered, which leads to huge problems in the delivery process of CRISPR/Cas9 and CRISPR/Cas12a to cells.
  • the extremely small nuclease CRISPR-AsCas12f1 gene editing system existing in the existing technology contains only 422 amino acids, and the gene size is less than half of Cas9 and Cas12a. It has natural gene editing capabilities and can be implemented in bacteria and mammalian cells. Efficient gene editing. Thanks to its compact molecular size, both the CRISPR-AsCas12f1 gene editing system itself and its derived gene editing tools can easily achieve the packaging and delivery of a single AAV.
  • the technical problem to be solved by the present invention is to overcome the low gene editing efficiency of extremely small nucleases in the prior art, and provide an engineering optimized gene editing system and its applications and methods: in particular, the engineering optimized new type of extremely small CRISPR/AsCas12f1 Genome editing systems and their applied editing methods.
  • the optimized gene editing system or method of the present invention can be used to precisely knock out or edit target genes in cells.
  • the present invention mainly solves the above technical problems through the following technical solutions.
  • One of the objects of the present invention is to provide a mutant AsCas12f1 nuclease, which has at least 50% identity with the wild-type AsCas12f1 nuclease, and contains position 80, One or several positions in the 104th and 364th amino acids are mutated.
  • Another object of the present invention is to provide a mutant guide RNA, which includes a tracrRNA sequence and a crRNA sequence; the crRNA sequence includes a gene targeting segment capable of hybridizing with a target sequence and a tracr partner sequence; the tracr RNA sequence and The tracr partner sequence constitutes the backbone sequence of the guide RNA;
  • the tracrRNA includes the nucleotide sequence shown in SEQ ID NO. 47 or a variant sequence thereof;
  • the tracr partner sequence includes the nucleotide sequence shown in SEQ ID NO. 48 or a variant sequence thereof.
  • the guide RNA further includes a connecting strand sequence.
  • Another object of the present invention is to provide an isolated polynucleotide encoding the mutant AsCas12f1 nuclease as described above or the guide RNA as described above.
  • Another object of the present invention is to provide a construct containing the isolated polynucleotides as described above alone or simultaneously. That is, the isolated polynucleotide encoding the mutant AsCas12f1 nuclease and the isolated polynucleotide encoding the guide RNA may be located on the same or different constructs.
  • Another object of the present invention is to provide an expression system, which contains the construct as described above or an exogenous polynucleotide as described above integrated into the genome.
  • Another object of the present invention is to provide a gene editing system, which includes the mutant AsCas12f1 nuclease or its encoding polynucleotide as described above, and guide RNA or its encoding polynucleotide.
  • the gene editing system of the present invention includes a nuclease or a polynucleotide encoding the nuclease, and a mutant guide RNA or a polynucleotide encoding the nuclease as described above.
  • Another object of the present invention is to provide a pharmaceutical composition, which includes the gene editing system as described above, and a pharmaceutically acceptable carrier.
  • Another object of the present invention is to provide a gene editing method that contacts the target gene with the gene editing system as described above to achieve editing of the target gene.
  • Another object of the present invention is to provide the mutant AsCas12f1 nuclease, mutant guide RNA, isolated polynucleotide, construct, expression system, gene editing system, pharmaceutical composition or method described above in vivo and in vitro cells. Or application in gene editing of target genes and/or related polypeptides in a cell-free environment.
  • Another object of the present invention is to provide a genetically modified cell, which is obtained by gene editing with the gene editing system, pharmaceutical composition or method described above.
  • the gene insertion/deletion efficiency of the engineered optimized guide RNA variant - sgRNA_T1 at the test target site is improved by 5-15% compared to the wild guide RNA - sgRNA_V1.
  • AsCas12f1 nuclease variants based on sgRNA_T1 such as AsCas12f1-K80R, AsCas12f1-A104R, AsCas12f1-D364R, AsCas12f1-A104+K80R, AsCas12f1-A104+D364R, AsCas12f1-K80R+A104+D364R, can all go further.
  • the engineering-optimized CRISPR/AsCas12f1 gene editing system in the present invention has significantly improved gene editing efficiency in 27 target sites of 11 genes in mammalian cells.
  • the present invention further improves the applicability of the extremely small CRISPR/Cas12f system in cell gene editing, and can realize precise gene editing in cells.
  • Figure 1 is a comparison chart of the mammalian cell gene editing results of the original wild AsCas12f1 nuclease combined with sgRNA_V1 and sgRNA_T1 respectively.
  • sgRNA_T1 can significantly improve gene editing efficiency at 6 target sites on APOB, HEXA, PDCD1, TP53 and VEGFA genes.
  • Figure 2 is a comparison chart of mammalian cell gene editing results based on sgRNA_T1, respectively combining the original wild AsCas12f1 nuclease and 6 AsCas12f1 nuclease variants. As shown in the figure, all six variants can improve gene editing efficiency at three target sites on the APOB, PDCD1 and VEGFA genes. Among them, AsCas12f1-Evo1 has the best effect.
  • Figure 3 is a comparison of the gene editing effects of the original wild CRISPR/AsCas12f1 system and the engineered optimized CRISPR/AsCas12f1 system in mammalian cells. As shown in the figure, the optimized CRISPR/AsCas12f1 gene editing system can significantly improve gene editing efficiency on 27 target sites of 11 genes.
  • Figure 4 is a comparison of the in vitro DNA cutting effects of the original wild CRISPR/AsCas12f1 system and the engineered optimized CRISPR/AsCas12f1 system. As shown in the figure, the engineered and optimized CRISPR/AsCas12f1 gene editing system can significantly increase the speed of DNA cutting in vitro.
  • WT represents the wild-type AsCas12f1 nuclease
  • V1 represents the wild-type guide RNA-sgRNA_V1
  • T1 represents the guide RNA variant-sgRNA_T1.
  • a single base editing system based on AsCas12f1 was developed by engineering optimized AsCas12f1 and base deaminase through fusion inactivation; a Prime editing system based on AsCas12f1 was developed by fusion inactivation AsCas12f1 and reverse transcriptase; a Prime editing system based on AsCas12f1 was developed by fusion inactivation AsCas12f1 and transcription activators to develop a transcription activation system based on AsCas12f1; by fusing the inactivated AsCas12f1 nucleic acid epigenetic modification enzyme, an epigenetic modification system based on AsCas12f1 was developed; using inactivated AsCas12f1, a transcription repression system based on AsCas12f1 was developed.
  • the CRISPR system mainly used in the present invention is the V-F type CRISPR/Cas12f system, in which the effector protein is mainly the original wild Acidibacillus sulfuroxidans Cas12f1 (AsCas12f1) nuclease and its various variants, and the guide RNA is mainly the original wild sgRNA_V1 and the variant sgRNA_T1 .
  • AsCas12f1 nuclease or its variants can accurately locate the target gene under the guidance of the corresponding guide RNA or variant, cut the genomic DNA, and achieve double-stranded breaks in the genomic DNA. Utilizing the host cell's own or exogenous repair mechanisms, this system can efficiently and accurately achieve gene editing in living cells.
  • One of the objects of the present invention is to provide a mutant AsCas12f1 nuclease, which has at least 50% identity with the wild-type AsCas12f1 nuclease, and contains position 80, One or several site mutations in the 104th and 364th amino acids; that is, the variant AsCas12f1 nuclease may include a mutation of the 80th amino acid, or a mutation of the 104th amino acid, or a mutation of the 364th amino acid
  • the amino acid mutation may include a mutation at the 80th and 104th amino acids at the same time, or a mutation at the 80th and 364th amino acids at the same time, or a mutation at the 80th, 104th and 364th amino acids at the same time.
  • the amino acids after mutation at each site may be the same or different.
  • the amino acids after mutation at each site are the same, that is, when the mutant AsCas12f1 nuclease contains two or three site mutations, the two mutations or Three mutation sites are mutated to the same or different amino acids, preferably the same amino acid; for example, two mutations or three mutation sites are mutated to any other amino acid different from the wild-type site.
  • the mutation is to arginine.
  • mutation of an amino acid is to arginine.
  • mutation of two or three amino acids is to arginine.
  • the invention provides one or several engineered AsCas12f1 nuclease variants, and expression constructs comprising these variant sequences.
  • the AsCas12f1 nuclease variants of the present invention include AsCas12f1-K80R, AsCas12f1-A104R, AsCas12f1-D364R, AsCas12f1-A104+K80R, AsCas12f1-A104+D364R, and AsCas12f1-K80R+A104+D364R.
  • the most preferred variant is AsCas12f1-K80R+A104+D364R (hereinafter referred to as AsCas12f1-Evo1).
  • variant AsCas12f1-K80R amino acid sequence preferably includes the sequence shown below:
  • the variant AsCas12f1-A104R amino acid sequence preferably contains the sequence shown below:
  • the variant AsCas12f1-D364R amino acid sequence preferably contains the sequence shown below:
  • the variant AsCas12f1-A104R+K80R amino acid sequence preferably includes the sequence shown below:
  • mutant AsCas12f1-A104R+D364R preferably includes the sequence shown below:
  • the amino acid sequence of the variant AsCas12f1-K80R+A104R+D364R (hereinafter referred to as AsCas12f1-Evo1) preferably includes the sequence shown below:
  • Another object of the present invention is to provide a mutant guide RNA (gRNA), which includes a tracrRNA sequence, a crRNA sequence, and a connecting strand sequence; the crRNA sequence includes a gene targeting segment capable of hybridizing to a target sequence and a tracr partner sequence. ;
  • the tracrRNA sequence and the tracr partner sequence constitute the backbone sequence of the guide RNA;
  • the tracrRNA includes the nucleotide sequence shown in SEQ ID NO.47 or its variant sequence; the tracr partner sequence includes the nucleotide sequence shown in SEQ ID NO.48 or its variant sequence. .
  • the tracrRNA sequence of the variant is SEQ ID NO.50, that is, the tracrRNA sequence corresponding to sgRNA_T1:
  • the tracrRNA partner sequence of the variant is SEQ ID NO.51, that is, the tracrRNA partner sequence corresponding to sgRNA_T1: 5'-uguggagugugaac (SEQ ID NO.51);
  • the tracr RNA sequence and the tracr partner sequence also include a connecting strand sequence; preferably, the connecting strand sequence includes 5'-AAGG, 5'-UACU or a variant sequence thereof.
  • the variant sequence of the connecting chain is a sequence obtained by adding, reducing or replacing one or more nucleotides on the basis of the 5'-AAGG and 5'-UACU sequences.
  • the connecting chain sequence between the tracr RNA sequence and the tracr partner sequence in the wild-type sgRNA_V1 of the present invention is: 5'-AAGG.
  • the connecting chain sequence between the tracr RNA sequence and the tracr partner sequence in mutant sgRNA_T1 is: 5'-UACU.
  • the mutant guide RNA of the present invention includes one or more of the complementary paired base pairs in the tracrRNA, and the tracrRNA is obtained by exchanging positions of the paired bases.
  • the base C at position 7 of tracrRNA is complementary to the base G at position 16.
  • the tracrRNA obtained after setting position 7 to G and position 16 to C can also achieve the technical effects of the present invention.
  • the mutant guide RNA of the present invention contains one or more base pairs of complementary pairs between tracrRNA and tracr partner sequence, and the tracrRNA and tracr partner sequence are obtained by exchanging positions of the paired bases.
  • base U at position 119 of tracrRNA is complementary to base A at base 21 of the tracr partner.
  • the tracrRNA and tracr obtained after setting base 119 of tracrRNA to A and position 21 of the tracr partner to U The counterpart sequence can also achieve the technical effects of the present invention.
  • the gene targeting segment is a nucleotide sequence complementary to the target sequence in the target gene and is located at the 3' end of the crRNA sequence; the gene targeting segment identifies PAM sequence on the targeting sequence; preferably the PAM sequence is 5'-TTR, where R represents A or G.
  • the gene targeting segment targets a nucleic acid fragment with a length of 12 to 40 bp after the PAM sequence; for example, it can be 13-20, 18-25, 22-32, 26-37, 30-38, 32-40 nucleotides
  • the length of the acid is preferably 20 bp.
  • the gene targeting segment is preferably an RNA sequence corresponding to a 20 bp length nucleic acid fragment following the PAM sequence.
  • the gene targeting segment is selected from SEQ ID NO. 19.
  • the complementarity percentage between the targeting segment of the guide RNA and the target sequence of the target gene can be at least 50% (for example, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100%).
  • the gene targeting segment targets at least one target sequence in the cell genome.
  • the mutant guide RNA further comprises a transcription terminator.
  • the guide RNA can be two strands, one strand includes a tracrRNA sequence, and the other strand includes a crRNA sequence, wherein the tracr partner sequence hybridizes with the tracrRNA sequence and forms a stem. -Ring structure.
  • the crRNA sequence and the tracrRNA sequence can be connected together through a connecting strand to form a single guide RNA skeleton sequence, that is, the guide RNA is a strand from the 5' end to the 3' end.
  • the end contains the tracr RNA sequence, the connecting strand sequence and the crRNA sequence in sequence.
  • the guide RNA is one strand, the 3' end of the tracr RNA sequence and the 5' end of the crRNA sequence are connected through a connecting strand.
  • the crRNA and tracrRNA sequences are two separate RNA sequences and can mediate the activity of nuclease such as AsCas12f1 endonuclease when they exist simultaneously.
  • nuclease such as AsCas12f1 endonuclease
  • the complete guide RNA expression construct for the target sequence obtained by connecting the guide RNA backbone sequence and the DNA-targeting segment that hybridizes to the target sequence can also mediate the activity of nucleases such as AsCas12f1 endonuclease.
  • the gene targeting segment includes a nucleotide sequence complementary to a sequence in the target gene, and the gene targeting sequence interacts with the target gene in a sequence-specific manner through hybridization (i.e., base pairing). interaction.
  • the gene targeting sequence of the gRNA can be modified, for example, by genetic engineering, so that the gRNA hybridizes to any desired sequence within the target gene.
  • gRNA guides the bound polypeptide to a specific nucleotide sequence within the target gene through the above-mentioned gene targeting sequence.
  • the target gene is a DNA sequence. In some embodiments, the target gene is an RNA sequence.
  • the invention also provides methods for modifying the guide RNA, including but not limited to individual modifications and combined modifications of trans-activating CRISPR RNA (tracrRNA) and CRISPR RNA (crRNA).
  • the modification methods include, but are not limited to, truncation, extension or replacement of different sequences of tracrRNA or crRNA.
  • the variant sequence of tracrRNA refers to adding, reducing or replacing part of the nucleosides at the 5' end and/or 3' end of the nucleotide sequence shown in SEQ ID NO. 47 Sequence obtained after acid.
  • the variant sequence of tracrRNA refers to a sequence obtained by reducing nucleotides at the 5' end and/or 3' end of the nucleotide sequence shown in SEQ ID NO.
  • the variant sequence of tracrRNA refers to a sequence obtained by reducing 1 to 50 nt nucleotides from the 5' end and/or 3' end of the nucleotide sequence shown in SEQ ID NO. 47. Further preferably, the variant sequence of tracrRNA refers to the sequence obtained by reducing the 3' end of the nucleotide sequence shown in SEQ ID NO. 47 by 14nt nucleotides.
  • the variant sequence of the tracr partner sequence refers to a sequence obtained by adding, reducing or replacing part of the nucleotides at the 5' end and/or 3' end of the nucleotide sequence shown in SEQ ID NO.48 ;
  • the variant sequence of the tracr partner sequence refers to a sequence obtained by reducing nucleotides at the 5' end and/or 3' end of the nucleotide sequence shown in SEQ ID NO. 48.
  • the variant sequence of the tracr partner sequence refers to a sequence obtained by reducing 1 to 29 nt nucleotides from the 5' end of the nucleotide sequence shown in SEQ ID NO. 48. Further preferably, the variant sequence of the tracr partner sequence refers to a sequence obtained by reducing 15 nt nucleotides from the 5' end of the nucleotide sequence shown in SEQ ID NO. 48.
  • the variant sequence of tracrRNA refers to a sequence obtained by reducing 14nt nucleotides at the 3' end of the nucleotide sequence shown in SEQ ID NO. 47, with a sequence such as SEQ ID NO. 50 shown.
  • the variant sequence of the tracr partner sequence refers to a sequence obtained by reducing 15nt nucleotides at the 5' end of the nucleotide sequence shown in SEQ ID NO. 48; the sequence is as follows Shown as SEQ ID NO.51.
  • the nucleotide sequence of the backbone sequence of the mutant guide RNA is shown in SEQ ID NO. 52.
  • the preferred guide RNA shows higher gene deletion and/or gene cleavage efficiency for the same target sequence in mammalian cell genome editing.
  • the present invention provides a new engineering optimized guide RNA (also known as sgRNA_T1) of the CRISPR/AsCas12f1 gene editing system, and an expression construct containing the variant sequence.
  • a new engineering optimized guide RNA also known as sgRNA_T1
  • the engineering optimized guide RNA sequence of the present invention is truncated by 29nt in total.
  • the complete sequence of the preferred guide RNA is as follows:
  • the sequence in 9 that does not contain the target gene segment is 5'-auucgucgguucagcgacgauaagccgagaagugccaauaaacuguuaagugguuugguaacgcucgguaagguagccaaaggcugaaacuccgcacaaagaccgcacggacgcuucacuacuuguggagugugaac (SEQ ID NO. 52).
  • the underlined part is the targeting sequence: 5’-CUCUCAAGACCCACAAUCCA-3’ (SEQ ID NO. 19); it can perform complementary base pairing with a DNA fragment of 20 bp in length after the preferred PAM sequence on the target gene.
  • a preferred PAM sequence is 5'-TTR, where R represents A or G.
  • the underlined sequence can be replaced accordingly according to the target gene site.
  • the complete sequence of the wild-type guide RNA sequence (sgRNA_V1) is shown in SEQ ID NO.10: 5'-auucgucgguucagcgacgauaagccgagaagugccaauaaaacuguuaagugguuugguaacgcucgguaagguagccaaaaggcugaaacuccgcacaaagaccgcacggacgcuucacauauagcucauaaaacAAGG guuugcgagcuagcuug uggagugugaac CUCUCAAGACCCACAAUCCA -3'; wherein, the tracrRNA sequence is shown in SEQ ID NO.47; the tracr partner sequence is shown in SEQ ID Shown in NO.48; the backbone sequence is 5'-auucgucgguucagcgacgauaagccgagaagugccaauaaacuguuaagugguuuggua
  • the present invention also provides modified mutant guide RNA, which can be used to achieve hybridization with any desired sequence within the target gene through modification; or, by modifying the gRNA to change the characteristics of the gRNA itself, such as enhancing the stability of the gRNA through modification , including but not limited to by increasing its resistance to ribonuclease (RNase) degradation present in the cell, thereby extending its half-life in the cell; alternatively, it can be modified to enhance the composition of gRNA and endonuclease ( The formation of the CRISPR-AsCas12f1 genome editing complex such as AsCas12f1 nuclease) or its stability; alternatively, it can be used to enhance the specificity of the genome editing complex through modification; alternatively, it can be used to enhance the genome editing complex through modification The starting site, stability or kinetics of the interaction with the target sequence in the genome; alternatively, it can be modified to reduce the likelihood or extent of the innate immune response triggered by the RNA introduced into the cell, etc.
  • RNA can be modified using modification methods known in the art, including but not limited to 2'-fluoro and 2'-amino modifications on the ribose and base residues of the pyrimidine or the reverse base at the 3' end of the RNA. .
  • any modification or combination of modifications can be used to modify gRNA.
  • the sgRNA introduced into the cell is modified to edit any one or more genomic loci.
  • Another object of the present invention is to provide an isolated polynucleotide encoding the mutant AsCas12f1 nuclease as described above or the guide RNA as described above.
  • Another object of the present invention is to provide a construct containing an isolated polynucleotide as described above.
  • the construct can usually be constructed by inserting the isolated polynucleotide into a suitable expression vector, and those skilled in the art can select a suitable expression vector.
  • the construct may, for example, be a recombinant expression vector, and any suitable expression vector may be used as long as it is compatible with the host cell, including, but not limited to, viral vectors (e.g., vaccinia virus-based viral vectors; poliovirus; Adenovirus; adeno-associated virus; SV40; herpes simplex virus; human immunodeficiency virus; retroviral vectors (e.g., murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses, such as Rous sarcoma virus, Harvey Sarcoma virus, avian leukemia virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus and mammary tumor virus), etc.
  • viral vectors e.g., vaccinia virus-based viral vectors; poliovirus; Adenovirus; adeno-associated virus; SV40; herpes simplex virus; human immunodefic
  • multiple nucleases or gRNAs are used simultaneously in the same cell to simultaneously regulate transcription of different locations on the same target gene or different target genes.
  • they can exist on the same expression vector or on different vectors, or they can be expressed at the same time; when they exist on the same vector, they can be expressed under the same control element .
  • a nucleotide sequence encoding a nuclease or gRNA is operably linked to a control element, such as a transcription control element, such as a promoter.
  • a nucleotide sequence encoding a nuclease or gRNA is operably linked to an inducible promoter.
  • a nucleotide sequence encoding a nuclease or gRNA is operably linked to a constitutive promoter.
  • Transcriptional control elements may function in eukaryotic cells, such as mammalian cells (HEK293T cells); or in prokaryotic cells, such as bacterial or archaeal cells.
  • a nucleotide sequence encoding a nuclease or gRNA is operably linked to a plurality of control elements that permit expression of the nucleotide sequence encoding a nuclease or gRNA in both prokaryotic and eukaryotic cells.
  • the nuclease or gRNA can be synthesized by artificial synthesis, so that it can be easily modified in various ways.
  • the modification can adopt any modification method known in the art, for example, using a polyA tail, adding a 5' cap analog, a 5' or 3' untranslated region (UTR), and the 5' or 3' end including phosphorothioate 2 '-O-methyl nucleotide or treated with phosphatase to remove the 5' terminal phosphate, etc.
  • the nucleotide sequence encoding a nuclease or gRNA contains one or more modifications that may be used, for example, to enhance activity, stability or specificity, alter delivery, reduce the innate immune response in the host cell or for other enhancements.
  • one or more targeting moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the nucleotide sequence encoding the nuclease or gRNA are chemically linked to the nuclease or gRNA.
  • the targeting moiety or conjugate may include a conjugate group covalently bonded to a functional group; the conjugate group includes reporter molecules, polyamines, polyethylene glycol.
  • a group that enhances pharmacodynamic properties, including improved uptake, increased resistance to degradation, and/or enhanced sequence specificity to the target nucleic acid is attached to a nuclease or gRNA. Sexually hybridized groups.
  • a nucleic acid containing a polynucleotide encoding a nuclease or gRNA may be a nucleic acid mimetic.
  • polynucleotide mimetic peptide nucleic acids with excellent hybridization properties may be provided.
  • the nuclease or gRNA, or the polynucleotide encoding the nuclease or gRNA is applicable to any biological or in vitro environment, including but not limited to bacteria, archaea, fungi, protists, plants or animals.
  • suitable target cells include, but are not limited to, bacterial cells, archaeal cells, fungal cells, protist cells, plant cells or animal cells.
  • Applicable target cells can be any type of cells, including stem cells, somatic cells, etc.
  • Another object of the present invention is to provide an expression system, which contains the construct as described above or an exogenous polynucleotide as described above integrated into the genome.
  • the host cell of the expression system is selected from eukaryotic cells or prokaryotic cells; preferably, the host cell is selected from mouse cells and human cells.
  • Another object of the present invention is to provide a gene editing system that includes the mutant AsCas12f1 nuclease or its encoding polynucleotide as described above, and a guide RNA or its encoding polynucleotide.
  • the gene editing system of the present invention includes a nuclease or a polynucleotide encoding the nuclease, and a mutant guide RNA or a polynucleotide encoding the nuclease as described above.
  • the engineering-optimized CRISPR/AsCas12f1 gene editing system is characterized by the use of any combination of wild guide RNA or guide RNA variants and wild AsCas12f1 nuclease or variants thereof. When any of the following conditions are met, it is considered to belong to the system:
  • Methods of combined use include but are not limited to routine use in the art, for example, simultaneously expressing wild-type guide RNA or guide RNA variants and wild-type AsCas12f1 nuclease or variants thereof in the same construct; expressing wild-type separately in different constructs Guide RNA or guide RNA variant and wild-type AsCas12f1 nuclease or its variant; in vitro compatibility of wild-type guide RNA or guide RNA variant and wild-type AsCas12f1 nuclease or its variant, etc.
  • the polynucleotide encoding the nuclease includes: a coding sequence encoding only a nuclease; a nuclease coding sequence and various additional coding sequences; a nuclease coding sequence (and optional additional coding sequences) and non-coding sequences.
  • the polynucleotide encoding the guide RNA includes: a coding sequence encoding only the guide RNA; a coding sequence of the guide RNA and various additional coding sequences; a coding sequence of the guide RNA (and optional additional coding sequences) and non-coding sequences.
  • the gene editing system includes one or more vectors; the one or more vectors include (i) a first regulatory element operably linked to the nuclease a coding polynucleotide; and (ii) a second regulatory element operably linked to the coding polynucleotide of the guide RNA nucleotide sequence; said (i) and (ii) are located on the same or on different carriers.
  • the gene editing system comprises (i) a nuclease or a variant thereof, and (ii) a vector comprising the coding sequence of the guide RNA.
  • the system includes a gRNA and nuclease complex.
  • the first regulatory element may regulate the transcription of a polynucleotide encoding the nuclease or a variant thereof.
  • the polynucleotide encoding the nuclease or its variant may be one or more, and the first regulatory element may be one or more.
  • the second regulatory element can regulate the transcription of the polynucleotide encoding the guide RNA.
  • the guide RNA encoding polynucleotide may be one or more, and the second regulatory element may be one or more.
  • the system of the present invention may contain one gRNA or multiple gRNAs simultaneously.
  • the system includes multiple gRNAs simultaneously to simultaneously modify different positions on the same target DNA or different target DNAs.
  • two or more guide RNAs target the same gene or transcript or locus.
  • two or more guide RNAs target different unrelated loci.
  • two or more guide RNAs target different but related loci.
  • the nuclease is a CRISPR nuclease; preferably, the nuclease is selected from the Cas9, Cas12, and Cas13 protein families Or a variant thereof; further preferably, the Cas nuclease is selected from SpCas9 and its mutants, SaCas9 and its mutants, Cas12a and its mutants, or Cas12f and its mutants; further preferably, Cas12f and its mutants.
  • the nuclease is provided directly as a protein; for example, spheroplast transformation may be used to transform the fungus with exogenous proteins and/or nucleic acids.
  • the nuclease can be introduced into the cells by any suitable method, such as injection.
  • the gene editing system of the present invention recognizes the PAM sequence on the target sequence; preferably, the PAM sequence is 5'-TTR, where R represents A or G.
  • the gene editing system targets nucleic acid fragments with a length of 12 to 40 bp after the PAM sequence, and the preferred length is 20 bp.
  • the gene editing system targets at least one target sequence in the genome of the cell.
  • the nucleic acid encoding a nuclease is DNA. In certain embodiments, the nucleic acid encoding a nuclease is RNA. In certain embodiments, the nucleic acid encoding a nuclease is an expression vector, such as a recombinant expression vector.
  • Any suitable expression vector may be used so long as it is compatible with the host cell, including, but not limited to, viral vectors (e.g., vaccinia virus-based viral vectors; poliovirus; adenovirus; adeno-associated virus; SV40; herpes simplex Viruses; human immunodeficiency virus; retroviral vectors (e.g., murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses, such as Rous sarcoma virus, Harvey sarcoma virus, avian leukemia virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus and breast tumor virus), etc.
  • viral vectors e.g., vaccinia virus-based viral vectors; poliovirus; adenovirus; adeno-associated virus; SV40; herpes simplex Viruses; human immunodeficiency virus; retroviral vectors (e
  • the nuclease is wild-type AsCas12f1, and the nucleic acid encoding the AsCas12f1 nuclease is shown in SEQ ID NO.7.
  • the invention provides a codon-optimized polynucleotide sequence of AsCas12f1 nuclease, having at least 90%, 92%, 93%, 94%, 95%, 96%, 97% with SEQ ID NO:7 , 98%, 99%, 99.2%, 99.5%, 99.8%, 99.9% or 100% sequence homology, which encodes a polypeptide having the same function as the polypeptide encoded by the original native nucleotide sequence.
  • a nucleotide sequence encoding a nuclease is operably linked to a control element, such as a transcription control element, such as a promoter. In certain embodiments, a nucleotide sequence encoding a nuclease is operably linked to an inducible promoter. In certain embodiments, a nucleotide sequence encoding a nuclease is operably linked to a constitutive promoter.
  • Transcriptional control elements may function in eukaryotic cells, such as mammalian cells (HEK293T cells); or in prokaryotic cells, such as bacterial or archaeal cells.
  • a nuclease-encoding nucleotide sequence is operably linked to a plurality of control elements that permit expression of the nuclease-encoding nucleotide sequence in both prokaryotic and eukaryotic cells.
  • the polynucleotide sequence encoding a nuclease is operably linked to a suitable nuclear localization signal for expression in a cellular or in vitro environment.
  • the polynucleotide encoding a nuclease can be synthesized by artificial synthesis, for example, by chemical methods, so that it can be easily modified in various ways.
  • the modification may adopt any modification method known in the art.
  • the nuclease-encoding polynucleotide contains one or more modifications, thereby enabling the easy incorporation of a number of modifications, such as enhancing transcriptional activity, altering enzymatic activity, improving its translation or stability (e.g. Increase its resistance to proteolysis, degradation) or specificity, alter solubility, alter delivery, and reduce the innate immune response in host cells.
  • the modification may adopt any modification method known in the art.
  • any one or more genomic loci are edited by modifying the nuclease-encoding DNA or RNA introduced into the cell.
  • the nucleic acid sequence encoding a nuclease is a modified nucleic acid, eg, codon optimized.
  • the modification may be a single modification or a combination of modifications.
  • the nucleic acid comprising a polynucleotide encoding a nuclease may be a nucleic acid mimetic.
  • polynucleotide mimetic peptide nucleic acids with excellent hybridization properties may be provided.
  • the nuclease, or the polynucleotide encoding the nuclease is suitable for use in any biological or in vitro environment, including but not limited to bacteria (such as Escherichia coli, Klebsiella pneumoniae), archaea, fungi, native Living things, plants or animals.
  • suitable target cells include but are not limited to eukaryotic cells and prokaryotic cells, such as bacterial cells, archaeal cells, fungal cells, protist cells, plant cells or animal cells; the eukaryotic cells include mammalian cells and Plant cells, the prokaryotic cells include Escherichia coli and Klebsiella pneumoniae.
  • Applicable target cells can be any type of cells, including stem cells, somatic cells, etc.
  • the present invention is preferably used for mammalian cells HEK293T cells.
  • the cells may be in vivo or ex vivo.
  • the nuclease or nucleic acid encoding a nuclease is formulated in liposomes or lipid nanoparticles.
  • nuclease and gRNA can form a complex in the host cell to identify the PAM sequence on the target gene (such as target DNA) sequence; the target sequence of the CRISPR/AsCas12f1 gene editing system is PAM The sequence is followed by a nucleic acid fragment (such as a DNA fragment) of 20 bp in length.
  • the complex can selectively regulate the transcription of target DNA in a host cell.
  • Gene editing systems can cut the double strands of targeted DNA, causing DNA breaks.
  • the system includes a recombinant expression vector.
  • the system comprises a recombinant expression vector comprising (i) a nucleotide sequence encoding a gRNA, wherein the gRNA comprises: (a) a core comprising a sequence complementary to a sequence in the target DNA a first segment of a nucleotide sequence; and (b) a second segment that interacts with a nuclease; and (ii) a nucleotide sequence encoding a nuclease, wherein the nuclease comprises: (a) An interacting RNA-binding portion of the gRNA; and (b) an active portion that regulates transcription within the target DNA, wherein the site of regulated transcription within the target DNA is determined by the gRNA.
  • nuclease variants can also be formed through modification, mutation, DNA shuffling, etc., so that the nuclease variants have improved desired characteristics, such as function, activity, kinetics, half-life, etc.
  • the modification may be, for example, deletion, insertion or substitution of amino acids, or may be, for example, the replacement of a nuclease such as AsCas12f1 with a homologous or heterologous cleavage domain from a different nuclease (for example, the HNH domain of a CRISPR-related nuclease).
  • “Cleaving domain” through any modification method of DNA binding and/or DNA modifying proteins known in the art, such as methylation, demethylation, acetylation, etc., for example, the DNA of AsCas12f1 nuclease can be changed Targeting.
  • the DNA shuffling refers to the exchange of sequence fragments between DNA sequences of AsCas12f1 nuclease from different sources to generate chimeric DNA sequences encoding synthetic proteins with RNA-guided endonuclease activity.
  • the modification, mutation, DNA shuffling, etc. may be used singly or in combination.
  • the Cas protein and its mutants are selected from:
  • (II) A variant having at least 50% sequence homology with the amino acid sequence of (I) and having RNA-guided nucleic acid binding activity;
  • the Cas protein has endonuclease activity.
  • nucleases such as AsCas12f1 can be used in combination with other enzyme components or other components to further develop various potential applications of nucleases such as AsCas12f1.
  • a single base editing system based on AsCas12f1 nuclease is developed by fusing an inactive nuclease such as AsCas12f1 and a base deaminase;
  • Reverse transcriptase develops a Prime editing system based on AsCas12f1 nuclease; develops a transcription activation system based on AsCas12f1 nuclease by fusing inactivated AsCas12f1 and a transcription activator; develops a transcription activation system based on AsCas12f1 by fusing inactivated AsCas12f1 with a nucleic acid epigenetic modification enzyme Nuclease epigenetic modification system; using inactivated AsCa
  • AsCas12f1 nuclease variants may have the following specific properties, including but not limited to:
  • deaminase activity which can act on cytosine, guanine or adenine bases and then replicate and repair them within the cell through the deamination site to produce guanine, thymine and guanine respectively;
  • the enzymatic activity can be methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin activity, etc.
  • peptide ligase activity deubiquitinating activity, ribosylation activity, etc.
  • the AsCas12f1 nuclease variant has no cleavage activity. In some embodiments, AsCas12f1 nuclease variants have single-strand cleavage activity. In some embodiments, AsCas12f1 nuclease variants have double-stranded cleavage activity.
  • Having enhanced activity or ability means having an activity or ability that is increased by at least 1%, 5%, 10%, 20%, 30%, 40%, or 50% compared to wild-type AsCas12f1 nuclease.
  • Having reduced activity and ability refers to having an activity or ability of less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% relative to the wild-type AsCas12f1 nuclease.
  • AsCas12f1 and “AsCas12f1 nuclease” include wild-type AsCas12f1 nuclease and all variants thereof. Those skilled in the art can determine the type of AsCas12f1 nuclease variant by routine means without limitation. Those cited above.
  • Each component in the system of the present invention can be transported by means of carriers.
  • methods that can be used include, but are not limited to, nanoparticles, liposomes, ribonucleoproteins, small molecule RNA-conjugates, chimeras, and RNA-fusion protein complexes, etc.
  • the system of the present invention may further include one or more donor templates.
  • the donor template includes donor sequences for insertion of a target gene.
  • the system of the present invention can edit or modify DNA at multiple locations in cells for gene therapy, including but not limited to gene therapy for diseases, biological research, and improvement or enhancement of crop resistance. Yield etc.
  • the present invention also provides a composition, which contains one or more of the above-mentioned nuclease or polynucleotide encoding the same, gRNA or polynucleotide encoding the same, recombinant expression vector, system, and can also Includes acceptable carriers, media, etc.
  • the acceptable carriers and media such as sterile water or physiological saline, stabilizers, excipients, antioxidants (ascorbic acid, etc.), buffers (phosphoric acid, citric acid, other organic acids, etc.), preservatives, surface Active agents (PEG, Tween, etc.), chelating agents (EDTA, etc.), adhesives, etc.
  • polypeptides such as serum albumin, gelatin or immunoglobulin; amino acids such as glycine, glutamine, asparagine, arginine and lysine; sugars such as polysaccharides and monosaccharides or Carbohydrates; sugar alcohols such as mannitol or sorbitol.
  • aqueous solutions for injection such as physiological saline, isotonic solutions containing glucose or other auxiliary drugs, such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride
  • appropriate Solubilizers include alcohols (ethanol, etc.), polyols (propylene glycol, PEG, etc.), nonionic surfactants (Tween 80, HCO-50), etc.
  • the composition includes gRNA and a buffer for stabilizing the nucleic acid.
  • the present invention also provides a kit comprising the system or composition as described above.
  • the kit may further include one or more additional reagents, for example selected from: dilution buffer; wash buffer; control reagents, etc.
  • the kit includes (a) an AsCas12f1 nuclease or a nucleic acid encoding an AsCas12f1 nuclease as described above; and (b) a gRNA or a nucleic acid encoding the gRNA, wherein the gRNA is capable of converting The AsCas12f1 nuclease or variant thereof is directed to a target polynucleotide sequence.
  • the kit further contains a donor template comprising a heterologous polynucleotide sequence capable of being inserted into the target polynucleotide sequence.
  • the present invention also provides a gene editing method, which involves contacting the target gene with the gene editing system as described above to achieve editing of the target gene.
  • the method of the present invention can be used to target, edit, modify or manipulate target genes (such as target DNA) in cells or in vivo, ex vivo cells or cell-free systems.
  • the method includes: adding the AsCas12f1 nuclease as described above Or the polynucleotide encoding it, gRNA or the polynucleotide encoding it, recombinant expression vector, system, composition, etc. are introduced into the kit in vivo, in vitro cells or cell-free systems to target, edit and modify the target gene. or manipulation.
  • the method includes the following:
  • sgRNA gRNA
  • a nucleic acid e.g., DNA
  • the gene editing method of the present invention includes the following steps:
  • the present invention provides an in vitro DNA cutting method based on the engineering optimized CRISPR/AsCas12f1 gene editing system, including: wild guide RNA or guide RNA variant and wild AsCas12f1 nuclease or variant thereof, and The target DNA sequence is mixed in a specific buffer for the reaction.
  • the in vitro DNA cleavage method described in the present invention can be routine in the art.
  • the dosage of the AsCas12f nuclease can be used in the reaction with other Cas nucleases in the field.
  • the AsCas12f nuclease and the complete guide RNA The molar ratio is 1:2; the reaction temperature of the cutting method is preferably 45°C, and the time gradient of the reaction rate comparison is preferably 0, 0.5, 1, 2, 4, 8, 16, 32, 64 minutes .
  • the targeting sequence is preferably a DNA fragment with a length of 20 bp after the PAM sequence.
  • the AsCas12f1 nuclease is guided to the target gene through a processed or unprocessed form of guide RNA.
  • the AsCas12f1 nuclease and guide RNA form a complex to recognize the PAM sequence on the target gene.
  • the method further includes the step of introducing a donor template comprising a heterologous polynucleotide sequence into the cell.
  • the present invention also provides the above-mentioned guide RNA, isolated polynucleotide, construct, expression system, gene editing system, pharmaceutical composition or said method for targeting genes and genes in vivo, ex vivo cells or cell-free environment. /or its related peptides for use in gene editing.
  • the in vitro cells are selected from at least one of bacterial cells, archaeal cells, fungal cells, protist cells, viral cells, plant cells and animal cells.
  • the gene editing is selected from the group consisting of: gene cutting, gene deletion, gene insertion, point mutation, transcription inhibition, transcription activation, base editing and guided editing, including but not limited to:
  • AsCas12f1 nuclease has an enzymatic activity that modifies the target gene in a manner other than introducing a double-strand break; the enzymatic activity may be possessed by AsCas12f1 itself, or by, for example, adding an enzyme with The active heterologous polypeptide is fused to the AsCas12f1 nuclease to form a chimeric AsCas12f1 nuclease.
  • the enzyme activities include but are not limited to methyltransferase activity, deamination activity, dismutase activity, alkylation activity, and demethylation. Enzyme activity, DNA repair activity, transposase activity, recombinase activity, DNA damage activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, etc.).
  • the gene editing is gene deletion or gene cutting; the gene editing can be used to achieve one or more of the following including but not limited to the correction of pathogenic sites, gene function research, enhancement of cell function, cell therapy, etc. .
  • the AsCas12f1 nuclease of the present invention or the polynucleotide encoding the same, gRNA or the polynucleotide encoding the same, recombinant expression vector, system, composition and kit can be applied in the research field, diagnostic field, industrial field (such as microbial engineering), Drug discovery (such as high-throughput screening), target identification, imaging fields, and therapeutic areas, etc.
  • the target gene is target DNA.
  • the target DNA can be in vitro naked DNA that is not bound to DNA-associated proteins.
  • the target DNA is chromosomal DNA in cells in vitro.
  • the target gene is target RNA.
  • the target DNA is contacted with a targeting complex comprising the AsCas12f1 nuclease and a gRNA, the gRNA providing target specificity to the targeting complex by comprising a nucleotide sequence complementary to the target DNA; AsCas12f1 nucleic acid Enzymes provide site-specific activity.
  • the targeting complex modifies the target DNA, resulting in, for example, DNA cleavage, DNA methylation, DNA damage, DNA repair, and the like.
  • the targeting complex modifies a polypeptide associated with the target DNA (e.g., a histone, a DNA-binding protein, etc.), resulting in, for example, methylation of the target DNA-associated polypeptide-histone, histone acetylation, histone acetylation, etc. Protein ubiquitination, etc.
  • AsCas12f1 nuclease or a nucleic acid containing a nucleotide sequence encoding a polypeptide of AsCas12f1 nuclease can be introduced into cells by known methods.
  • gRNA or a nucleic acid comprising a nucleotide sequence encoding gRNA can be introduced into a cell by well-known methods.
  • Well-known methods include DEAE-dextran-mediated transfection, liposome-mediated transfection, virus or phage infection, lipofection, transfection, conjugation, protoplast fusion, polyethylenimine-mediated guided transfection, electroporation, calcium phosphate precipitation, gene gun, calcium phosphate precipitation, microinjection, nanoparticle-mediated nucleic acid delivery, etc. Plasmids are delivered, for example, by electroporation, calcium chloride transfection, microinjection, and lipofection.
  • cells are contacted with viral particles comprising nucleic acid encoding gRNA and/or AsCas12f1 nuclease and/or chimeric AsCas12f1 nuclease and/or donor polynucleotide.
  • a nuclease cleaves target DNA in a cell to create a double-stranded break, which is then repaired by the cell, typically in the following manner: non-homologous end joining (NHEJ) and homologous end joining The Restoration of Sex Guidance.
  • NHEJ non-homologous end joining
  • homologous end joining The Restoration of Sex Guidance.
  • the invention also provides a genetically modified cell, including a host cell that has been genetically modified with the above-mentioned AsCas12f1 nuclease or a polynucleotide encoding the same, gRNA or a polynucleotide encoding the same, a recombinant expression vector, a system, or a composition. Grooming.
  • the effective dosage of gRNA and/or AsCas12f1 nuclease and/or recombinant expression vector and/or donor polynucleotide is routine for those skilled in the art. This may vary depending on the route of administration and the nature of the condition being treated.
  • the bacteria or prokaryotic bacteria may be Escherichia coli, Klebsiella pneumoniae, Bacteroides ovatus, Campylobacter jejuni, Staphylococcus saprophyticus, Enterococcus faecalis, Bacteroides thetaiotaomicron, Bacteroides vulgaris, Bacteroides monomorpha, Bacteroides, Lactobacillus casei, Bacteroides fragilis, Acinetobacter reuteri, Fusobacterium nucleatum, Bacteroides johnsonii, Bacteroides arabidopsis, Lactobacillus rhamnosus, Bacteroides marseillei, Parabacteroides faecalis, Clostridium death Bacillus and Bifidobacterium breve, etc.
  • the eukaryotic cells include but are not limited to mammalian cells, fungi and other eukaryotic cells.
  • the fungi include yeast and Aspergillus, such as Saccharomyces cerevisiae, Hansenula polymorpha, Pichia pastoris, Kluyveromyces fragilis, Kluyveromyces lactis, Schizosaccharomyces pombe and Candida albicans.
  • a novel genome editing method based on extremely small CRISPR/AsCas12f1 nuclease is disclosed.
  • the invention shows that, through the guidance and positioning functions of guide RNA, AsCas12f1 can precisely cut genomic DNA and achieve double-stranded breaks in genomic DNA. Utilizing the host cell's own or exogenous repair mechanisms, this system can efficiently and accurately achieve gene editing in living cells.
  • AsCas12f1 "AsCas12f1 nuclease”
  • AsCas12f1 polypeptide AsCas12f1 protein
  • AsCas12f1 protein AsCas12f1 protein
  • guide RNA guide RNA
  • gRNA single gRNA
  • chimeric gRNA chimeric gRNA
  • homology refers to the sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing corresponding positions in different polypeptides or nucleic acid molecules. When the same position in the sequence of the molecule being compared is occupied by the same base or amino acid in different sequences, then the molecule is at that position. Homogenous. The degree of homology between sequences is determined as a function of the number of matches or homologous positions shared by the sequences. An "unrelated" or “non-homologous" sequence should have less than 20% homology to one of the sequences disclosed herein.
  • a polynucleotide or polynucleotide region has a certain percentage of sequence homology (e.g., 20%, 30%) with another polynucleotide or polynucleotide region (or polypeptide or polypeptide region). , 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99%) refers to the percentage of bases (or amino acids) are the same.
  • sequence homology e.g. 20%, 30%
  • polynucleotide and “oligonucleotide” are used interchangeably and they refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides or its analogues. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown.
  • polynucleotides include, but are not limited to, the following: genes or gene fragments (including probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, Ribozymes, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers.
  • Polynucleotides also include modified nucleotides, such as methylated nucleotides and nucleotide analogs.
  • any embodiment of a polynucleotide disclosed herein includes its double-stranded form and any of the two complementary single-stranded forms known or predicted to constitute the double-stranded form.
  • the term “encoding” means that the polynucleotide "encodes" a polypeptide, meaning that in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or Or translated to produce a polypeptide of interest and/or a fragment thereof, or to produce an mRNA capable of encoding the polypeptide of interest and/or a fragment thereof.
  • the antisense strand refers to the sequence that is complementary to the polynucleotide and from which the coding sequence can be deduced.
  • genomic DNA refers to the DNA of the genome of an organism, including the DNA of the genome of bacteria, archaea, fungi, protists, viruses, plants or animals.
  • Manipulating DNA includes binding, nicking one strand, or cleaving both strands of DNA, or includes modifying or editing DNA or polypeptides that bind to DNA.
  • Manipulating DNA can silence, activate or regulate the expression of RNA or polypeptide encoded by the DNA (so that it is not transcribed, or reduces the transcription activity, or makes it not translated, or reduces the level of translation), or prevents or enhances the interaction of the polypeptide with the DNA. combine.
  • Cleavage can be performed by a variety of methods, such as enzymatic or chemical hydrolysis of phosphodiester bonds; cleavage can be single-stranded or double-stranded; DNA cleavage can result in blunt or staggered ends.
  • hybridizable or “complementary” or “substantially complementary” means that a nucleic acid (e.g., RNA) contains a nucleotide sequence that enables it to react at appropriate in vitro and/or in vivo temperatures and solution ionic strengths. Non-covalently bind to another nucleic acid under conditions in a sequence-specific, antiparallel manner, i.e., form Watson-Crick base pairs and/or G/U base pairs, “anneal” or “hybridize” ".
  • sequence of a polynucleotide need not be 100% complementary to the sequence of a target nucleic acid to which it specifically hybridizes.
  • Polynucleotides can hybridize on one or more segments.
  • the polynucleotide may comprise at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence complementarity to a target region within the target nucleic acid sequence to which it is targeted.
  • peptide refers to polymeric forms of amino acids of any length, which may include both coded and non-coded amino acids, chemically or biochemically modified or derivatized Amino acids, and polypeptides with modified peptide backbones.
  • RNA RNA that is translated into a protein
  • RNA RNA that is not translated into a protein
  • a "protein coding sequence” or a sequence encoding a specific protein or polypeptide is a nucleic acid sequence that is transcribed into mRNA (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo or in vitro under the control of appropriate regulatory sequences .
  • vector or "expression vector” is a replicon, such as a plasmid, phage, virus or cosmid, to which another DNA segment, i.e., an "insert” can be attached so that the attached segment can be expressed in a cell copy in.
  • expression cassette encompasses a DNA coding sequence operably linked to a promoter.
  • "Operably connected” means connected in parallel, with the components in a relationship that allows them to function in their intended manner.
  • recombinant expression vector or "DNA construct” are used interchangeably in the present invention to refer to a DNA molecule comprising a vector and at least one insert. Recombinant expression vectors are typically produced for the purpose of expressing and/or amplifying inserts or for the construction of other recombinant nucleotide sequences.
  • the cell When foreign DNA, such as a recombinant expression vector, has been introduced into a cell, the cell has been "genetically modified” or “transformed” or “transfected” by the DNA. The presence of foreign DNA results in permanent or transient genetic changes.
  • the transforming DNA may or may not be integrated into the cell's genome.
  • target DNA is a DNA polynucleotide comprising a "target site” or “target sequence”.
  • target site a DNA polynucleotide comprising a "target site” or "target sequence”.
  • target site a DNA polynucleotide comprising a "target site” or "target sequence”.
  • target site a DNA polynucleotide comprising a "target site” or "target sequence”.
  • target site RNA molecules contain sequences that bind, hybridize, or are complementary to target sequences within target DNA, thereby targeting the bound polypeptide to a specific location within the target DNA (target sequence).
  • “Cleaving” refers to the break of the covalent backbone of the DNA molecule.
  • nuclease and “endonuclease” are used interchangeably to refer to enzymes having endonucleic acid degrading catalytic activity for polynucleotide cleavage.
  • the "cleavage domain” or “active domain” or “nuclease domain” of a nuclease refers to a polypeptide sequence or domain within a nuclease that has catalytic activity for DNA cleavage.
  • the cleavage domain may be contained in a single polypeptide chain, or the cleavage activity may result from the association of two or more polypeptides.
  • targeting polypeptide or "RNA-binding site directed polypeptide” refers to a polypeptide that binds RNA and is targeted to a specific DNA sequence.
  • leader sequence or DNA-targeting segment (or “DNA-targeting sequence”) encompasses a nucleotide sequence complementary to a specific sequence within the target DNA, referred to in the present invention as a “protospacer-like” sequence (Complementary strand of target DNA).
  • HDR Homology-directed repair
  • homology-directed repair may result in changes to the target molecule sequence (e.g., insertions, deletions, mutation).
  • nonhomologous end joining refers to the repair of double-stranded breaks in DNA by directly joining the broken ends to each other without the need for homologous templates. NHEJ often results in deletions of nucleotide sequences near the double-strand break site.
  • treatment includes preventing the occurrence of a disease or symptom; inhibiting a disease or symptom or alleviating a disease.
  • the terms "individual”, “subject”, “host” and “patient” are used interchangeably in the present invention and refer to any mammalian subject, particularly a human, for whom diagnosis, treatment or therapy is desired. .
  • Example 1 Gene editing of original wild-type AsCas12f1 nuclease and guide RNA variant—sgRNA_T1 in mammalian cells
  • the coding gene sequence of the original wild-type guide RNA-sgRNA_V1 described in this example is:
  • the coding gene sequence of the engineered optimized guide RNA variant—sgRNA_T1 described in this example is:
  • the underlined 20 bp sequence in the coding gene sequence of the guide RNA can be rationally designed and replaced according to the target site that needs to be edited.
  • the target site should be located on the target gene, and the 5’ upstream of the target site should contain a preferred PAM sequence, whose sequence feature should be 5’-TTR, where R represents A or G.
  • the original wild-type AsCas12f1 described in this example uses the better AsCas12f1 encoding gene optimized for human codons, and its sequence is
  • the preferred pCMV-AsCas12f1-T1 plasmid sequence (expressing wild-type AsCas12f1 nuclease and guide RNA variant-sgRNA_T1) is:
  • the plasmid expressing wild-type AsCas12f1 nuclease and wild-type RNA-sgRNA_V1 was obtained using a construction method similar to the pCMV-AsCas12f1-T1 plasmid.
  • the underlined 20 bp sequence in the plasmid sequence can be edited and the target site reasonably designed and replaced as needed.
  • the target site should be located on the target gene, and the 5’ upstream of the target site should contain a preferred PAM sequence, whose sequence feature should be 5’-TTR, where R represents A or G.
  • the target sequence in the plasmid can be replaced by conventional technical methods in the field, such as Golden gate assembly or Gibson assembly.
  • the target sequence is as follows:
  • APOB_L1 CTGTCGACACCCAGAATCAT(SEQ ID NO.13);
  • PDCD1_L1 CTGTGAGCTCTAGTCCCCAC(SEQ ID NO.15);
  • VEGFA_L1 CTCTCAAGACCCACAATCCA(SEQ ID NO.17);
  • VEGFA_L2 AAGAAGGGGATGTGGTGCATT(SEQ ID NO.18);
  • human embryonic kidney cell HEK293T was used as the cell used in the experiment.
  • the activated HEK293T cells were cultured in DMEM medium containing 10% FBS. When the cell growth density reached about 90%, they were passaged into a 24-well plate. The number of cells in each well was about 1.0 ⁇ 10 5 . 16-18 hours later, 1.5 ⁇ L of lipofectamine3000 was used to transfect 1000 ng of gene editing plasmids containing different target sequences expressing wild-type AsCas12f1 nuclease, sgRNA_V1 or sgRNA_T1 into the cells. After 24 hours, puromycin was added to reach a final concentration of 2 ⁇ g/ml for screening. After continuing to culture for 48 hours, the adherent cells were digested and genomic DNA was extracted.
  • PCR amplifies the target gene fragment of the target sequence, and the PCR product after gel recovery is annealed with NEBuffer2 (NEB). Then T7 endonuclease 1 was added to the reaction system, digested at 37°C for 15 minutes, and then 6 ⁇ Gel Loading Dye was added to terminate the reaction. The reaction products were separated by 6% TBE-PAGE and stained and imaged with 4S Red dye.
  • Figure 1 is a comparison chart of the mammalian cell gene editing results of the original wild-type AsCas12f1 nuclease combined with sgRNA_V1 and sgRNA_T1 respectively.
  • sgRNA_T1 can significantly improve gene editing efficiency at 6 target sites on APOB, HEXA, PDCD1, TP53 and VEGFA genes.
  • primers used to construct plasmids for AsCas12f1 nuclease variants are as follows:
  • A104R-Fw gcgggtctcaaTCTcttgaacctgtctgtggctctc(SEQ ID NO.23);
  • D364R-Fw gcgggtctcagTCTgatcttgatcactttgatgccgg(SEQ ID NO.25);
  • the PCR product is digested by Dpn1 and recovered from the solution. After being assembled by Golden gate assembly technology, it is transformed into Escherichia coli DH5 ⁇ competent cells, coated on an LBA plate with ampicillin, single clones are picked for expansion culture, plasmids are extracted, and sequenced and identified.
  • the target sequences APOB_L1, PDCD1_L1 and VEGFA_L1 were inserted into each variant plasmid respectively.
  • Example 1 human embryonic kidney cells HEK293T were used as experimental cells to conduct gene editing tests.
  • Figure 2 is a comparison chart of mammalian cell gene editing results based on sgRNA_T1, combining the original wild-type AsCas12f1 nuclease and six AsCas12f1 nuclease variants. As shown in the figure, the six AsCas12f1 nuclease variants can improve gene editing efficiency at three target sites on the APOB, PDCD1 and VEGFA genes; among them, AsCas12f1-Evo1 has the best effect.
  • the engineered optimized guide RNA variant described in this example is sgRNA_T1.
  • the engineered optimized AsCas12f1 nuclease variant described in this example is AsCas12f1-Evo1.
  • the engineering-optimized CRISPR/AsCas12f1 gene editing system described in this example is a combination of AsCas12f1-Evo1 and sgRNA_T1.
  • the target sequence replacement method in Example 1 insert the target sequences AAVS1-L1 ⁇ L8, APOB_L1, DNMT1_L1, HBG_L1 ⁇ L2, HEXA_L1, IFN ⁇ _L1 into the pCMV-AsCas12f1-T1-K80R+A104+D364R (Evo1) plasmid respectively.
  • PCSK9_L1, PDCD1_L1, PRNP_L1, TP53_L1 ⁇ L6, VEGFA_L1 ⁇ L4; the target sequences are as follows:
  • AAVS1_L1 AGGAAAGAAGGATGGAGAAA(SEQ ID NO.26);
  • AAVS1_L2 CCTGGACACCCCGTTCTCCT(SEQ ID NO.27);
  • AAVS1_L3 CTTACGATGGAGCCAGAGAG(SEQ ID NO.28);
  • AAVS1_L4 CCTGTGAGATAAGGCCAGTA(SEQ ID NO.29);
  • AAVS1_L5 CTGCCTCCAGGGATCCTGTG(SEQ ID NO.30);
  • AAVS1_L7 TCTGTCCCCTCCACCCCACA(SEQ ID NO.32);
  • AAVS1_L8 GGCAGCTCCCCTACCCCCCT(SEQ ID NO.33);
  • APOB_L1 CTGTCGACACCCAGAATCAT(SEQ ID NO.13);
  • DNMT1_L1 TGTGGCCACAAGGCTCAGTT(SEQ ID NO.34);
  • HBG_L1 CCTTGTCAAGGCTATTGGTC(SEQ ID NO.35);
  • HBG_L2 CCTTGTTCCGATTCAGTCAT(SEQ ID NO.36);
  • IFN ⁇ _L1 ACGATGAGACAGACCCATTA(SEQ ID NO.37);
  • PCSK9_L1 CCCAGAGCATCCCGTGGAAC(SEQ ID NO.38);
  • PDCD1_L1 CTGTGAGCTCTAGTCCCCAC(SEQ ID NO.15);
  • PRNP_L1 TGGCCACATGGAGTGACCTG(SEQ ID NO.39);
  • TP53_L1 ATAAGAGGTCCCAAGACTTA(SEQ ID NO.40);
  • TP53_L3 TCCTGCTTGCTTACCTCGCT(SEQ ID NO.41);
  • TP53_L4 CCTCTTTCCTAGCACTGCCC(SEQ ID NO.42);
  • TP53_L5 GCTGGGGAGAGGAGCTGGTG(SEQ ID NO.43);
  • TP53_L6 CTTACCTCGCTTAGTGCTCC(SEQ ID NO.44);
  • VEGFA_L1 CTCTCAAGACCCACAATCCA(SEQ ID NO.17);
  • VEGFA_L2 AAGAAGGGGATGTGGTGCATT(SEQ ID NO.18);
  • VEGFA_L3 CTGTGATTTCCCCACAAAAG(SEQ ID NO.45);
  • VEGFA_L4 CCTCTTCCGGCCTGGATTGT(SEQ ID NO.46);
  • Example 1 human embryonic kidney cells HEK293T were used as experimental cells to conduct gene editing tests.
  • Figure 3 is a comparison of the gene editing effects of the original wild CRISPR/AsCas12f1 system and the engineered optimized CRISPR/AsCas12f1 system in mammalian cells. As shown in the figure, the optimized CRISPR/AsCas12f1 gene editing system can significantly improve gene editing efficiency on 27 target sites of 11 genes.
  • the sgRNA_T1 in this example is prepared by conventional technical methods in the art, such as in vitro transcription. Can be prepared using HiScribe T7 High Yield RNA Synthesis Kit (NEB). The prepared sgRNA_T1 was purified through phenol-chloroform extraction and ethanol precipitation.
  • the wild original AsCas12f1 nuclease and variant nuclease in this example were prepared by conventional technical methods in the art, such as E. coli recombinant expression and affinity chromatography technology.
  • the nuclease expression construct was transformed into E. coli expression strain BL21(DE3). The next day, the transformants were transferred into 1 L of LB medium and cultured with shaking at 37°C. When OD 600 reaches 0.6, add 0.25 mL of 1M IPTG to the culture solution and continue culturing at 16°C overnight. The strains obtained overnight were collected, disrupted by sonication, and purified using a HisTrap Ni-NTA (Cytiva) chromatography column.
  • the purified protein was labeled using HRV3c protease and then further purified using HiLoad 16/600 Superdex 200pg molecular sieve (Cytiva).
  • the DNA substrate was prepared by conventional technical methods in the art, such as polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the cleavage substrate contains the target sequence in sgRNA_T1, and the upstream of the target sequence contains a PAM sequence, whose sequence feature is 5’-TTR, where R represents A or G.
  • the engineering-optimized CRISPR/AsCas12f1 gene editing system described in this example is a combination of AsCas12f1-Evo1 and sgRNA_T1.
  • Figure 4 is a comparison of the in vitro DNA cutting effects of the original wild CRISPR/AsCas12f1 system and the engineered optimized CRISPR/AsCas12f1 system. As shown in the figure, the optimized CRISPR/AsCas12f1 gene editing system can significantly improve the efficiency and speed of DNA cutting in vitro.

Abstract

提供了一种工程优化的极小型CRISPR/AsCas12f1的新型基因组编辑系统及其方法和应用，其包括：一种新型工程优化向导RNA变体，及包含该变体序列的表达构建体；多种工程优化的AsCas12f1核酸酶变体，所述变体包含第80位、第104位、第364位氨基酸中的一个或几个位点突变，及包含这些变体序列的表达构建体。提供了工程优化的CRISPR/AsCas12f1基因编辑系统，其特征在于野生向导RNA或向导RNA变体与野生AsCas12f1核酸酶或其变体的任意组合。

Description

一种工程优化的核酸酶、向导RNA、编辑系统和应用 技术领域
本发明属于生物技术领域，具体涉及一种一种工程优化的核酸酶、向导RNA、编辑系统和应用，特别涉及一种工程优化的极小型CRISPR/AsCas12f1的新型基因组编辑系统及其应用和编辑方法。
背景技术
基因组编辑技术是指利用基因编辑机器，例如可编程的核酸酶(分子剪刀)，打断特定的基因序列，进而引入基因的插入、缺失或置换，实现对生物体基因组DNA特定片段进行改造，从而达到对目标基因进行编辑的一种基因工程技术。
自CRISPR/Cas(Clustered regularly interspaced short palindromic repeats)基因组编辑系统问世以来，由于其简便性和高效性，已经被广泛应用于生物学、医学、农业等各领域的基础与应用研究。Cas核酸酶利用向导RNA可在多种细胞的基因组特定靶点定位，对其进行切割从而产生DNA双链断裂，然后利用细胞内源或外加的DNA修复机制，例如同源重组和非同源重组末端连接的修复机制实现编辑。根据不同DNA修复通路的激活，基因组编辑将会导致基因的失活或者突变的校正。通过融合失活的Cas核酸酶与碱基脱氨酶或者逆转录酶，CRISPR/Cas系统还可以进一步被改造成碱基编辑系统和先导编辑系统，广泛应用于生物学、农学研究以及疾病治疗。
目前广泛使用的CRISPR/Cas基因组编辑系统主要包括以CRISPR/Cas9和CRISPR/Cas12a两种类型。在这两种类型的基因组编辑系统中，CRISPR效应蛋白核酸酶Cas9和Cas12a均是含有超过1000个氨基酸的大型蛋白，以其为核心开发的基因编辑器均具有较大的分子尺寸，难以被腺相关病毒等常用载体有效包装和递送，这导致CRISPR/Cas9和CRISPR/Cas12a在向细胞递送过程中存在巨大问题。现有技术中存在的极小型核酸酶CRISPR-AsCas12f1基因编辑系统其核酸酶仅包含422个氨基酸，基因尺寸不到Cas9和Cas12a的一半，天然具有基因编辑能力，可在细菌和哺乳动物细胞中实现高效的基因编辑。得益于其紧凑的分子尺寸，无论CRISPR-AsCas12f1基因编辑系统自身，还是其衍生基因编辑工具都能十分容易的实现单AAV的包装与递送。但目前天然AsCas12f1核酸酶的基 因编辑能力仍相对低于广泛应用的Cas9和Cas12a核酸酶，限制了AsCas12f1核酸酶在基因编辑领域的应用。因此，对CRISPR-AsCas12f1基因编辑系统进行工程化改造提高基因编辑的活性意义重大。
发明内容
本发明所要解决的技术问题是克服现有技术中极小型核酸酶的低基因编辑效率，提供一种工程优化的基因编辑系统及其应用和方法：特别是工程优化的极小型CRISPR/AsCas12f1的新型基因组编辑系统及其应用编辑方法。利用本发明中的优化基因编辑系统或者方法可以在细胞内的目标基因上进行精准敲除或精准编辑。
本发明主要通过以下技术方案解决上述技术问题。
本发明的目的之一是提供一种突变型AsCas12f1核酸酶，所述突变型AsCas12f1核酸酶与野生型AsCas12f1核酸酶具有至少50％以上同一性，且相对于野生型AsCas12f1核酸酶包含第80位、第104位、第364位氨基酸中的一个或几个位点突变。
本发明另一目的是提供一种突变型向导RNA，其包含tracrRNA序列和crRNA序列；所述crRNA序列包含能够与靶序列杂交的基因靶向区段和tracr配对物序列；所述tracr RNA序列和tracr配对物序列构成向导RNA的骨架序列；
其中，所述tracrRNA包含如SEQ ID NO.47所示的核苷酸序列或其变体序列；
所述tracr配对物序列包含如SEQ ID NO.48所示的核苷酸序列或其变体序列。
优选地，所述向导RNA还包括连接链序列。
本发明的另一目的是提供一种分离的多核苷酸，其编码如上所述的突变型AsCas12f1核酸酶或如上所述的向导RNA。
本发明的另一目的是提供一种构建体，所述构建体单独或同时含有如上所述的分离的多核苷酸。即，编码所述突变型AsCas12f1核酸酶的分离的多核苷酸和编码所述向导RNA的分离的多核苷酸可以位于相同或不同构建体上。
本发明的另一目的是提供一种表达系统，所述表达系统含有如上所述的构建体或基因组中整合有外源的如上所述的多核苷酸。
本发明的另一目的是提供一种基因编辑系统，所述基因编辑系统包括如上所述的突变型AsCas12f1核酸酶或其编码多核苷酸，以及向导RNA或其编码多核 苷酸。或，本发明所述的基因编辑系统包含核酸酶或其编码多核苷酸，以及如上所述的突变型向导RNA或其编码多核苷酸。
本发明的另一目的是提供一种药物组合物，其包含如上所述的基因编辑系统，以及药学上可接受的载体。
本发明的另一目的是提供一种基因编辑方法，将靶基因与如上所述的基因编辑系统接触，以实现靶基因的编辑。
本发明的另一目的是提供如上所述的突变型AsCas12f1核酸酶、突变型向导RNA、分离的多核苷酸、构建体、表达系统、基因编辑系统、药物组合物或方法在体内、离体细胞或无细胞环境中对靶基因和/或其相关多肽进行基因编辑中的应用。
本发明的另一目的是提供一种遗传修饰的细胞，其通过如上所述的基因编辑系统、药物组合物或所述的方法进行基因编辑获得。
本发明的积极进步效果：
本发明中，经工程优化的向导RNA变体—sgRNA_T1在测试靶点上的基因插入/删除效率相比野生向导RNA—sgRNA_V1，性能提升了5-15％。在sgRNA_T1基础上组合AsCas12f1核酸酶变体，例如AsCas12f1-K80R，AsCas12f1-A104R，AsCas12f1-D364R，AsCas12f1-A104+K80R，AsCas12f1-A104+D364R，AsCas12f1-K80R+A104+D364R均能更进一步提升基因插入/删除效率20-30％。本发明中工程优化的CRISPR/AsCas12f1基因编辑系统相比原始野生系统，在哺乳动物细胞中的11个基因的27个靶位点的基因编辑效率均有明显提高。本发明进一步提高了极小型CRISPR/Cas12f系统在细胞基因编辑中的适用性，可以实现细胞内的精准编辑基因编辑。
附图说明
图1为原始野生AsCas12f1核酸酶分别组合sgRNA_V1和sgRNA_T1的哺乳动物细胞基因编辑结果比较图。如图所示，sgRNA_T1可在APOB、HEXA、PDCD1、TP53和VEGFA基因上6个靶位点明显提升基因编辑效率。
图2为在sgRNA_T1基础上，分别组合原始野生AsCas12f1核酸酶和6种AsCas12f1核酸酶变体的哺乳动物细胞基因编辑结果比较图。如图所示，6种变体均可在APOB、PDCD1和VEGFA基因上3个靶位点上提升基因编辑效率。其 中AsCas12f1-Evo1效果最佳。
图3为原始野生CRISPR/AsCas12f1系统与工程优化的CRISPR/AsCas12f1系统在哺乳动物细胞中基因编辑效果的比较图。如图所示，工程优化后的CRISPR/AsCas12f1基因编辑系统可11个基因的27个靶位点上显著提升基因编辑效率。
图4为原始野生CRISPR/AsCas12f1系统与工程优化的CRISPR/AsCas12f1系统在体外切割DNA效果的比较图。如图所示，工程优化后的CRISPR/AsCas12f1基因编辑系统能显著提升体外DNA切割的速度。
图1～4中，WT代表野生型AsCas12f1核酸酶，V1代表野生型向导RNA-sgRNA_V1，T1代表向导RNA变体-sgRNA_T1。
具体实施方式
为更好的说明本发明的目的、技术方案和积极进步的效果，下面将结合附图和具体实施例对本发明作进一步阐述。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解，在阅读了本发明讲授的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。例如，通过融合失活的工程优化AsCas12f1和碱基脱氨酶开发基于AsCas12f1的单碱基编辑系统；通过融合失活的AsCas12f1和逆转录酶开发基于AsCas12f1的Prime编辑系统；通过融合失活的AsCas12f1和转录激活因子，开发基于AsCas12f1的转录激活系统；通过融合失活的AsCas12f1核酸表观修饰酶，开发基于AsCas12f1的表观修饰系统；利用失活的AsCas12f1，开发基于AsCas12f1的转录抑制系统。
本发明中主要使用的CRISPR系统是V-F型CRISPR/Cas12f系统，其中效应蛋白主要为原始野生Acidibacillus sulfuroxidans Cas12f1(AsCas12f1)核酸酶及其各种变体，其中向导RNA主要为原始野生sgRNA_V1和变体sgRNA_T1。AsCas12f1核酸酶或其变体在对应的向导RNA或变体的指导下能准确定位到目标基因，对基因组DNA进行切割，实现基因组DNA双链断裂。利用宿主细胞自身或外源的修复机制，该系统能够高效和精确地实现活细胞内基因编辑。
本发明的目的之一是提供一种突变型AsCas12f1核酸酶，所述突变型AsCas12f1核酸酶与野生型AsCas12f1核酸酶具有至少50％以上同一性，且相对 于野生型AsCas12f1核酸酶包含第80位、第104位、第364位氨基酸中的一个或几个位点突变；即所述变异型AsCas12f1核酸酶可以包括一个第80位氨基酸的突变，或一个第104位氨基酸的突变，或一个第364位氨基酸的突变，或同时包含第80位和第104位氨基酸的突变，或同时包含第80位和第364位氨基酸的突变，或同时包含第80位、第104位和第364位氨基酸的突变。所述各位点突变后的氨基酸可以相同或不同，优选地，各位点突变后的氨基酸相同，即当所述突变型AsCas12f1核酸酶包含两个或三个位点突变时，所述两个突变或三个突变位点突变为相同或不同的氨基酸，优选突变为相同的氨基酸；例如，两个突变或三个突变位点突变为与野生型位点不同的其他任一氨基酸。在一些实施方式中，所述突变是指突变为精氨酸。在一些实施方式中，一个氨基酸的突变是指突变为精氨酸。在另一些实施方式中，两个或三个氨基酸的突变是指突变为精氨酸。在一些实施方式中，本发明提供一种或几种工程优化的AsCas12f1核酸酶变体，及包含这些变体序列的表达构建体。
本发明所述的AsCas12f1核酸酶变体有AsCas12f1-K80R，AsCas12f1-A104R，AsCas12f1-D364R，AsCas12f1-A104+K80R，AsCas12f1-A104+D364R，AsCas12f1-K80R+A104+D364R。其中最优选的变体为AsCas12f1-K80R+A104+D364R(以下简称AsCas12f1-Evo1)。
其中，变体AsCas12f1-K80R氨基酸序列优选包含如下所示的序列：
Figure PCTCN2022113357-appb-000001
变体AsCas12f1-A104R氨基酸序列优选包含如下所示的序列：
Figure PCTCN2022113357-appb-000002
Figure PCTCN2022113357-appb-000003
变体AsCas12f1-D364R氨基酸序列优选包含如下所示的序列：
Figure PCTCN2022113357-appb-000004
变体AsCas12f1-A104R+K80R氨基酸序列优选包含如下所示的序列：
Figure PCTCN2022113357-appb-000005
突变体AsCas12f1-A104R+D364R氨基酸序列优选包含如下所示的序列：
Figure PCTCN2022113357-appb-000006
变体AsCas12f1-K80R+A104R+D364R(后称AsCas12f1-Evo1)氨基酸序列优选包含如下所示的序列：
Figure PCTCN2022113357-appb-000007
本发明另一目的是提供一种突变型向导RNA(gRNA)，其包含tracrRNA序列和crRNA序列和连接链序列；所述crRNA序列包含能够与靶序列杂交的基因靶向区段和tracr配对物序列；所述tracrRNA序列和tracr配对物序列构成向导RNA的骨架序列；
其中，所述tracrRNA包含如SEQ ID NO.47所示的核苷酸序列或其变体序列；所述tracr配对物序列包含如SEQ ID NO.48所示的核苷酸序列或其变体序列。
野生型sgRNA_V1对应的tracrRNA序列：
Figure PCTCN2022113357-appb-000008
Figure PCTCN2022113357-appb-000009
在一实施方式中，所述变体的tracrRNA序列为SEQ ID NO.50，即sgRNA_T1对应的tracrRNA序列：
Figure PCTCN2022113357-appb-000010
野生型sgRNA_V1对应的tracrRNA配对物序列：
5’-guuugcgagcuagcuuguggagugugaac(SEQ ID NO.48)；
在一实施方式中，所述变体的tracrRNA配对物序列为SEQ ID NO.51，即sgRNA_T1对应的tracrRNA配对物序列：5’-uguggagugugaac(SEQ ID NO.51)；
本发明所述向导RNA中，所述tracr RNA序列和tracr配对物序列之间还包括连接链序列；优选地，所述连接链序列包含5’-AAGG、5’-UACU或其变体序列。所述连接链的变体序列是在5’-AAGG、5’-UACU序列的基础上，增加、减少或替换一个或多个核苷酸后获得的序列。本发明所述的野生型sgRNA_V1中tracr RNA序列和tracr配对物序列之间的连接链序列为：5’-AAGG。在一实施方式中，突变型sgRNA_T1中tracr RNA序列和tracr配对物序列之间的连接链序列为：5’-UACU。
本发明所述突变型向导RNA包含在所述tracrRNA中互补配对的碱基对的一个或多个中，配对碱基互换位置后获得的tracrRNA。例如，tracrRNA的第7位碱基C与第16位碱基G互补配对，在将7位设置为G，第16位设置为C后获得的tracrRNA也能实现本发明的技术效果。
本发明所述突变型向导RNA包含在tracrRNA与tracr配对物序列互补配对的碱基对的一个或多个中，配对碱基互换位置后获得的tracrRNA和tracr配对物序列。例如，tracrRNA第119位碱基U与tracr配对物的第21位碱基A互补配对，在将tracrRNA第119位碱基设置为A，tracr配对物第21位设置为U后获得的tracrRNA和tracr配对物序列也能实现本发明的技术效果。
本发明所述突变型向导RNA中，所述基因靶向区段为与靶基因中的靶序列互补的核苷酸序列，位于所述crRNA序列的3’端；所述基因靶向区段识别靶向序列上的PAM序列；优选PAM序列为5’-TTR，其中R代表A或G。所述基因 靶向区段靶向PAM序列之后长度为12～40bp的核酸片段；例如可以是13-20、18-25、22-32、26-37、30-38、32-40个核苷酸的长度，优选的长度为20bp。在一实施方式中，所述的基因靶向区段较佳地为PAM序列之后长度为20bp的核酸片段所对应的RNA序列。在一优选实施方式中，所述基因靶向区段选自SEQ ID NO.19。本发明中，所述向导RNA的靶向区段与靶基因的靶序列之间的互补性百分比可以为至少50％(例如，至少55％，至少60％，至少65％，至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少97％，至少98％，至少99％或100％)。本发明所述突变型向导RNA中，所述基因靶向区段靶向细胞基因组中的至少一个靶序列。
在一些实施方式中，所述突变型向导RNA还包含转录终止子。
本发明所述突变型向导RNA中，所述向导RNA可以为两条链，一条链包含tracrRNA序列，另一条链包含crRNA序列，其中所述tracr配对物序列与所述tracrRNA序列杂交，并形成茎-环结构。本发明所述向导RNA中，所述crRNA序列与所述tracrRNA序列能够通过连接链连接在一起，形成单条的向导RNA骨架序列，即所述向导RNA为一条链，其从5’端至3’端依次包含所述tracr RNA序列、连接链序列和crRNA序列。当所述向导RNA为一条链时，所述tracr RNA序列的3’端与crRNA序列的5’端通过连接链连接。
本发明所述向导RNA中，crRNA与tracrRNA序列作为两条单独的RNA序列，在同时存在情况下可以介导核酸酶如AsCas12f1核酸内切酶活性。或向导RNA骨架序列和与靶序列杂交的DNA-靶向区段连接后得到的针对靶序列的完整向导RNA表达构建体，同样可以介导核酸酶如AsCas12f1核酸内切酶活性。
本发明所述向导RNA中，所述基因靶向区段包含与靶基因中的序列互补的核苷酸序列，该基因靶向序列通过杂交(即碱基配对)以序列特异性方式与靶基因相互作用。可以通过例如基因工程的方式修饰gRNA的基因靶向序列，以使得gRNA与靶基因内的任何所需序列杂交。gRNA通过上述基因靶向序列将结合的多肽引导至靶基因内的特定核苷酸序列。
在一些实施方式中，靶基因是DNA序列。在一些实施方式中，靶基因是RNA序列。
本发明还提供了对所述向导RNA进行改造的方法，包括但不限于对 trans-activating CRISPR RNA(tracrRNA)和CRISPR RNA(crRNA)的单独改造以及组合改造。所述的改造方法包括但不限于对tracrRNA或crRNA的截断、延长或替换不同序列。
本发明所述向导RNA中，所述tracrRNA的变体序列是指在所述SEQ ID NO.47所示的核苷酸序列的5’端和/或3’端增加、减少或替换部分核苷酸后获得的序列。优选地，所述tracrRNA的变体序列是指在所述SEQ ID NO.47所示的核苷酸序列的5’端和/或3’端减少核苷酸后获得的序列，即，可以只在5’端减少或截断一定个数的核苷酸，或只在3’端减少或截断一定数量的核苷酸，或同时在5’端和3’端减少或截断一定个数的核苷酸。进一步优选地，所述tracrRNA的变体序列是指在所述SEQ ID NO.47所示的核苷酸序列的5’端和/或3’端减少1～50nt核苷酸后获得的序列。进一步优选地，所述tracrRNA的变体序列是指在所述SEQ ID NO.47所示的核苷酸序列的3’端减少14nt核苷酸后获得的序列。
所述tracr配对物序列的变体序列是指在所述SEQ ID NO.48所示的核苷酸序列的5’端和/或3’端增加、减少或替换部分核苷酸后获得的序列；优选地，所述tracr配对物序列的变体序列是指在所述SEQ ID NO.48所示的核苷酸序列的5’端和/或3’端减少核苷酸后获得的序列。即，可以只在5’端减少或截断一定个数的核苷酸，或只在3’端减少或截断一定数量的核苷酸，或同时在5’端和3’端减少或截断一定个数的核苷酸。进一步优选地，所述tracr配对物序列的变体序列是指在所述SEQ ID NO.48所示的核苷酸序列的5’端减少1～29nt核苷酸后获得的序列。进一步优选地，所述tracr配对物序列的变体序列是指在所述SEQ ID NO.48所示的核苷酸序列的5’端减少15nt核苷酸后获得的序列。
在一些优选实施方式中，所述tracrRNA的变体序列是指在所述SEQ ID NO.47所示的核苷酸序列的3’端减少14nt核苷酸获得的序列，序列如SEQ ID NO.50所示。
在另一些优选实施方式中，所述tracr配对物序列的变体序列是指在所述SEQ ID NO.48所示的核苷酸序列的5’端减少15nt核苷酸获得的序列；序列如SEQ ID NO.51所示。
在另一些优选实施方式中，所述突变型向导RNA的骨架序列的核苷酸序列如SEQ ID NO.52所示。优选的向导RNA在哺乳动物细胞基因组编辑中，对同一靶序列显示有更高的基因删除和/或基因切割效率。
在一优选实施方式中，本发明提供一种CRISPR/AsCas12f1基因编辑系统的新型工程优化向导RNA(又称为sgRNA_T1)，及包含该变体序列的表达构建体。本发明所述的工程优化的向导RNA序列相比原始野生型向导RNA序列(sgRNA_V1)共截短了29nt，优选的向导RNA的完整序列如下所示：
5’-auucgucgguucagcgacgauaagccgagaagugccaauaaaacuguuaagugguuugguaacgcucgguaagguagccaaaaggcugaaacuccgugcacaaagaccgcacggacgcuucacauacuuguggagugugaac C UCUCAAGACCCACAAUCCA-3’(SEQ ID NO.9)；其中，tracrRNA序列如SEQ ID NO.50所示；tracr配对物序列如SEQ ID NO.51所示；骨架序列为SEQ ID NO.9中不含靶向基因区段的序列，为5’-auucgucgguucagcgacgauaagccgagaagugccaauaaaacuguuaagugguuugguaacgcucgguaagguagccaaaaggcugaaacuccgugcacaaagaccgcacggacgcuucacauacuuguggagugugaac(SEQ ID NO.52)。
其中下划线部分为所述的靶向序列：5’-CUCUCAAGACCCACAAUCCA-3’(SEQ ID NO.19)；可与靶基因上优选的PAM序列之后长度为20bp的DNA片段进行碱基互补配对。优选的PAM序列为5’-TTR，其中R代表A或G。划线序列可根据靶基因位点的不同进行相应的替换。
野生型向导RNA序列(sgRNA_V1)的完整序列如SEQ ID NO.10所示：5’-auucgucgguucagcgacgauaagccgagaagugccaauaaaacuguuaagugguuugguaacgcucgguaagguagccaaaaggcugaaacuccgugcacaaagaccgcacggacgcuucacauauagcucauaaacAAGG guuugcgagcuagcuuguggagugugaac CUCUCAAGACCCACAAUCCA-3’；其中，tracrRNA序列如SEQ ID NO.47所示；tracr配对物序列如SEQ ID NO.48所示；骨架序列为5’-auucgucgguucagcgacgauaagccgagaagugccaauaaaacuguuaagugguuugguaacgcucgguaagguagccaaaaggcugaaacuccgugcacaaagaccgcacggacgcuucacauauagcucauaaacAAGG guuugcgagcuagcuuguggagugugaac(SEQ ID NO.49)。
本发明还提供了修饰型突变向导RNA，其可以通过修饰用于实现与靶基因 内的任何所需序列杂交；或者，通过对gRNA修饰来改变gRNA本身的特性，如通过修饰增强gRNA的稳定性，包括但不限于通过增加其对细胞中存在的核糖核酸酶(RNase)降解的抗性，从而延长其在细胞中的半衰期；或者，其可以通过修饰用于增强包含gRNA和核酸内切酶(例如AsCas12f1核酸酶)的CRISPR-AsCas12f1基因组编辑复合体的形成或其稳定性；或者，其可以通过修饰用于增强基因组编辑复合体的特异性；或者，其可以通过修饰用于增强基因组编辑复合体与基因组中靶序列之间相互作用的起始位点，稳定性或动力学；或者，其可以通过修饰用于降低引入细胞中的RNA引发先天免疫反应的可能性或程度等。本发明中，可以通过对gRNA修饰来改变CRISPR-AsCas12f1系统(如下文中所述)的多种特性，如增强CRISPR-AsCas12f1基因组编辑复合物的形成、中靶活性、特异性、稳定性或动力学特性。可以采用本领域已知的修饰方式对RNA进行修饰，包括但不限于在嘧啶的核糖、碱基残基或RNA的3'端的反向碱基上的2'-氟、2'-氨基修饰等。本发明中，可以采用任意一种修饰或多种修饰组合对gRNA进行修饰。在一些实施方式中，通过修饰引入细胞中的sgRNA，以编辑任何一个或多个基因组的基因座。
本发明的另一目的是提供一种分离的多核苷酸，其编码如上所述的突变型AsCas12f1核酸酶或如上所述的向导RNA。
本发明的另一目的是提供一种构建体，所述构建体含有如上所述的分离的多核苷酸。所述构建体通常可以通过将所述分离的多核苷酸插入合适的表达载体中构建获得，本领域技术人员可选择合适的表达载体。所述构建体例如可以是重组表达载体，可以使用任何合适的表达载体，只要它与宿主细胞相容即可，包括但不限于，病毒载体(例如基于痘苗病毒的病毒载体；脊髓灰质炎病毒；腺病毒；腺相关病毒；SV40；单纯疱疹病毒；人免疫缺陷病毒；反转录病毒载体(例如鼠白血病病毒，脾脏坏死病毒，以及衍生自反转录病毒的载体，例如劳斯肉瘤病毒，Harvey肉瘤病毒，禽类白血病病毒，慢病毒，人免疫缺陷病毒，骨髓增生肉瘤病毒和乳腺肿瘤病毒)等。
在某些实施方式中，在相同细胞中同时使用多种核酸酶或gRNA以同时调节相同靶基因或不同靶基因上的不同位置的转录。当多个核酸酶或gRNA同时使用时，它们可以存在于相同的表达载体上或不同的载体上，也可以同时表达； 当存在于相同的载体上时，它们可以在相同的控制元件下进行表达。
在某些实施方式中，编码核酸酶或gRNA的核苷酸序列可操作地连接至控制元件，例如转录控制元件，例如启动子。在某些实施方式中，编码核酸酶或gRNA的核苷酸序列可操作地连接至诱导型启动子。在某些实施方式中，编码核酸酶或gRNA的核苷酸序列可操作地连接至组成型启动子。转录控制元件可以在真核细胞，例如哺乳动物细胞(HEK293T细胞)；或原核细胞(例如细菌或古细菌细胞)中起作用。在某些实施方式中，编码核酸酶或gRNA的核苷酸序列可操作地连接至多个控制元件，其允许在原核和真核细胞两者中表达编码核酸酶或gRNA的核苷酸序列。
本发明中，所述核酸酶或gRNA可以通过人工合成方式进行合成，从而使得能够容易地对其进行多种修饰。所述修饰可以采用本领域公知的任何修饰方式，例如，使用polyA尾巴，添加5'帽类似物，5'或3'非翻译区(UTR)，5'或3'端包括硫代磷酸化2’-O-甲基核苷酸或用磷酸酶处理以去除5'末端磷酸酯等。
在一些实施方案中，所述编码核酸酶或gRNA的核苷酸序列包含一种或多种修饰，其可用于例如增强活性，稳定性或特异性，改变递送，减少宿主细胞中的先天免疫应答或用于其他增强。
在一些实施方式中，将增强编码核酸酶或gRNA的核苷酸序列的活性，细胞分布或细胞摄取的一个或多个靶向部分或缀合物化学连接至核酸酶或gRNA。所述靶向部分或缀合物可包括与功能基团共价结合的缀合物基团；缀合物基团包括报告分子，多胺，聚乙二醇。在一些实施方式中，将增强药效学性质的基团连接至核酸酶或gRNA，增强药效学性质的基团包括改善摄取，增强对降解的抗性和/或增强与靶核酸的序列特异性杂交的基团。
本发明中，包含编码核酸酶或gRNA的多核苷酸的核酸可以是核酸模拟物。例如具有优良杂交性质的多核苷酸模拟物肽核酸等。
本发明中，所述核酸酶或gRNA，或编码核酸酶或gRNA的多核苷酸适用任何生物或体外环境，包括但不限于是细菌、古细菌、真菌、原生生物、植物或动物。相应地，适用的靶细胞包括但不限于细菌细胞、古细菌细胞、真菌细胞、原生生物细胞、植物细胞或动物细胞。适用的靶细胞可以是任何类型的细胞，包括干细胞，体细胞等。
本发明的另一目的是提供一种表达系统，所述表达系统含有如上所述的构建体或基因组中整合有外源的如上所述的多核苷酸。所述表达系统的宿主细胞选自真核细胞或原核细胞；优选地，所述宿主细胞选自小鼠细胞、人细胞。
本发明的另一目的是提供一种基因编辑系统，所述基因编辑系统包括如上所述的突变型AsCas12f1核酸酶或其编码多核苷酸，以及向导RNA或其编码多核苷酸。或，本发明所述的基因编辑系统包含核酸酶或其编码多核苷酸，以及如上所述的突变型向导RNA或其编码多核苷酸。本发明提供的一种工程优化的CRISPR/AsCas12f1基因编辑系统特征在于野生向导RNA或向导RNA变体与野生AsCas12f1核酸酶或其变体的任意组合使用。当满足以下任一条件即可视为属于该系统范畴：
(1)野生型AsCas12f1核酸酶与优选的向导RNA变体—sgRNA_T1的组合使用。
(2)任意优选的AsCas12f1核酸酶变体与野生型向导RNA—sgRNA_V1的组合使用。
(3)任意优选的AsCas12f1核酸酶变体与优选的向导RNA变体—sgRNA_T1的组合使用。
组合使用的方式包括但不限于本领域常规，例如，在同一构建体中同时表达野生型向导RNA或向导RNA变体与野生型AsCas12f1核酸酶或其变体；在不同构建体中分别表达野生型向导RNA或向导RNA变体与野生型AsCas12f1核酸酶或其变体；体外配伍野生型向导RNA或向导RNA变体与野生型AsCas12f1核酸酶或其变体等。
本发明所述编辑系统中，编码所述核酸酶的多核苷酸包括：只编码核酸酶的编码序列；核酸酶的编码序列和各种附加编码序列；核酸酶的编码序列(和任选的附加编码序列)以及非编码序列。编码所述向导RNA的多核苷酸包括：只编码向导RNA的编码序列；向导RNA的编码序列和各种附加编码序列；向导RNA的编码序列(和任选的附加编码序列)以及非编码序列。在一些实施方式中，所述基因编辑系统包含一个或多个载体；所述一个或多个载体包含(i)第一调控元件，所述第一调控元件可操作地连接至所述核酸酶的编码多核苷酸；以及(ii)第二调控元件，所述第二调控元件可操作地连接至所述向导RNA核苷酸序列的编码多 核苷酸；所述(i)和(ii)位于相同或不同载体上。在一些实施方式中，所述基因编辑系统包含(i)核酸酶或其变体，以及(ii)包含所述向导RNA的编码序列的载体。在另一实施方式中，所述系统包含gRNA和核酸酶复合物。
所述第一调控元件可以调控所述核酸酶或其变体的编码多核苷酸的转录。所述核酸酶或其变体的编码多核苷酸可以是一个或多个，所述第一调控元件可以是一个或多个。所述第二调控元件可以调控所述向导RNA的编码多核苷酸的转录。所述向导RNA的编码多核苷酸可以是一个或多个，所述第二调控元件可以是一个或多个。
本发明所述系统可以包含一个gRNA或同时包含多个gRNA。在一实施方式中，所述系统同时包含多个gRNA以同时修饰相同靶标DNA或不同靶标DNA上的不同位置。在一实施方案中，两个或更多个向导RNA靶向相同的基因或转录本或基因座。在一实施方案中，两个或更多个向导RNA靶向不同的不相关基因座。在一些实施方案中，两个或更多个向导RNA靶向不同但相关的基因座。
本发明所述基因编辑系统中，当所述向导RNA为如上所述的突变型向导RNA时，所述核酸酶是CRISPR核酸酶；优选地，所述核酸酶选自Cas9、Cas12、Cas13蛋白家族或其变体；进一步优选地，所述Cas核酸酶选自SpCas9及其突变体、SaCas9及其突变体、Cas12a及其突变体或Cas12f及其突变体；更进一步优选为Cas12f及其突变体。在一些实施方式中，核酸酶直接作为蛋白提供；例如可以使用原生质球转化用外源蛋白和/或核酸转化真菌的方式。可以通过任何合适的方法将核酸酶引入细胞，如注射方式等。本发明所述基因编辑系统识别靶向序列上的PAM序列；优选地PAM序列为5’-TTR，其中R代表A或G。所述基因编辑系统靶向PAM序列之后长度为12～40bp的核酸片段，优选的长度为20bp。所述基因编辑系统靶向细胞基因组中的至少一个靶序列。
在某些实施方式中，编码核酸酶的核酸为DNA。在某些实施方式中，编码核酸酶的核酸为RNA。在某些实施方式中，编码核酸酶的核酸是表达载体，例如重组表达载体。可以使用任何合适的表达载体，只要它与宿主细胞相容即可，包括但不限于，病毒载体(例如基于痘苗病毒的病毒载体；脊髓灰质炎病毒；腺病毒；腺相关病毒；SV40；单纯疱疹病毒；人免疫缺陷病毒；反转录病毒载体(例如鼠白血病病毒，脾脏坏死病毒，以及衍生自反转录病毒的载体，例如劳斯 肉瘤病毒，Harvey肉瘤病毒，禽类白血病病毒，慢病毒，人免疫缺陷病毒，骨髓增生肉瘤病毒和乳腺肿瘤病毒)等。
在一实施方式中，所述核酸酶为野生型AsCas12f1，编码所述AsCas12f1核酸酶的核酸如SEQ ID NO.7所示。在一实施方式中，本发明提供了AsCas12f1核酸酶密码子优化的多核苷酸序列，与SEQ ID NO:7具有至少90％、92％、93％、94％、95％、96％、97％、98％、99％、99.2％、99.5％、99.8％、99.9％或100％序列同源性，其编码具有与原始天然核苷酸序列编码的多肽相同功能的多肽。
在某些实施方式中，编码核酸酶的核苷酸序列可操作地连接至控制元件，例如转录控制元件，例如启动子。在某些实施方式中，编码核酸酶的核苷酸序列可操作地连接至诱导型启动子。在某些实施方式中，编码核酸酶的核苷酸序列可操作地连接至组成型启动子。转录控制元件可以在真核细胞，例如哺乳动物细胞(HEK293T细胞)；或原核细胞(例如细菌或古细菌细胞)中起作用。在某些实施方式中，编码核酸酶的核苷酸序列可操作地连接至多个控制元件，其允许在原核和真核细胞两者中表达编码核酸酶的核苷酸序列。在一些实施方案中，所述编码核酸酶的多核苷酸序列可操作地连接至用于在细胞或体外环境中表达的合适的核定位信号。
本发明中，所述编码核酸酶的多核苷酸可以通过人工合成方式进行合成，例如通过化学方法合成，从而使得能够容易地对其进行多种修饰。所述修饰可以采用本领域公知的任何修饰方式。在一些实施方案中，所述编码核酸酶的多核苷酸包含一种或多种修饰，从而使得能够容易地并入许多修饰，例如增强转录活性，改变酶活性，提高其翻译或稳定性(例如增加其对蛋白水解、降解的抗性)或特异性，改变溶解性，改变递送，减少宿主细胞中的先天免疫应答。所述修饰可以采用本领域公知的任何修饰方式。在一些实施方式中，通过修饰引入细胞中的编码核酸酶的DNA或RNA，以编辑任何一个或多个基因组的基因座。在一些实施方式中，编码核酸酶的核酸序列是修饰的核酸，例如密码子优化的。所述修饰可以是单一修饰，或组合修饰。
本发明中，包含编码核酸酶的多核苷酸的核酸可以是核酸模拟物。例如具有优良杂交性质的多核苷酸模拟物肽核酸等。
本发明中，所述核酸酶，或编码核酸酶的多核苷酸适用于任何生物或体外环 境，包括但不限于是细菌(如大肠杆菌、肺炎克雷伯氏菌)、古细菌、真菌、原生生物、植物或动物。相应地，适用的靶细胞包括但不限于真核细胞和原核细胞，例如是细菌细胞、古细菌细胞、真菌细胞、原生生物细胞、植物细胞或动物细胞；所述真核细胞包含哺乳动物细胞和植物细胞，所述原核细胞包括大肠杆菌和肺炎克雷伯氏菌。适用的靶细胞可以是任何类型的细胞，包括干细胞，体细胞等。本发明优选用于哺乳动物细胞HEK293T细胞。所述细胞可以是体内的或离体的。在某些实施方式中，所述核酸酶或编码核酸酶的核酸配制在脂质体或脂质纳米颗粒中。
本发明所述系统中，核酸酶和gRNA可以在宿主细胞中形成复合物，识别靶向基因(如靶向DNA)序列上的PAM序列；所述CRISPR/AsCas12f1基因编辑系统的靶向序列为PAM序列之后长度为20bp的核酸片段(如DNA片段)。在一实施方式中，所述复合物可以选择性地调节宿主细胞中靶DNA的转录。基因编辑系统能够切割靶向DNA的双链，造成DNA断裂。
在一实施方式中，所述系统包含重组表达载体。在一实施方式中，所述系统包含重组表达载体，所述重组表达载体包含(i)编码gRNA的核苷酸序列，其中所述gRNA包含：(a)包含与靶DNA中的序列互补的核苷酸序列的第一区段；和(b)与核酸酶相互作用的第二区段；和(ii)编码核酸酶的核苷酸序列，其中所述核酸酶包含：(a)与所述gRNA相互作用的RNA-结合部分；和(b)调节靶DNA内转录的活性部分，其中靶DNA内调节的转录的位点由所述gRNA确定。
本发明中，还可以通过修饰、突变、DNA改组等方式形成核酸酶变体，使得核酸酶变体具有改善的所期望的特征，例如功能、活性、动力学、半衰期等。所述修饰例如可以是氨基酸的缺失、插入或取代，再例如可以是用来自不同核酸酶的同源或异源切割结构域(例如CRISPR-相关核酸酶的HNH结构域)置换核酸酶如AsCas12f1的“切割结构域”)；通过本领域已知的DNA结合和/或DNA修饰蛋白的任何修饰方法，如甲基化作用、脱甲基作用、乙酰化作用等，例如可以改变AsCas12f1核酸酶的DNA靶向性。所述DNA改组是指在不同来源的AsCas12f1核酸酶的DNA序列之间交换序列片段，以产生编码具有RNA-指导的内切核酸酶活性的合成蛋白的嵌合DNA序列。所述修饰、突变、DNA改组等可 以是单一使用或组合使用。
在一些实施方式中，所述Cas蛋白及其突变体选自：
(I)野生型Cas蛋白或其片段，具有受RNA引导的核酸结合活性；
(II)与(I)的氨基酸序列具有至少50％序列同源性的变体，且具有受RNA引导的核酸结合活性；
(III)根据(I)或(II)，其进一步包括核定位信号片段；
(IV)根据(I)或(II)或(III)，其进一步包含：
(a)一种或多种修饰或突变，其产生具有相比修饰或突变前显著减小的核酸内切酶活性，或使核酸内切酶活性丧失；和/或
(b)具有其他功能活性的多肽或结构域；
(V)根据(I)或(II)或(III)，所述Cas蛋白具有核酸内切酶活性。
在一些实施方式中，核酸酶如AsCas12f1可以通过与其他酶成分或其他成分结合使用，以进一步开发核酸酶如AsCas12f1的各种潜在应用。作为(IV)下的变体非限制性的例子，例如是通过融合失活的核酸酶如AsCas12f1和碱基脱氨酶开发基于AsCas12f1核酸酶的单碱基编辑系统；通过融合失活的AsCas12f1和逆转录酶开发基于AsCas12f1核酸酶的Prime编辑系统；通过融合失活的AsCas12f1和转录激活因子，开发基于AsCas12f1核酸酶的转录激活系统；通过融合失活的AsCas12f1和核酸表观修饰酶，开发基于AsCas12f1核酸酶的表观修饰系统；利用失活的AsCas12f1，开发基于AsCas12f1核酸酶的转录抑制系统。
AsCas12f1核酸酶变体可以具有如下特定性质，包括但不限于：
具有增强的或降低的与靶位结合的能力，或保留了与靶位结合的能力；
具有增强的或降低的核糖核酸内切酶和/或核酸内切酶活性，或保留了核糖核酸内切酶和/或核酸内切酶活性；
具有脱氨酶活性，其可作用于胞嘧啶、鸟嘌呤或腺嘌呤碱基，并随后通过脱氨基位点复制并在细胞内修复，分别产生鸟嘌呤、胸腺嘧啶和鸟嘌呤；
具有调节靶DNA的转录的活性，可以是增加也可以是减少靶DNA中特定位置处的靶DNA转录；
具有改变的DNA靶向性；
增加或降低或维持的稳定性；
可以切割靶DNA的互补链，但具有降低的切割靶DNA的非互补链的能力；
可以切割靶DNA的非互补链，但具有降低的切割靶DNA的互补链的能力；
具有降低的切割靶DNA的互补链和非互补链两者的能力；
具有修饰与DNA相关的多肽(例如组蛋白)的酶活性，酶活性可以是甲基转移酶活性、脱甲基酶活性、乙酰转移酶活性、脱乙酰酶活性、激酶活性、磷酸酶活性、泛素连接酶活性、脱泛素活性、核糖基化活性等中的一种或几种(通过这些酶活性催化对蛋白的共价修饰；例如，AsCas12f1核酸酶变体通过甲基化作用、乙酰化作用、泛素化、磷酸化作用等，修饰组蛋白，以引起组蛋白相关DNA的结构变化，从而控制DNA的结构和特性)。
在一些实施方式中，AsCas12f1核酸酶变体无切割活性。在一些实施方式中，AsCas12f1核酸酶变体具有单链切割活性。在一些实施方式中，AsCas12f1核酸酶变体具有双链切割活性。
具有增强的活性或能力是指，相对于野生型AsCas12f1核酸酶，具有提升至少1％、5％、10％、20％、30％、40％、50％的活性或能力。
具有降低的活性和能力是指相对于野生型AsCas12f1核酸酶，具有小于50％、小于40％、小于30％、小于20％、小于10％、小于5％或小于1％的活性或能力。
本发明所述的这些小的AsCas12f1及其变体可用作本发明下文所述的任何系统、组合物、试剂盒和方法中。
如果没有另外说明，术语“AsCas12f1”、“AsCas12f1核酸酶”包括野生型AsCas12f1核酸酶以及其所有变体，本领域技术人员可以通过常规手段可以确定AsCas12f1核酸酶变体的类型，而不受限于上文所例举的那些。
本发明所述系统中的各组成部分可通过载体方式来运送。例如，对于多核苷酸，可以采用的方法包括但不限于纳米颗粒、脂质体、核糖核蛋白、小分子RNA-缀合物、嵌合体和RNA-融合蛋白复合物等。
本发明所述的系统还可以进一步包括一个或多个供体模板。在某些实施方式中，所述供体模板包含用于插入靶基因的供体序列。
本发明所述系统可以在细胞中的多个位置处编辑或修饰DNA，以用于基因治疗，包括但不限于用于疾病的基因治疗，用于生物学研究，用于农作物抗性改 进或提高产量等。
本发明还提供了一种组合物，其包含如上所述的核酸酶或编码其的多核苷酸、gRNA或编码其的多核苷酸、重组表达载体、系统中的一种或几种，还可以包括可接受的载体、介质等。所述可接受的载体、介质例如无菌水或生理盐水、稳定剂、赋形剂、抗氧化剂(抗坏血酸等)、缓冲剂(磷酸、枸橼酸、其它的有机酸等)、防腐剂、表面活性剂(PEG、Tween等)、螯合剂(EDTA等)、粘合剂等。而且，也可含有其它低分子量的多肽；血清白蛋白、明胶或免疫球蛋白等蛋白质；甘氨酸、谷酰胺、天冬酰胺、精氨酸和赖氨酸等氨基酸；多糖和单糖等糖类或碳水化物；甘露糖醇或山梨糖醇等糖醇。当制备用于注射的水溶液时，例如生理盐水、含有葡萄糖或其它的辅助药物的等渗溶液，如D-山梨糖醇、D-甘露糖、D-甘露糖醇、氯化钠，可并用适当的增溶剂例如醇(乙醇等)、多元醇(丙二醇，PEG等)、非离子表面活性剂(吐温80，HCO-50)等。在一些实施方式中，组合物包含gRNA和用于稳定核酸的缓冲液。
本发明还提供了一种试剂盒，其包括如上所述的系统或组合物。所述试剂盒还可以进一步包括一种或多种例如选自：稀释缓冲液；洗涤缓冲液；对照试剂等的额外试剂。在一些实施方式中，所述试剂盒包括(a)根据上文所述的AsCas12f1核酸酶或编码AsCas12f1核酸酶的核酸；和(b)gRNA或编码所述gRNA的核酸，其中所述gRNA能够将所述AsCas12f1核酸酶或其变体指导至靶多核苷酸序列。在某些实施方式中，所述试剂盒进一步含有包含异源多核苷酸序列的供体模板，其中所述异源多核苷酸序列能够被插入所述靶多核苷酸序列中。
本发明还提供了一种基因编辑方法，将靶基因与如上所述的基因编辑系统接触，以实现靶基因的编辑。本发明方法可以用于在细胞中或在体内、离体细胞或无细胞系统中靶向、编辑、修饰或操纵靶基因(如靶DNA)，所述方法包括：将如上所述的AsCas12f1核酸酶或编码其的多核苷酸、gRNA或编码其的多核苷酸、重组表达载体、系统、组合物等引入试剂盒体内、离体细胞或无细胞系统中，对靶基因进行靶向、编辑、修饰或操纵。在一实施方式中，所述方法包括如下：
(a)将所述AsCas12f1核酸酶或编码AsCas12f1核酸酶的核酸引入体内、离体细胞或无细胞系统中；和
(b)引入所述gRNA(sgRNA)或适合于原位产生这种sgRNA的核酸(例 如，DNA)；和
(c)使细胞或靶基因与AsCas12f1核酸酶或编码AsCas12f1核酸酶的核酸、gRNA(sgRNA)或适合于原位产生这种sgRNA的核酸接触，以在所述靶基因中产生一个或多个切割、切口或编辑；其中所述AsCas12f1核酸酶通过其加工的或未加工形式的gRNA被指导至所述靶基因。
在一些实施方式中，本发明所述基因编辑方法包括下列步骤：
i)将所述AsCas12f1核酸酶或其编码多核苷酸、以及所述向导RNA或其编码多核苷酸引入细胞中；
ii)由所述AsCas12f1核酸酶介导，在靶基因中产生一种或多种切口，或靶向、编辑、修饰或操纵所述靶基因。
在一优选实施方式中，本发明提供一种基于工程优化的CRISPR/AsCas12f1基因编辑系统的体外DNA的切割方法，包括：野生向导RNA或向导RNA变体与野生AsCas12f1核酸酶或其变体，以及靶DNA序列在特定缓冲液内混合反应。
本发明中所述的体外DNA切割方法可为本领域常规，例如，在本发明一较佳实施例中：所述的切割方法在含有50mM NaCl、5mM MgCl 2、10mM Tris-HCl，pH＝7.5的缓冲溶液中进行。本发明中，所述AsCas12f核酸酶的用量可同本领域中其他Cas核酸酶进行反应时的用量，例如，在本发明一较佳实施例中：所述AsCas12f核酸酶与所述完整的向导RNA的摩尔比为1:2；所述切割方法的反应温度较佳地为45℃、反应速率比较的时间梯度较佳地为0、0.5、1、2、4、8、16、32、64分钟。所述的靶向序列较佳地为PAM序列之后长度为20bp的DNA片段。
本发明所述基因编辑方法中，所述AsCas12f1核酸酶通过加工或未加工形式的向导RNA引导至靶基因。所述AsCas12f1核酸酶和向导RNA形成复合物，识别所述靶基因上的PAM序列。在一些优选实施方式中，所述方法进一步包括将包含异源多核苷酸序列的供体模板引入细胞中的步骤。
本发明还提供如上所述的向导RNA、分离的多核苷酸、构建体、表达系统、基因编辑系统、药物组合物或所述的方法在体内、离体细胞或无细胞环境中对靶基因和/或其相关多肽进行基因编辑中的应用。所述离体细胞选自细菌细胞、古细菌细胞、真菌细胞、原生生物细胞、病毒细胞、植物细胞和动物细胞中的至少 一种。所述基因编辑选自由：基因切割、基因删除、基因插入、点突变、转录抑制、转录激活、碱基编辑和引导编辑构成的群组，包括但不限于：
切割靶基因；
操控靶基因的表达；
遗传修饰靶基因；
遗传修饰靶基因相关多肽；
用于在靶基因的任何所需位置处的有意和受控的损伤；
用于在靶基因的任何所需位置处的有意和受控的修复；
引入双链断裂以外的方式修饰靶基因(AsCas12f1核酸酶具有酶活性，其以除引入双链断裂以外的方式修饰靶基因；所述酶活性可以是AsCas12f1本身所具有的，或通过例如将具有酶活性的异源多肽融合到AsCas12f1核酸酶形成嵌合AsCas12f1核酸酶获得的，所述酶活性包括但不限于甲基转移酶活性、脱氨作用活性、歧化酶活性、烷基化活性、脱甲基酶活性、DNA修复活性、转座酶活性、重组酶活性、DNA损伤活性、脱嘌呤活性、氧化活性、嘧啶二聚体形成活性等)。
优选地，所述基因编辑为基因删除或者基因切割；所述基因编辑可用于实现包括但不限于致病位点的修正、基因功能研究、增强细胞功能、细胞治疗等中的一种或几种。
本发明AsCas12f1核酸酶或编码其的多核苷酸、gRNA或编码其的多核苷酸、重组表达载体、系统、组合物和试剂盒可以应用于研究领域，诊断领域，工业领域(例如微生物工程)，药物发现(例如高通量筛选)，靶标确认，影像学领域以及治疗领域等。
在一些实施方式中，所述靶基因为靶DNA。在一些实施方式中，靶DNA可以是未与DNA相关蛋白结合的体外裸DNA。在一些实施方式中，靶DNA是体外细胞中的染色体DNA。在一些实施方式中，所述靶基因为靶RNA。在一些实施方式中，将靶DNA与包含所述AsCas12f1核酸酶和gRNA的靶向复合物接触，gRNA通过包含与靶DNA互补的核苷酸序列，为靶向复合物提供靶特异性；AsCas12f1核酸酶提供位点特异性活性。在一些实施方式中，靶向复合物修饰靶DNA，从而导致例如DNA切割、DNA甲基化作用、DNA损伤、DNA修复等。在一些实施方式中，靶向复合物修饰与靶DNA相关多肽(例如，组蛋白、DNA- 结合蛋白等)，从而导致例如靶DNA相关多肽-组蛋白的甲基化、组蛋白乙酰化、组蛋白泛素化等。
本发明所述方法中，可以通过公知的方法将AsCas12f1核酸酶或包含编码AsCas12f1核酸酶的多肽的核苷酸序列的核酸引入细胞。同样地，可以通过公知的方法将gRNA或包含编码gRNA的核苷酸序列的核酸引入细胞。公知的方法包括，DEAE-葡聚糖介导的转染、脂质体介导的转染、病毒或噬菌体感染、脂质转染法、转染、接合、原生质体融合、聚乙烯亚胺介导的转染、电穿孔、磷酸钙沉淀、基因枪、磷酸钙沉淀、显微注射、纳米颗粒介导的核酸递送等。例如通过电穿孔、氯化钙转染、显微注射和脂质转染法等递送质粒。对于病毒载体递送，使细胞与包含编码gRNA和/或AsCas12f1核酸酶和/或嵌合AsCas12f1核酸酶的核酸和/或供体多核苷酸的病毒颗粒接触。
在一些实施方式中，本发明所述应用或方法中，核酸酶切割细胞中的靶DNA以产生双链断裂，然后由细胞通常以下列方式进行修复：非同源末端连接(NHEJ)和同源性指导的修复。
本发明还提供了一种遗传修饰的细胞，包括已用上述AsCas12f1核酸酶或编码其的多核苷酸、gRNA或编码其的多核苷酸、重组表达载体、系统、组合物对宿主细胞进行了遗传修饰。
本发明中，gRNA和/或AsCas12f1核酸酶和/或重组表达载体和/或供体多核苷酸的有效剂量对于本领域技术人员而言是常规的。可以根据不同的施用途径以及治疗的病症的特性来确定。
本发明中，所述细菌或原核细菌可以是大肠杆菌、肺炎克雷伯氏菌、卵形拟杆菌、空肠弯曲菌、腐生葡萄球菌、粪肠球菌、多形拟杆菌、普通拟杆菌、单形拟杆菌、干酪乳杆菌、脆弱拟杆菌、鲁氏不动杆菌、具核梭杆菌、乔氏拟杆菌、拟南芥拟杆菌、鼠李糖乳杆菌、马赛拟杆菌、粪副拟杆菌、死亡梭杆菌和短双歧杆菌等。
本发明中，所述真核细胞包括但不限于是哺乳动物细胞、真菌等真核生物细胞。所述真菌包括酵母、曲霉，例如可以是酿酒酵母、多形汉逊酵母、毕赤酵母、脆壁克鲁维氏酵母、乳酸克鲁维氏酵母，以及栗酒裂殖酵母、白假丝酵母、杜氏假丝酵母、光滑假丝酵母、季也蒙假丝酵母、乳酒假丝酵母、克鲁斯假丝酵母、 葡萄牙假丝酵母、梅林假丝酵母、嗜油假丝酵母、近平滑假丝酵母、热带假丝酵母和产朊假丝酵母、烟曲霉、黄曲霉、黑曲霉、棒曲霉、灰绿曲霉群、构巢曲霉、米曲霉、土曲霉、焦曲霉和杂色曲霉等。
在本发明的实施例中，公开了一种基于极小型CRISPR/AsCas12f1核酸酶的新型基因组编辑方法。该发明显示，通过向导RNA的导向和定位功能，AsCas12f1能够精确切割基因组DNA，实现基因组DNA双链断裂。利用宿主细胞自身或外源的修复机制，该系统能够高效和精确地实现活细胞内基因编辑。
术语“AsCas12f1”、“AsCas12f1核酸酶”、“AsCas12f1多肽”、“AsCas12f1蛋白”、“AsCas12f1蛋白质”可互换使用。
术语“向导RNA”、“向导RNA”、“gRNA”、“单一gRNA”和“嵌合gRNA”可互换使用。
术语“一”或“一个”实体是指一个或多个该实体；因此，术语“一”(或“一个”)，“一个或多个”和“至少一个”在本文中可互换使用。
术语“同源性”或“同一性”或“相似性”是指两个肽之间或两个核酸分子之间的序列相似性。同源性可以通过比对不同多肽或核酸分子中的相对应位置来确定，当被比较分子序列中的同一位置在不同序列中被相同的碱基或氨基酸占据时，那么该分子在该位置是同源的。序列之间的同源程度由序列共有的匹配或同源位置的数目的函数决定。“不相关的”或“非同源的”序列与本发明所公开的序列之一的同源性应小于20％。
多核苷酸或多核苷酸区域(或多肽或多肽区域)与另一多核苷酸或多核苷酸区域(或多肽或多肽区域)具有一定百分比的序列同源性(例如，20％、30％、40％、50％、60％、70％、80％、90％、95％、98％或99％)是指比对时，被比对的两个序列中有该百分比的碱基(或氨基酸)是相同的。该比对和百分比同源性或序列同一性可以使用本领域已知的软件程序和方法来确定。
在本发明中，术语“多核苷酸”和“寡核苷酸”可互换使用，并且它们是指任何长度的核苷酸的聚合形式，无论是脱氧核糖核苷酸还是核糖核苷酸或其类似物。多核苷酸可以具有任何三维结构并且可以执行已知或未知的任何功能。多核苷酸的实例包括但不限于如下这些：基因或基因片段(包括探针，引物，EST或SAGE标签)，外显子，内含子，信使RNA(mRNA)，转运RNA，核糖体RNA， 核酶，cDNA，dsRNA，siRNA，miRNA，重组多核苷酸，分支多核苷酸，质粒，载体，任何序列的分离的DNA，任何序列的分离的RNA，核酸探针和引物。多核苷酸也包含经过修饰的核苷酸，例如甲基化的核苷酸和核苷酸类似物。如果多核苷酸上存在修饰，该修饰可以在组装多核苷酸之前或之后赋予。核苷酸序列可以被非核苷酸组分打断。多核苷酸可以在聚合后被进一步修饰，例如通过偶联被标记组分所标记。该术语同时指双链和单链多核苷酸分子。除非另有说明或要求，否则本发明公开的多核苷酸的任何实施方案包括其双链形式和已知或预测能构成双链形式的两条互补单链形式中的任一种。
当将其应用于多核苷酸时，术语“编码”是指多核苷酸“编码”多肽，意即在其天然状态下或当通过本领域技术人员公知的方法操作时，其可以通过转录和/或翻译以产生目的多肽和/或其片段，或产生能够编码该目的多肽和/或其片段的mRNA。反义链是指与该多核苷酸互补的序列，并且可以从中推导出编码序列。
术语“基因组DNA”表示生物的基因组的DNA，包括细菌、古细菌、真菌、原生生物、病毒、植物或动物的基因组的DNA。
术语“操纵”DNA包括结合、在一条链产生切口、或切割DNA的两条链，或包括修饰或编辑DNA或与DNA结合的多肽。操纵DNA可以沉默、激活或调节由所述DNA编码的RNA或多肽的表达(使其不转录，或降低转录活性，或使其不翻译，或降低翻译水平)，或防止或增强多肽与DNA的结合。切割可通过多种方法进行，例如磷酸二酯键的酶促或化学水解；可单链切割或双链切割；DNA切割可导致平头末端或交错末端的产生。
术语“可杂交的”或“互补的”或“基本上互补的”是指，核酸(例如RNA)包含核苷酸序列，该序列使得其能够在适当的体外和/或体内温度和溶液离子强度条件下以序列特异性的、反平行的方式与另一核酸非共价地结合，即，形成沃森-克里克碱基对和/或G/U碱基对、“退火”或“杂交”。
在本领域中可以理解，多核苷酸的序列不需要与其可特异性杂交的靶核酸的序列100％互补。多核苷酸可在一个或多个区段上杂交。多核苷酸可包含与其所靶向的靶核酸序列内的靶区域的至少70％、至少80％、至少90％、至少95％、至少99％或100％的序列互补性。
术语“肽”、“多肽”和“蛋白”在本发明中可互换地使用，且表示任何长度 的氨基酸的聚合形式，其可以包括编码和非编码的氨基酸，化学或生物化学修饰或衍生的氨基酸，和具有修饰的肽主链的多肽。
术语“编码”特定RNA的DNA序列是转录为RNA的DNA核酸序列。DNA多核苷酸可以编码被翻译成蛋白的RNA(mRNA)，或者DNA多核苷酸可以编码不被翻译成蛋白质的RNA(例如，tRNA、rRNA或gRNA；也称为“非编码”RNA或“ncRNA”)。“蛋白编码序列”或编码特定蛋白或多肽的序列是在合适调节序列的控制下，在体内或体外转录成mRNA(在DNA的情况下)并翻译(在mRNA的情况下)成多肽的核酸序列。
术语“载体”或“表达载体”是复制子，诸如质粒、噬菌体、病毒或粘粒，可以在其上附着另一种DNA区段，即，“插入片段”，以便实现附着的区段在细胞中的复制。
术语“表达盒”包含可操作地连接至启动子的DNA编码序列。“可操作地连接的”表示并列连接，各组分处于允许它们以其预期方式起作用的关系中。术语“重组表达载体”或“DNA构建体”在本发明中互换地用于表示包含载体和至少一个插入片段的DNA分子。重组表达载体通常是为了表达和/或扩增插入片段的目的或为了构建其它重组核苷酸序列而产生。
当外源DNA例如重组表达载体已经被引入细胞内时，细胞已被所述DNA“遗传上修饰”或“转化”或“转染”。外源DNA的存在导致永久或短暂的遗传改变。转化DNA整合或不整合到细胞的基因组中。
术语“靶DNA”是包含“靶位点”或“靶序列”的DNA多核苷酸。术语“靶位点”、“靶序列”、“靶原间隔区DNA”或“原间隔区样序列”在本发明中互换地用于表示存在于靶DNA中的核酸序列，如果存在足够的用于结合的条件，则gRNA的DNA-靶向区段将与其结合。RNA分子包含与靶DNA内的靶序列结合、杂交或互补的序列，从而将结合的多肽靶向至靶DNA内的特定位置(靶序列)。“切割”是指DNA分子的共价主链的断裂。
术语“核酸酶”和“内切核酸酶”可互换使用，用于指具有用于多核苷酸切割的内切核酸降解催化活性的酶。核酸酶的“切割结构域”或“活性结构域”或“核酸酶结构域”是指在核酸酶内具有用于DNA切割的催化活性的多肽序列或结构域。切割结构域可以包含在单个多肽链中，或者切割活性可以由两个或多个 多肽的缔合产生。
术语“定位多肽”或“RNA-结合位点指导的多肽”是指结合RNA并靶向至特定DNA序列的多肽。
术语“引导序列”或DNA-靶向区段(或“DNA-靶向序列”)包含与在本发明中称为“原间隔区样”序列的靶DNA内的特定序列互补的核苷酸序列(靶DNA的互补链)。
术语“重组”是指在两个多核苷酸之间交换遗传信息的过程。本发明中使用的“同源性指导的修复(HDR)”表示例如在细胞中双链断裂修复期间发生的特殊化形式的DNA修复。这个过程需要核苷酸序列同源性，使用“供体”分子为“靶”分子(即，经历双链断裂的分子)的修复提供模板，并导致遗传信息从供体转移至靶。如果供体多核苷酸不同于靶分子，并且供体多核苷酸的部分或全部序列被并入到靶DNA中，则同源性指导的修复可能导致靶分子序列的改变(例如插入、缺失、突变)。
术语“非同源末端连接(NHEJ)”是指在不需要同源模板的情况下通过将断裂末端彼此直接连接来修复DNA中的双链断裂。NHEJ经常导致双链断裂位点附近的核苷酸序列缺失。
术语“治疗”包括防止疾病或症状的发生；抑制疾病或症状或减轻疾病。
术语“个体”、“受试者”、“宿主”和“患者”在本发明中可互换地使用，并且表示期望对其进行诊断、治疗或疗法的任何哺乳动物受试者，特别是人。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的常规技术或条件，或者按照产品制造商的说明书进行。实施例中所用引物均由上海生工生物工程股份有限公司和苏州金唯智生物科技有限公司合成，所用试剂或仪器未注明生产厂商者，视为可通过正规渠道商购得的常规产品。
实施例1原始野生型AsCas12f1核酸酶与向导RNA变体—sgRNA_T1在哺乳动物细胞中的基因编辑
本实施例中所述的原始野生型向导RNA—sgRNA_V1的编码基因序列为：
Figure PCTCN2022113357-appb-000011
Figure PCTCN2022113357-appb-000012
本实施例中所述的工程优化后的向导RNA变体—sgRNA_T1的编码基因序列为：
Figure PCTCN2022113357-appb-000013
向导RNA的编码基因序列中下划线的20bp序列可根据需要编辑的靶位点进行合理的设计和替换。靶位点应位于靶基因上，靶位点的5’上游应包含有优选的PAM序列，其序列特征应为5’-TTR，其中R代表A或G。
本实施例中所述的原始野生型AsCas12f1使用了较佳的针对人密码子优化的AsCas12f1编码基因，其序列为
Figure PCTCN2022113357-appb-000014
优选的pCMV-AsCas12f1-T1质粒序列(表达野生型AsCas12f1核酸酶与向 导RNA变体—sgRNA_T1)为：
Figure PCTCN2022113357-appb-000015
Figure PCTCN2022113357-appb-000016
Figure PCTCN2022113357-appb-000017
表达野生型AsCas12f1核酸酶与野生型RNA—sgRNA_V1的质粒采用pCMV-AsCas12f1-T1质粒相似的构建方法获得。
质粒序列中下划线的20bp序列可根据需要编辑靶位点进行合理的设计和替换。靶位点应位于靶基因上，靶位点的5’上游应包含有优选的PAM序列，其序列特征应为5’-TTR，其中R代表A或G。质粒中靶序列的替换方法可采用本领域内常规的技术方法，例如Golden gate assembly或Gibson assembly。
本实施例中，靶序列为如下：
APOB_L1:CTGTCGACACCCAGAATCAT(SEQ ID NO.13)；
HEXA_L1:AGTATACGCTTCCACAGAAA(SEQ ID NO.14)；
PDCD1_L1:CTGTGAGCTCTAGTCCCCAC(SEQ ID NO.15)；
TP53_L2:AGGCATCACTGCCCCCTGAT(SEQ ID NO.16)；
VEGFA_L1:CTCTCAAGACCCACAATCCA(SEQ ID NO.17)；
VEGFA_L2:AAGAAGGGATGTGGTGCATT(SEQ ID NO.18)；
本实施例中，以人胚胎肾细胞HEK293T为实验所用细胞。
在含有10％FBS的DMEM培养基中培养活化后的HEK293T细胞，待细胞生长密度至90％左右传代至24孔板中，每孔细胞数量约为1.0×10 5个。16-18小时后，用1.5μL的lipofectamine3000分别转染1000ng的含不同靶序列的表达野生型AsCas12f1核酸酶、sgRNA_V1或sgRNA_T1的基因编辑质粒至细胞中。24小时后，加入嘌呤霉素达到终浓度为2μg/ml进行筛选。继续培养48小时后，消化贴壁细胞，提取基因组DNA。PCR扩增靶向序列的目的基因片段，胶回收后的PCR产物用NEBuffer2(NEB)进行退火。随后在反应体系加入T7 endonuclease 1，37℃酶切15min后加入6×Gel Loading Dye终止反应。反应产物通过6％的TBE-PAGE分离，并通过4S Red dye进行染色成像。
图1为原始野生型AsCas12f1核酸酶分别组合sgRNA_V1和sgRNA_T1的哺乳动物细胞基因编辑结果比较图。如图所示，sgRNA_T1可在APOB、HEXA、PDCD1、TP53和VEGFA基因上6个靶位点明显提升基因编辑效率。
实施例2AsCas12f1核酸酶变体与sgRNA_T1在哺乳动物细胞中的基因编辑
以实施例1中构建的pCMV-AsCas12f1-T1质粒为模板，分别使用针对不同氨基酸残基的点突变引物进行环式聚合酶延伸克隆，获得6种带有一种或多种氨基酸残基点突变的AsCas12f1核酸酶变体质粒，其可以用于哺乳动物细胞基因编 辑。
用于构建AsCas12f1核酸酶变体的质粒的引物如下：
K80R-Rv：gcgggtctcacAGAacaaaggcctacagactgaactc(SEQ ID NO.20)；
K80R-Fw：gcgggtctcaTCTgatggtgtggtaggcgtagccg(SEQ ID NO.21)；
A104R-Rv：gcgggtctcaAGAtaccagaaggagatcctgcgcg(SEQ ID NO.22)；
A104R-Fw：gcgggtctcaaTCTcttgaacctgtctgtggctctc(SEQ ID NO.23)；
D364R-Rv：gcgggtctcaAGAccccagtacaccagccagagatg(SEQ ID NO.24)；
D364R-Fw：gcgggtctcagTCTgatcttgatcactttgatgccgg(SEQ ID NO.25)；
PCR产物经Dpn1消化处理后进行溶液回收，由Golden gate assembly技术组装后，转化大肠杆菌DH5α感受态细胞，涂布带有氨苄青霉素的LBA平板，挑取单克隆扩大培养，提取质粒，测序鉴定后，获得pCMV-AsCas12f1-T1-K80R，pCMV-AsCas12f1-T1-A104R，pCMV-AsCas12f1-T1-D364R，pCMV-AsCas12f1-T1-A104+K80R，pCMV-AsCas12f1-T1-A104+D364R，pCMV-AsCas12f1-T1-K80R+A104+D364R(Evo1)质粒，其编码的核酸酶变体分别为AsCas12f1-K80R，AsCas12f1-A104R，AsCas12f1-D364R，AsCas12f1-A104+K80R，AsCas12f1-A104+D364R，AsCas12f1-K80R+A104+D364R，氨基酸分别为SEQ ID NO.1～6。
参考实施例1中的靶序列替换方法，在各变体质粒中分别插入靶序列APOB_L1，PDCD1_L1和VEGFA_L1。
参考实施例1，以人胚胎肾细胞HEK293T为实验细胞，进行基因编辑测试。
图2为在sgRNA_T1基础上，分别组合原始野生型AsCas12f1核酸酶和6种AsCas12f1核酸酶变体的哺乳动物细胞基因编辑结果比较图。如图所示，6种AsCas12f1核酸酶变体均可在APOB、PDCD1和VEGFA基因上3个靶位点上提升基因编辑效率；其中AsCas12f1-Evo1效果最佳。
实施例3工程优化的CRISPR/AsCas12f1基因编辑系统在哺乳动物细胞中的基因编辑应用
本实施例中所述的工程优化的向导RNA变体为sgRNA_T1。
本实施例中所述的工程优化的AsCas12f1核酸酶变体为AsCas12f1-Evo1。
本实施例中所述的工程优化的CRISPR/AsCas12f1基因编辑系统为AsCas12f1-Evo1和sgRNA_T1的组合。
参考实施例1中的靶序列替换方法，在pCMV-AsCas12f1-T1-K80R+A104+D364R(Evo1)质粒中分别插入靶序列AAVS1-L1～L8，APOB_L1，DNMT1_L1，HBG_L1～L2，HEXA_L1，IFNγ_L1，PCSK9_L1，PDCD1_L1，PRNP_L1，TP53_L1～L6，VEGFA_L1～L4；靶序列如下：
AAVS1_L1：AGGAAAGAAGGATGGAGAAA(SEQ ID NO.26)；
AAVS1_L2：CCTGGACACCCCGTTCTCCT(SEQ ID NO.27)；
AAVS1_L3：CTTACGATGGAGCCAGAGAG(SEQ ID NO.28)；
AAVS1_L4：CCTGTGAGATAAGGCCAGTA(SEQ ID NO.29)；
AAVS1_L5：CTGCCTCCAGGGATCCTGTG(SEQ ID NO.30)；
AAVS1_L6：GCCACCTCTCCATCCTCTTG(SEQ ID NO.31)；
AAVS1_L7：TCTGTCCCCTCCACCCCACA(SEQ ID NO.32)；
AAVS1_L8：GGCAGCTCCCCTACCCCCCT(SEQ ID NO.33)；
APOB_L1：CTGTCGACACCCAGAATCAT(SEQ ID NO.13)；
DNMT1_L1：TGTGGCCACAAGGCTCAGTT(SEQ ID NO.34)；
HBG_L1：CCTTGTCAAGGCTATTGGTC(SEQ ID NO.35)；
HBG_L2：CCTTGTTCCGATTCAGTCAT(SEQ ID NO.36)；
HEXA_L1：AGTATACGCTTCCACAGAAA(SEQ ID NO.14)；
IFNγ_L1：ACGATGAGACAGACCCATTA(SEQ ID NO.37)；
PCSK9_L1：CCCAGAGCATCCCGTGGAAC(SEQ ID NO.38)；
PDCD1_L1：CTGTGAGCTCTAGTCCCCAC(SEQ ID NO.15)；
PRNP_L1：TGGCCACATGGAGTGACCTG(SEQ ID NO.39)；
TP53_L1：ATAAGAGGTCCCAAGACTTA(SEQ ID NO.40)；
TP53_L2：AGGCATCACTGCCCCCTGAT(SEQ ID NO.16)；
TP53_L3：TCCTGCTTGCTTACCTCGCT(SEQ ID NO.41)；
TP53_L4：CCTCTTTCCTAGCACTGCCC(SEQ ID NO.42)；
TP53_L5：GCTGGGGAGAGGAGCTGGTG(SEQ ID NO.43)；
TP53_L6：CTTACCTCGCTTAGTGCTCC(SEQ ID NO.44)；
VEGFA_L1：CTCTCAAGACCCACAATCCA(SEQ ID NO.17)；
VEGFA_L2：AAGAAGGGATGTGGTGCATT(SEQ ID NO.18)；
VEGFA_L3：CTGTGATTTCCCCACAAAAG(SEQ ID NO.45)；
VEGFA_L4：CCTCTTCCGGCCTGGATTGT(SEQ ID NO.46)；
参考实施例1，以人胚胎肾细胞HEK293T为实验细胞，进行基因编辑测试。
图3为原始野生CRISPR/AsCas12f1系统与工程优化的CRISPR/AsCas12f1系统在哺乳动物细胞中基因编辑效果的比较图。如图所示，工程优化后的CRISPR/AsCas12f1基因编辑系统可在11个基因的27个靶位点上显著提升基因编辑效率。
实施例4工程优化的CRISPR/AsCas12f1基因编辑系统在体外DNA切割中的应用
本实施例中的sgRNA_T1由本领域内常规技术方法，例如体外转录法进行制备。可使用HiScribe T7 High Yield RNA Synthesis Kit(NEB)试剂盒进行制备。制备后的sgRNA_T1经过酚氯仿抽提和乙醇沉淀进行纯化。
本实施例中的野生原始AsCas12f1核酸酶和变体核酸酶由本领域内常规技术方法，例如大肠杆菌重组表达和亲和层析技术进行制备。将核酸酶表达构建体转化入大肠杆菌表达菌株BL21(DE3)。次日将转化子转接入1L的LB培养基中，37℃条件下进行震荡培养。当OD 600到达0.6时，在培养液中加入0.25mL 1M IPTG，在16℃继续培养过夜。收集过夜后的菌株，超声破碎后，用HisTrap Ni-NTA(Cytiva)层析柱进行纯化。纯化后的蛋白，使用HRV3c蛋白酶去除标签，后经过HiLoad 16/600 Superdex 200pg分子筛(Cytiva)进一步纯化。纯化后的蛋白保存在1000mM NaCl，10mM Tris-HCl，pH＝7.5，1mM DTT的缓冲液中。
本实施例中DNA底物由本领域内常规技术方法，例如聚合酶链式反应PCR进行制备。切割底物中包含sgRNA_T1中的靶序列，且靶序列上游包含有PAM序列，其序列特征为5’-TTR，其中R代表A或G。
本实施例中所述的工程优化的CRISPR/AsCas12f1基因编辑系统为AsCas12f1-Evo1和sgRNA_T1的组合。
体外DNA切割实验在50mM NaCl，10mM MgCl 2，10mM Tris-HCl，pH＝7.5条件中进行。总反应体积为20μL，其中包括10nM切割底物，500nM AsCas12f1和1000nM向导RNA。反应时间为0、0.5、1、2、4、8、16、32、64分钟，反应温度为45℃。反应结束后加入6×Gel Loading Dye终止反应。反应产物通过1％的琼脂糖胶分离，并通过4S Red dye进行染色成像。
图4为原始野生CRISPR/AsCas12f1系统与工程优化的CRISPR/AsCas12f1系统在体外切割DNA效果的比较图。如图所示，工程优化后的CRISPR/AsCas12f1基因编辑系统能显著提升体外DNA切割的效率和速度。
最后应当说明的是，虽然以上详细描述了本发明实施例的具体方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对本发明的实施方式进行修改或者等同替换。因此，本发明的保护范围由所附权利要求书限定。

Claims (25)

  1. 一种突变型AsCas12f1核酸酶，其特征在于，所述突变型AsCas12f1核酸酶与野生型AsCas12f1核酸酶具有至少50％以上同一性，且相对于野生型AsCas12f1核酸酶包含第80位、第104位、第364位氨基酸中的一个或几个位点突变。
  2. 如权利要求1所述的AsCas12f1核酸酶变体，其特征在于，当所述突变型AsCas12f1核酸酶包含两个或三个位点突变时，所述突变是指突变为相同或不同的氨基酸；优选地，突变为相同的氨基酸。
  3. 如权利要求1所述的AsCas12f1核酸酶变体，其特征在于，所述突变型AsCas12f1核酸酶的氨基酸序列如SEQ ID NO.1～6所示。
  4. 一种突变型向导RNA，其特征在于，其包含tracrRNA序列和crRNA序列；所述crRNA序列包含能够与靶序列杂交的基因靶向区段和tracr配对物序列；所述tracr RNA序列和tracr配对物序列和连接链序列构成向导RNA的骨架序列；其中，所述tracrRNA包含如SEQ ID NO.47所示的核苷酸序列或其变体序列；所述tracr配对物序列包含如SEQ ID NO.48所示的核苷酸序列或其变体序列。
  5. 如权利要求4所述的向导RNA，其特征在于，包含以下至少任一项：
    1)所述tracr RNA序列和tracr配对物序列之间还包括连接链序列；优选地，所述连接链序列包含5’-AAGG、5’-UACU或其变体序列；
    2)所述基因靶向区段位于所述crRNA序列的3’端；
    3)所述向导RNA包含在所述tracrRNA中互补配对的碱基对的一个或多个互换位置后获得的tracrRNA；
    4)所述向导RNA包含在tracrRNA与tracr配对物序列互补配对的碱基对的一个或多个互换位置后获得的tracrRNA和tracr配对物序列；
    5)所述基因靶向区段识别靶向序列上的PAM序列；优选地PAM序列为5’-TTR，其中R代表A或G；
    6)所述基因靶向区段靶向PAM序列之后长度为12～40bp的核酸片段；优选的长度为20bp；
    7)所述基因靶向区段靶向细胞基因组中的至少一个靶序列；
    8)所述基因靶向区段靶向的靶序列DNA和/或RNA；
    9)所述向导RNA为两条链，一条链包含所述tracrRNA序列，另一条链包含所述crRNA序列；
    10)所述向导RNA为一条链，从5’端至3’端依次包含所述tracrRNA序列、连接链序列和crRNA序列。
  6. 如权利要求4或5所述的向导RNA，其特征在于，包括以下至少任一项：
    1)所述tracrRNA的变体序列是指在所述SEQ ID NO.47所示的核苷酸序列的5’端和/或3’端增加、减少或替换部分核苷酸后获得的序列；优选地，所述tracrRNA的变体序列是指在所述SEQ ID NO.47所示的核苷酸序列的5’端和/或3’端减少核苷酸后获得的序列；
    2)所述tracr配对物序列的变体序列是指在所述SEQ ID NO.48所示的核苷酸序列的5’端和/或3’端增加、减少或替换部分核苷酸后获得的序列；优选地，所述tracr配对物序列的变体序列是指在所述SEQ ID NO.48所示的核苷酸序列的5’端和/或3’端减少核苷酸后获得的序列；
    3)所述连接链序列的变体序列是指在5’-AAGG、5’-UACU的5’端和/或3’端增加、减少或替换部分核苷酸后获得的序列；
    4)所述tracrRNA与tracr配对物序列之间的连接链序列为5’-AAGG或5’-UACU；优选地，为5’-UACU。
  7. 如权利要求6所述的向导RNA，其特征在于，包括以下至少任一项：
    1)所述tracrRNA的变体序列是指在所述SEQ ID NO.47所示的核苷酸序列的5’端和/或3’端减少1～50nt核苷酸后获得的序列；优选地，在3’端减少14nt核苷酸；优选地，所述tracrRNA的变体序列如SEQ ID NO.50所示；
    2)所述tracr配对物序列的变体序列是指在所述SEQ ID NO.48所示的核苷酸序列的5’端减少1～29nt核苷酸后获得的序列；优选地，减少15nt核苷酸；优选地，所述tracrRNA配对物序列如SEQ ID NO.51所示。
  8. 如权利要求7所述的向导RNA，其特征在于，所述向导RNA的骨架序列的核苷酸序列如SEQ ID NO.52所示。
  9. 一种分离的多核苷酸，其特征在于，其编码如权利要求1～3任一项所述的突变型AsCas12f1核酸酶。
  10. 一种分离的多核苷酸，其特征在于，其编码如权利要求4～8任一项所述的向 导RNA。
  11. 一种构建体，其特征在于，所述构建体单独或同时含有如权利要求9所述的分离的多核苷酸、如权利要求10所述的分离的多核苷酸。
  12. 一种表达系统，其特征在于，所述表达系统含有如权利要求11所述的构建体，或基因组中整合有外源的如权利要求11所述的构建体。
  13. 如权利要求12所述的表达系统，其特征在于，所述表达系统的宿主细胞选自真核细胞或原核细胞；优选地，所述宿主细胞选自小鼠细胞、人细胞。
  14. 一种基因编辑系统，其特征在于，选自以下至少任一项：
    1)所述基因编辑系统包含如权利要求1～3任一项所述的突变型AsCas12f1核酸酶或其编码多核苷酸以及向导RNA或其编码多核苷酸；
    2)所述基因编辑系统包含核酸酶或其编码多核苷酸如权利要求4～8任一项所述的突变型向导RNA或其编码多核苷酸。
  15. 如权利要求14所述的基因编辑系统，其特征在于，
    1)中，所述向导RNA为野生型向导RNA或其变体；优选地，所述野生型向导RNA为sgRNA_V1，所述野生型向导RNA的变体为sgRNA_T1；和/或，
    2)中，所述核酸酶为Cas蛋白及其突变体；优选地，选自Cas9、Cas12、Cas13蛋白家族及其突变体；进一步优选地，选自Cas12f及其突变体。
  16. 如权利要求15所述的基因编辑系统，其特征在于，所述Cas蛋白及其突变体选自：
    (I)野生型Cas蛋白或其片段，具有受RNA引导的核酸结合活性；
    (II)与(I)的氨基酸序列具有至少50％序列同源性的变体，且具有受RNA引导的核酸结合活性；
    (III)根据(I)或(II)，其进一步包括核定位信号片段；
    (IV)根据(I)或(II)或(III)，其进一步包含：
    (a)一种或多种修饰或突变，其产生具有相比修饰或突变前显著减小的核酸内切酶活性，或使核酸内切酶活性丧失；和/或
    (b)具有其他功能活性的多肽或结构域；
    (V)根据(I)或(II)或(III)，所述Cas蛋白具有核酸内切酶活性。
  17. 如权利要求14所述的基因编辑系统，其特征在于，包括以下至少任一项：
    1)所述基因编辑系统包含一个或多个载体；所述一个或多个载体包含(i)第一调控元件，所述第一调控元件可操作地连接至所述核酸酶的编码多核苷酸；以及(ii)第二调控元件，所述第二调控元件可操作地连接至所述向导RNA核苷酸序列的编码多核苷酸；
    所述(i)和(ii)位于相同或不同载体上；
    2)所述基因编辑系统包含(i)核酸酶，以及(ii)所述向导RNA的编码多核苷酸的载体；
    3)所述基因编辑系统包含(i)核酸酶的编码多核苷酸的载体，以及(ii)所述向导RNA；
    4)所述基因编辑系统包含(i)核酸酶，以及(ii)向导RNA。
  18. 如权利要求14-17任一项所述的基因编辑系统，其特征在于，包括以下至少任一项：
    1)所述基因编辑系统识别靶向序列上的PAM序列；优选地PAM序列为5’-TTR，其中R代表A或G；
    2)所述基因编辑系统靶向PAM序列之后长度为12～40bp的核酸片段，优选的长度为20bp；
    3)所述基因编辑系统靶向细胞基因组中的至少一个靶序列；
    4)所述基因编辑系统靶向的靶序列DNA和/或RNA。
  19. 一种药物组合物，其特征在于，其包含如权利要求14-18任一所述的基因编辑系统，以及药学上可接受的载体。
  20. 一种基因编辑方法，其特征在于，将靶基因与如权利要求14-18任一项所述的基因编辑系统接触，以实现靶基因的编辑。
  21. 如权利要求20所述的基因编辑方法，其特征在于，包括下列步骤：
    i)将所述核酸酶或其编码多核苷酸、以及所述向导RNA或其编码多核苷酸引入细胞中；
    ii)由所述核酸酶介导，在靶基因中产生一种或多种切口，或靶向、编辑、修饰或操纵所述靶基因。
  22. 如权利要求20或21所述的基因编辑方法，其特征在于，包括以下至少任一项：
    1)所述靶基因为体内基因组上的靶基因、或离体细胞中的靶基因、或在体外无细胞环境中的靶基因；
    2)所述核酸酶通过加工或未加工形式的向导RNA引导至靶基因；
    3)所述核酸酶和向导RNA形成复合物，识别所述靶基因上的PAM序列；
    4)所述基因编辑系统的靶向序列为PAM序列之后长度为12～40bp的核酸片段，优选PAM序列之后长度为20bp的核酸片段；
    5)所述方法进一步包括将包含异源多核苷酸序列的供体模板引入细胞中的步骤。
  23. 如权利要求1～3任一项所述的突变型AsCas12f1核酸酶或如权利要求4～8任一项所述的突变型向导RNA或如权利要求9或10所述的分离的多核苷酸或如权利要求11所述的构建体或如权利要求12或13所述的表达系统或如权利要求14～18任一项所述的基因编辑系统或如权利要求19所述的药物组合物或如权利要求20-22任一所述的方法在体内、离体细胞或无细胞环境中对靶基因和/或其相关多肽进行基因编辑中的应用。
  24. 如权利要求23所述的应用，其特征在于，包括以下至少任一项：
    1)所述基因编辑选自由：基因切割、基因删除、基因插入、点突变、转录抑制、转录激活、先导编辑、碱基编辑和引导编辑构成的群组；优选地，所述基因编辑为基因删除和/或基因切割；
    2)所述离体细胞选自细菌细胞、古细菌细胞、真菌细胞、原生生物细胞、病毒细胞、植物细胞和动物细胞中的至少一种；
    3)所述基因编辑用于实现致病位点的修正、基因功能研究、增强细胞功能、细胞治疗至少任一。
  25. 一种遗传修饰的细胞，其通过如权利要求14～18任一项所述的基因编辑系统或如权利要求19所述的药物组合物或如权利要求20～22任一项所述的方法进行基因编辑获得。
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