CRISPR-Cas gene editing system and application thereof

Abstract

The invention discloses a CRISPR-Cas gene editing system and an application thereof. The invention discloses a CRISPR-Cas12i gene editing system which comprises RNA (Ribonucleic Acid) for coding a Cas12i editor and engineered guide RNA, a nucleic acid composition for coding the RNA and the engineered guide RNA, a composition, a kit and a method for modifying nucleic acid by utilizing the components.

Description

Technical Field

[0001] The present invention relates to the field of nucleic acid editing, particularly the technical field of regularly clustered interspaced short palindromic repeats (CRISPR). Specifically, the present invention relates to a CRISPR-Cas12i gene editing system, which comprises RNA encoding a Cas12i editor and an engineered guide RNA, and a nucleic acid composition encoding them. The present invention further relates to a composition or a kit for nucleic acid modification (e.g., gene or genome editing), comprising the CRISPR-Cas12i gene editing system of the present invention encapsulated in a lipid nanoparticle (LNP), or the CRISPR-Cas12i gene editing system of the present invention encapsulated in a virus-like particle (VLP). The present invention further relates to a method for nucleic acid modification (e.g., gene or genome editing), which uses a CRISPR-Cas12i gene editing system comprising the present invention. Background Art

[0002] CRISPR-Cas related gene editing tools have become one of the most cutting-edge technologies in the field of life sciences and medicine due to their high efficiency and simplicity. The CRISPR-Cas12i system can cut dsDNA and complete the maturation of pre-crRNA by itself, with the potential for multiple gene editing; compared with CRISPR Cas9 and Cas12a, Cas12i has a smaller molecular weight and has certain advantages in in vivo delivery, showing that the Cas12i system has great potential in gene editing applications. However, the natural Cas12i system shows low editing efficiency at certain targets, limiting its application. Summary of the invention

[0003] One aspect of the present invention provides a CRISPR-Cas gene editing system, comprising: (a) a ribonucleotide sequence encoding a Cas12i editor, wherein the Cas12i editor comprises a Cas12i protein; the Cas12i protein comprises an amino acid sequence having at least 99% sequence identity compared to the amino acid sequence shown in any one of SEQID NO.1 to 6; and (b) an engineered guide RNA, wherein the engineered guide RNA comprises a first direct repeat sequence, a spacer sequence, and a second direct repeat sequence connected in sequence from the 5' end to the 3' end; the spacer sequence is specifically complementary to the target nucleic acid, and the engineered guide RNA is complexed with the Cas12i editor to guide the Cas12i protein to bind to the target nucleic acid.

[0004] Cas12i proteins can be independently selected from those Cas12i proteins and gene editing fusion proteins disclosed in CN202311464815.0 or CN202311851352.3, and their disclosed contents are fully incorporated herein by reference.

[0005] In other embodiments, the CRISPR-Cas gene editing system of the present invention does not include the Cas12i editor protein polypeptide.

[0006] In some embodiments, the ribonucleotide sequence encoding the Cas12i editor of the present invention is codon optimized. This type of optimization may require mutations in the ribonucleotide sequence encoding the Cas12i editor to simulate the codon preference of the expected host organism or cell when encoding the same protein at the same time. Therefore, the codon can be changed, but the encoded protein remains unchanged. For example, if the expected target cell is a human cell, a ribonucleotide sequence encoding the Cas12i editor optimized by human codons can be used. As another non-limiting example, if the expected host cell is a mouse cell, a mouse codon-optimized ribonucleotide sequence encoding the Cas12i editor can be generated. As another non-limiting example, if the expected host cell is a plant cell, a plant codon-optimized ribonucleotide sequence encoding the Cas12i editor can be generated. As another non-limiting example, if the expected host cell is an insect cell, an insect codon-optimized ribonucleotide sequence encoding the Cas12i editor can be generated.

[0007] Methods for introducing nucleic acids (e.g., comprising one or more ribonucleotide sequences encoding Cas12i editors and / or engineered guide RNAs, etc.) into host cells are known in the art, and any convenient method can be used to introduce nucleic acids (e.g., expression constructs) into cells. In some embodiments, the ribonucleotide sequence encoding the Cas12i editor can be provided by direct chemical synthesis, or can be transcribed in vitro from DNA (e.g., DNA encoding the Cas12i editor). Once synthesized, RNA can be introduced into cells by any well-known technique for introducing nucleic acids into cells (e.g., microinjection, electroporation, transfection, viral vector delivery, etc.).

[0008] In other embodiments, the components of the CRISPR-Cas gene editing system of the present invention or the nucleic acid composition molecules thereof can be delivered into suitable mammalian host cells using RNA delivery techniques well known to those skilled in the art, examples of which include but are not limited to electroporation, microinjection, liposome transfection, lipid nanoparticles (LNP) or virus-like particles (VLP) and the like.

[0009] In a preferred embodiment, the ribonucleotide sequence encoding the Cas12i editor is mRNA, and the mRNA further comprises a 5' cap sequence or / and a 3' poly A sequence (polyA); in addition to the 5' cap sequence or / and the 3' poly A sequence (polyA) element, the mRNA may also contain a stabilizing element, including but not limited to an untranslated region (UTR) at its 5' end (5'UTR) and / or at its 3' end (3'UTR). Both 5'UTR and 3'UTR are typically transcribed from genomic DNA and are elements of pre-mature mRNA.

[0010] In a preferred embodiment, the ribonucleotide sequence encoding the Cas12i editor is codon-optimized for expression in a target cell; preferably, the ribonucleotide sequence is codon-optimized for expression in eukaryotic cells.

[0011] In other embodiments, the ribonucleotide sequence encoding the Cas12i editor is an mRNA encoding the Cas12i editor. The mRNA encoding the Cas12i editor can be generated using in vitro transcription (IVT, in vitro transcription) or in vitro synthesis. For example, the mRNA encoding the Cas12i editor can be synthesized using a nucleic acid expression vector obtained by comprising the following sequentially connected elements, including: a promoter (which can be a promoter of any RNA polymerase, such as a T7 promoter or an SP6 promoter), a 5'UTR (such as a 5'UTR from a human β-globin gene), an optional kozak sequence, a Cas12i editor, a 3'UTR (such as a 3'UTR from a human β-globin) and a poly A sequence (polyA). In addition to the above elements, the nucleic acid expression vector may also contain one or more nuclear localization signals (such as Bp NLS, SV40 NLS or myc NLS) or one or more epitope tags (such as 3×FLAG).

[0012] In a preferred embodiment, the nucleic acid expression vector comprises the following elements connected in sequence: a promoter (which can be a promoter of any RNA polymerase, such as a T7 promoter or an SP6 promoter), a 5'UTR (such as a 5'UTR from a human β-globin gene), an optional kozak sequence, a Cas12i protein, a 3'UTR (such as a 3'UTR from a human β-globin), and a poly A sequence (polyA).

[0013] In a preferred embodiment, the nucleic acid expression vector comprises the following sequentially connected elements: a promoter (which can be a promoter of any RNA polymerase, such as a T7 promoter or an SP6 promoter), a 5'UTR (such as a 5'UTR from a human β-globin gene), an optional kozak sequence, a nuclear localization signal (such as BPSV40 NLS), an epitope tag (such as 3×FLAG), a Cas12i protein, a nuclear localization signal (such as BPSV40NLS), a 3'UTR (such as a 3'UTR from a human β-globin) and a poly A sequence (polyA). In a preferred embodiment, the nucleic acid expression vector comprises or is a nucleotide sequence as shown in SEQ ID NO.104.

[0014] In a preferred embodiment, the nucleic acid expression vector comprises the following elements connected in sequence: a promoter (which can be a promoter of any RNA polymerase, such as a T7 promoter or an SP6 promoter), a 5'UTR (such as a 5'UTR from a human β-globin gene), an optional kozak sequence, a fusion protein of a Cas12i protein and a 5'-3' exonuclease domain, a 3'UTR (such as a 3'UTR from a human β-globin) and a poly A sequence (polyA).

[0015] In a preferred embodiment, the nucleic acid expression vector comprises the following sequentially connected elements: a promoter (which can be a promoter of any RNA polymerase, such as a T7 promoter or an SP6 promoter), a 5'UTR (such as a 5'UTR from a human β-globin gene), an optional kozak sequence, a nuclear localization signal (such as BPSV40 NLS), an epitope tag (such as 3×FLAG), a fusion protein of a Cas12i protein and a 5'-3' exonuclease functional domain, a nuclear localization signal (such as BPSV40 NLS), a 3'UTR (such as a 3'UTR from a human β-globin), and a poly A sequence (polyA). In a preferred embodiment, the nucleic acid expression vector comprises or is a nucleotide sequence as shown in SEQ ID NO.103.

[0016] In other embodiments, the ribonucleotide sequence encoding the Cas12i editor synthesized by in vitro transcription is subjected to a purification step to remove various contaminants that may be contained. The purification is well known to those skilled in the art, and examples thereof include but are not limited to silica gel membrane purification column purification method and RNase-free HPLC purification method.

[0017] In other embodiments, the engineered guide RNA (gRNA) can be generated using in vitro synthesis (such as chemical synthesis) or in vitro transcription (such as transcription using an in vitro transcription vector). Preferably, the engineered guide RNA obtained by in vitro synthesis or in vitro transcription is purified by a purification step to remove various contaminants that may be present, and the purification is well known to those skilled in the art, examples of which include but are not limited to RNase-free HPLC purification. Preferably, the 5' end or / and 3' end of the engineered guide RNA also includes a chemical modification.

[0018] In other embodiments, the Cas12i protein is mutated to have enhanced nucleic acid binding activity and enhanced nucleic acid cleavage activity. In such embodiments, the at least one amino acid substitution may result in an enhanced nucleic acid binding activity of the Cas12i protein, for example, an enhanced nucleic acid binding activity of at least 10% compared to the parent Cas12i protein, for example, 10% to 500%, 10% to 100%, 10% to 200%, 10% to 300%, 10% to 50%, 10% to 30%, 10% to 20%, 50% to 100%, 50% to 200%, 50% to 300%, 100% to 200%, or 200% to 300%. In such an embodiment, the at least one amino acid substitution may result in an enhancement of the nucleic acid cleavage activity of the Cas12i protein, for example, an enhancement of the nucleic acid cleavage activity by at least 10%, for example, 10% to 500%, 10% to 100%, 10% to 200%, 10% to 300%, 10% to 50%, 10% to 30%, 10% to 20%, 50% to 100%, 50% to 200%, 50% to 300%, 100% to 200%, or 200% to 300%. Any nucleotide sequence that retains the Cas12i protein binding activity after at least one nucleotide substitution, deletion and / or insertion is within the scope of the present invention.

[0019] In some embodiments, the Cas12i protein comprises or is an amino acid sequence shown in any one of SEQ ID NO.1 to 6, respectively referred to as "enCas12i-001", "enCas12i-002", "enCas12i-003", "enCas12i-004", "enCas12i-005", "enCas12i-006". In other embodiments, the Cas12i protein has an N229R mutation on the basis of the amino acid sequence shown in any one of SEQ ID NO.1 to 6 to enhance the nucleic acid binding activity and nucleic acid cleavage activity of these Cas12i proteins. In other embodiments, the Cas12i protein has an N229R mutation on the basis of the amino acid sequence shown in SEQ ID NO.1, referred to as "enCas12i-001-N229R".

[0020] In other embodiments, the Cas12i protein is mutated so that it has enhanced nucleic acid binding activity and enhanced nucleic acid cleavage activity. According to the sequence number shown in SEQ ID NO.1, the Cas12i protein has an amino acid substitution at position N229, preferably substituted by lysine, arginine or histidine, more preferably substituted by arginine; more preferably, the Cas12i protein comprises the amino acid sequence shown in SEQ ID NO.7; specifically, the enCas12i-001-N229R comprises the amino acid sequence shown in SEQ ID NO.7.

[0021] Specifically, the name of the mutant polypeptide in which the Cas12i protein is replaced by a specific amino acid at a specific position is named according to the mutation naming rules of the Human Genome Variation Society (HGVS), for example: "The Cas12i protein has an amino acid substitution at position N229" "N229R" means that the amino acid at position 229 of any one of SEQ ID NO.1 to 6 has replaced the original N with R.

[0022] In other embodiments, the Cas12i editor further comprises a 5'-3' exonuclease domain, which is fused to the N-terminus and / or C-terminus of the Cas12i protein; preferably, the 5'-3' exonuclease domain is from T5 phage; more preferably, the 5'-3' exonuclease domain comprises an amino acid sequence having at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO.8.

[0023] In some embodiments, the Cas12i protein can be fused to the 5'-3' exonuclease functional domain via one or more linker polypeptides (or connecting peptides).

[0024] In other embodiments, the fusion protein of the Cas12i protein and the 5'-3' exonuclease functional domain comprises or is the amino acid sequence shown in SEQ ID NO.100.

[0025] In some embodiments, the Cas12i editor further comprises one or more heterologous polypeptides, which are independently epitope tags, nuclear localization signals, reporter gene sequences, or DNA binding domains.

[0026] In some embodiments, the heterologous polypeptide is selected from an epitope tag. Such epitope tags are conventional tags, including but not limited to His, V5, FLAG, HA, Myc, VSV-G, Trx, etc., and those skilled in the art know how to select a suitable epitope tag according to the desired purpose (e.g., purification, detection or tracing).

[0027] In a preferred embodiment, the epitope tag is 3×FLAG, and the 3×FLAG comprises the amino acid sequence shown in SEQ ID NO.44.

[0028] In some embodiments, the heterologous polypeptide is selected from a nuclear localization signal. The Cas12i editor comprises (is fused with) a nuclear localization signal (NLS) (e.g., in some embodiments, 2 or more, 3 or more, 4 or more, or 5 or more NLS). Therefore, in some embodiments, the Cas12i protein includes one or more NLS (e.g., 2 or more, 3 or more, 4 or more, or 5 or more NLS). In some embodiments, the fusion protein of the Cas12i protein and the 5'-3' exonuclease functional domain includes one or more NLS (e.g., 2 or more, 3 or more, 4 or more, or 5 or more NLS). In some embodiments, one or more NLS (2 or more, 3 or more, 4 or more, or 5 or more NLS) are positioned at or near the N-terminus and / or C-terminus (e.g., within 50 amino acids). In some embodiments, one or more NLS (2 or more, 3 or more, 4 or more or 5 or more NLS) are positioned at or near the N-terminus (e.g., within 50 amino acids). In some embodiments, one or more NLS (2 or more, 3 or more, 4 or more or 5 or more NLS) are positioned at or near the C-terminus (e.g., within 50 amino acids). In some embodiments, one or more NLS (3 or more, 4 or more or 5 or more NLS) are positioned at or near both the N-terminus and the C-terminus (e.g., within 50 amino acids). In some embodiments, one or more NLS are positioned at the N-terminus, and one or more NLS are positioned at the C-terminus. Specifically, the nuclear localization signal (NLS) connection sequence can be: NH2-[Cas12i editor]-[NLS]-COOH; NH2-[NLS]-[Cas12i editor]-COOH; wherein]-[represents a connecting peptide (hereinafter the same) that may be optionally present according to the definition below.

[0029] In some embodiments, the Cas12i editor comprises (is fused with) 1 to 10 NLSs (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 2-10, 2-9, 2-8, 2-7, 2-6 or 2-5 NLSs). In some embodiments, the Cas12i editor comprises (is fused with) 2 to 5 NLSs (e.g., 2-4 or 2-3 NLSs).

[0030] Non-limiting examples of NLS include, but are not limited to, bipartite SV40 NLS (BPSV40 NLS), NLS of SV40 virus large T antigen, bipartite NLS of nucleoplasmin, c-myc NLS, hRNPA1 M9 NLS, IBB domain of importin-α, myoma T protein, human p53, mouse c-abl IV, influenza virus NS1, hepatitis virus delta antigen, mouse Mx1 protein, human poly (ADP-ribose) polymerase, steroid hormone receptor (human) glucocorticoid, etc. Commonly used NLS. Non-limiting examples of NLS include amino acid sequences as shown in any one of SEQ ID NO.9 to 25.

[0031] In some embodiments, the heterologous polypeptide is selected from a reporter gene sequence. Such reporter genes are well known to those skilled in the art, and examples thereof include but are not limited to GST, HRP, CAT, GFP, HcRed, DsRed, CFP, YFP, BFP, etc.

[0032] In other embodiments, the Cas12i editor fused with a heterologous polypeptide (BPSV40 NLS, SV40 NLS and His tag) comprises or is the amino acid sequence shown in SEQ ID NO.101 or 102.

[0033] In some embodiments, the heterologous polypeptide is selected from a DNA binding domain, which is well known to those skilled in the art, and examples thereof include but are not limited to maltose binding protein (MBP), the DNA binding domain (DBD) of LexA, the DBD of GAL4, etc.

[0034] In some embodiments, the structure of the Cas12i editor is selected from: NH2-[Cas12i protein]-COOH; NH2-[nuclear localization signal]-[Cas12i protein]-COOH; NH2-[Cas12i protein]-[nuclear localization signal]-COOH; NH2-[nuclear localization signal]-[Cas12i protein]-[nuclear localization signal]-COOH; NH2-[epitope tag]-[Cas12i protein]-COOH; NH2-[epitope tag]-[Cas12i protein]-[epitope tag]-COOH; NH2-[nuclear localization signal]-[epitope tag]-[Cas12i protein]-[epitope tag]-COOH; NH2-[nuclear localization signal]-[epitope tag]-[ Cas12i protein]-COOH; NH2-[nuclear localization signal]-[Cas12i protein]-[epitope tag]-COOH; NH2-[nuclear localization signal]-[epitope tag]-[Cas12i protein]-[epitope tag]-COOH; NH2-[epitope tag]-[Cas12i protein]-[nuclear localization signal]-COOH; NH2-[epitope tag]-[Cas12i protein]-[epitope tag]-[nuclear localization signal]-COOH; NH2-[epitope tag]-[Cas12i protein]-[epitope tag]-[nuclear localization signal]-COOH; NH2-[nuclear localization signal]-[epitope tag]-[Cas12i protein]-[epitope tag]-[nuclear localization signal]-COOH; NH2-[Nuclear localization signal]-[Cas12i protein]-[epitope tag]-[Nuclear localization signal]-COOH; NH2-[Nuclear localization signal]-[epitope tag]-[Cas12i protein]-[epitope tag]-[Nuclear localization signal]-COOH; NH2-[Cas12i protein]-[reporter gene sequence]-COOH; NH2-[reporter gene sequence]-[Cas12i protein]-COOH; NH2-[reporter gene sequence]-[Cas12i protein]-[reporter gene sequence]-COOH; NH2-[Nuclear localization signal]-[Cas12i protein]-[reporter gene sequence] -COOH; NH2-[nuclear localization signal]-[reporter gene sequence]-[Cas12i protein]-COOH; NH2-[nuclear localization signal]-[reporter gene sequence]-[Cas12i protein]-[reporter gene sequence]-COOH; NH2-[Cas12i protein]-[reporter gene sequence]-[nuclear localization signal]-COOH; NH2-[reporter gene sequence]-[Cas12i protein]-[nuclear localization signal]-COOH; NH2-[reporter gene sequence]-[Cas12i protein]-[reporter gene sequence]-[nuclear localization signal]-COOH; NH2-[nuclear localization signal]- [Cas12i protein]-[reporter gene sequence]-[nuclear localization signal]-COOH; NH2-[nuclear localization signal]-[reporter gene sequence]-[Cas12i protein]-[nuclear localization signal]-COOH; NH2-[nuclear localization signal]-[reporter gene sequence]-[Cas12i protein]-[reporter gene sequence]-[nuclear localization signal]-COOH; NH2-[Cas12i protein]-[DNA binding domain]-COOH; NH2-[DNA binding domain]-[Cas12i protein]-[ DNA binding domain]-COOH; NH2-[nuclear localization signal]-[Cas12i protein]-[DNA binding domain]-COOH; NH2-[nuclear localization signal]-[DNA binding domain]-[Cas12i protein]-COOH; NH2-[nuclear localization signal]-[DNA binding domain]-[Cas12i protein]-[DNA binding domain]-COOH; NH2-[Cas12i protein]-[DNA binding domain]-[nuclear localization signal]-COOH; NH2-[DNA binding domain]-[Cas12i protein]-[nuclear localization signal]-COOH; NH2-[DNA binding domain]-[Cas12i protein]-[DNA binding domain]-[nuclear localization signal]-COOH; NH2-[nuclear localization signal]-[Cas12i protein]-[DNA binding domain]-[nuclear localization signal]-COOH; NH2-[nuclear localization signal]-[DNA binding domain]-[Cas12i protein]-[nuclear localization signal]-COOH; NH2-[nuclear localization signal]-[DNA binding domain]-[Cas12i protein]-[DNA binding domain]-[nuclear localization signal]-COOH; NH2-[Cas12i protein]-[5'-3' exonuclease functional domain]-COOH; NH2-[Nuclear localization signal]-[Cas12i protein]-[5'-3' exonuclease domain]-COOH; NH2-[Cas12i protein]-[5'-3' exonuclease domain]-[Nuclear localization signal]-COOH; NH2-[Nuclear localization signal]-[Cas12i protein]-[5'-3' exonuclease domain]-[Nuclear localization signal]-COOH; NH2-[epitope tag]-[Cas12i protein]-[5'-3' exonuclease domain]-COOH; NH2-[Cas12i protein]-[5'-3' exonuclease domain]-[epitope tag]-COOH; NH2-[epitope tag]-[ Cas12i protein]-[5'-3' exonuclease domain]-[epitope tag]-COOH; NH2-[nuclear localization signal]-[epitope tag]-[Cas12i protein]-[5'-3' exonuclease domain]-COOH; NH2-[nuclear localization signal]-[Cas12i protein]-[5'-3' exonuclease domain]-[epitope tag]-COOH; NH2-[nuclear localization signal]-[epitope tag]-[Cas12i protein]-[5'-3' exonuclease domain]-[epitope tag]-COOH; NH2-[epitope tag]-[Cas12i protein]-[5'-3' exonuclease domain]-[epitope tag]-COOH; Localization signal]-COOH; NH2-[Cas12i protein]-[5'-3' exonuclease functional domain]-[epitope tag]-[nuclear localization signal]-COOH; NH2-[epitope tag]-[Cas12i protein]-[5'-3' exonuclease functional domain]-[epitope tag]-[nuclear localization signal]-COOH; NH2-[nuclear localization signal]-[epitope tag]-[Cas12i protein]-[5'-3' exonuclease functional domain]-[nuclear localization signal]-COOH; NH2-[nuclear localization signal]-[Cas12i protein]-[5'-3' exonuclease functional domain]-[epitope tag]-[nuclear localization signal]-COOH;NH2-[nuclear localization signal]-[epitope tag]-[Cas12i protein]-[5'-3' exonuclease functional domain]-[epitope tag]-[nuclear localization signal]-COOH; NH2-[Cas12i protein]-[5'-3' exonuclease functional domain]-[reporter gene sequence]-COOH; NH2-[reporter gene sequence]-[Cas12i protein]-[5'-3' exonuclease domain]-COOH; NH2-[reporter gene sequence]-[Cas12i protein]-[5'-3' exonuclease domain]-[reporter gene sequence]-COOH; NH2-[nuclear localization signal]-[Cas12i protein]-[5'-3' exonuclease domain]-[reporter gene sequence]-COOH; NH2-[nuclear localization signal]-[reporter gene sequence]-[Cas12i protein]-[5'-3' exonuclease domain]-COOH; NH2-[nuclear localization signal]-[reporter gene sequence]-[Cas12i protein]-[5'-3' exonuclease domain]-COOH; NH2-[nuclear localization signal]-[reporter gene sequence]-[Cas12i protein]-[5'-3' exonuclease domain]-[reporter gene sequence]-COOH; NH2-[Cas12i protein]-[5'-3' exonuclease domain]-[reporter gene sequence]-[nuclear localization signal]-COOH; NH2-[reporter gene sequence]-[Cas12i protein]-[5'-3' exonuclease functional domain]-[nuclear localization signal]-COOH; NH2-[reporter gene sequence]-[Cas12i protein]-[5'-3' exonuclease functional domain]-[reporter gene sequence]-[nuclear localization signal]-COOH; NH2-[nuclear localization signal]-[Cas12i protein]-[5'-3' exonuclease functional domain]-[reporter gene sequence]-[nuclear localization signal]-COOH; NH2-[nuclear localization signal]-[reporter gene sequence]-[Cas12i protein]-[5'-3' exonuclease functional domain]-[nuclear localization signal]-COOH; NH2-[nuclear localization signal]-[reporter gene sequence]-[Cas12i protein]-[5'-3' exonuclease domain]-[reporter gene sequence]-[nuclear localization signal]-COOH; NH2-[Cas12i protein]-[5'-3' exonuclease domain]-[DNA binding domain]-COOH; NH2-[DNA binding domain]-[Cas12i protein]-[5'-3' exonuclease domain]-COOH; NH2-[DNA binding domain]-[Cas12i protein]-[5' -3' exonuclease domain]-[DNA binding domain]-COOH; NH2-[nuclear localization signal]-[Cas12i protein]-[5'-3' exonuclease domain]-[DNA binding domain]-COOH; NH2-[nuclear localization signal]-[DNA binding domain]-[Cas12i protein]-[5'-3' exonuclease domain]-COOH; NH2-[nuclear localization signal]-[DNA binding domain]-[Cas12i protein]-[5'-3' exonuclease domain]-COOH; NH2-[nuclear localization signal]-[DNA binding domain]-[Cas12i protein]-[5'-3' exonuclease domain]-[DNA binding binding domain]-COOH; NH2-[Cas12i protein]-[5'-3' exonuclease domain]-[DNA binding domain]-[nuclear localization signal]-COOH; NH2-[DNA binding domain]-[Cas12i protein]-[5'-3' exonuclease domain]-[nuclear localization signal]-COOH; NH2-[DNA binding domain]-[Cas12i protein]-[5'-3' exonuclease domain]-[nuclear localization signal]-COOH; NH2-[DNA binding domain]-[Cas12i protein]-[5'-3' exonuclease domain]-[DNA binding domain]-[nuclear localization signal]-COOH; Localization signal]-[Cas12i protein]-[5'-3' exonuclease functional domain]-[DNA binding domain]-[nuclear localization signal]-COOH; NH2-[nuclear localization signal]-[DNA binding domain]-[Cas12i protein]-[5'-3' exonuclease functional domain]-[nuclear localization signal]-COOH; NH2-[nuclear localization signal]-[DNA binding domain]-[Cas12i protein]-[5'-3' exonuclease functional domain]-[DNA binding domain]-[nuclear localization signal]-COOH.

[0035] In some embodiments, the Cas12i editor further comprises one or more heterologous polypeptides, wherein the one or more heterologous polypeptides have one or more of the following enzymatic activities: reverse transcriptase activity, nuclease activity, methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitination activity, adenylation activity, deadenylation activity, SUMOylation activity, deSUMOylation activity, ribosylation activity, deribosylation activity, myristoylation activity, demyristoylation activity, glycosylation activity (e.g., from O-GlcNAc transferase) and deglycosylation activity, DNA repair activity, DNA damage activity, deaminase activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity and glycosylase activity; more preferably, the enzymatic activity domain has one or more of the following enzymatic activities: deaminase activity, methyltransferase activity, demethylase activity, acetyltransferase activity and deacetylase activity; preferably, the one or more heterologous polypeptides are independently a transcription repression domain, a transcription activation domain, an adenosine deaminase domain, a cytidine deaminase domain.

[0036] In a preferred embodiment, the Cas12i editor is an epigenetic editor. In a preferred embodiment, the Cas12i editor is a base editor.

[0037] In some embodiments, the Cas12i protein can be fused with a heterologous polypeptide via one or more linker polypeptides (or linker peptides, linkers). Specifically, the linker polypeptide (or linker peptide, linker) may have any of a variety of amino acid sequences. Proteins can be connected by spacer peptides, which are generally flexible, but other chemical bonds are not excluded. Suitable linkers include polypeptides with a length of between 4 and 40 amino acids or a length of between 4 and 25 amino acids. These linkers can be produced by using synthetic oligonucleotides encoding linkers to couple proteins, or can be encoded by a nucleic acid sequence encoding a fusion protein. Peptide linkers with a certain degree of flexibility can be used. The linker peptide can actually have any amino acid sequence, and it should be remembered that the preferred linker will have a sequence that produces a generally flexible peptide. The use of small amino acids (such as glycine and alanine) is used to produce flexible peptides. For those skilled in the art, it is conventional to produce such sequences. A variety of different linkers are commercially available and are considered suitable for use.

[0038] Specifically, examples of linker polypeptides include glycine polymers ((G)n, wherein n is selected from 1, 2, 3, 4, 5 or 6), glycine-serine polymers ((GGGGS)n, (GGGS)n, (GGS)n, (GS)n or (G)n, wherein n is selected from 1, 2, 3, 4, 5 or 6), glycine-alanine polymers, alanine-serine polymers and α-helical linkers ((EAAAK)n, wherein n is selected from 1, 2, 3, 4, 5 or 6). The connecting peptide can also be various XTEN linkers, etc. The length of the XTEN linker is about 16-80 amino acids, and the XTEN linker can be XTEN16 linker, XTEN18 linker, XTEN32 linker, XTEN80 linker. More specifically, the connecting peptide includes but is not limited to the amino acid sequence shown in any one of SEQ ID NOs. 26 to 43. Those skilled in the art will recognize that the design of the peptide conjugated to any desired element can include a fully or partially flexible linker, so that the linker can include a flexible linker and one or more parts that confer a less flexible structure.

[0039] In some embodiments, the first direct repeat sequence and the second direct repeat sequence are the same sequence or different sequences; the first direct repeat sequence comprises the nucleotide sequence shown in any one of SEQ ID NOs.45 to 52 or a nucleotide sequence having 1 to 10 nucleotide substitutions, deletions and / or insertions compared with the nucleotide sequence shown in any one of SEQ ID NOs.45 to 52; preferably, the first direct repeat sequence is the nucleotide sequence shown in any one of SEQ ID NOs.45 to 52; the second direct repeat sequence comprises the nucleotide sequence shown in any one of SEQ ID NOs.45 to 52 or a nucleotide sequence having 1 to 10 nucleotide substitutions, deletions and / or insertions compared with the nucleotide sequence shown in any one of SEQ ID NOs.45 to 52; preferably, the second direct repeat sequence is the nucleotide sequence shown in any one of SEQ ID NOs.45 to 52. In the following examples, the above-mentioned first direct repeat sequence is respectively referred to as "DR-1", "DR-2", "DR-3", "DR-4", "DR-5", "DR-6", "DR-7" and "DR-8", and the above-mentioned second direct repeat sequence is respectively referred to as "DR-1", "DR-2", "DR-3", "DR-4", "DR-5", "DR-6", "DR-7" and "DR-8".

[0040] In some embodiments, the engineered guide RNA comprises or is a nucleotide sequence as shown in any one of SEQ ID NO.53 to 60 or its reverse complementary sequence, wherein N is any nucleotide (A, G, C or U), and n is an integer from 15 to 40, such as 15 to 30, 15 to 20, 17 to 25, 17 to 22, 18 to 22, 18 to 20, 20 to 25 or 25 to 30, for example, it can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30. In some embodiments, the engineered guide RNA comprises or is a nucleotide sequence as shown in SEQ ID NO.53 or its reverse complementary sequence, wherein N is any nucleotide (A, G, C or U), and n is an integer from 15 to 40. In some embodiments, the engineered guide RNA comprises or is a nucleotide sequence as shown in SEQ ID NO.54 or its reverse complementary sequence, wherein N is any nucleotide (A, G, C or U), and n is an integer from 15 to 40. In some embodiments, the engineered guide RNA comprises or is a nucleotide sequence as shown in SEQ ID NO.55 or its reverse complementary sequence, wherein N is any nucleotide (A, G, C or U), and n is an integer from 15 to 40. In some embodiments, the engineered guide RNA comprises or is a nucleotide sequence as shown in SEQ ID NO.56 or its reverse complementary sequence, wherein N is any nucleotide (A, G, C or U), and n is an integer from 15 to 40. In some embodiments, the engineered guide RNA comprises or is a nucleotide sequence as shown in SEQ ID NO.57 or its reverse complementary sequence, wherein N is any nucleotide (A, G, C or U), and n is an integer from 15 to 40. In some embodiments, the engineered guide RNA comprises or is a nucleotide sequence shown in SEQ ID NO.58 or its reverse complementary sequence, wherein N is any nucleotide (A, G, C or U), and n is an integer from 15 to 40. In some embodiments, the engineered guide RNA comprises or is a nucleotide sequence shown in SEQ ID NO.59 or its reverse complementary sequence, wherein N is any nucleotide (A, G, C or U), and n is an integer from 15 to 40. In some embodiments, the engineered guide RNA comprises or is a nucleotide sequence shown in SEQ ID NO.60 or its reverse complementary sequence, wherein N is any nucleotide (A, G, C or U), and n is an integer from 15 to 40. In the following examples, the above-mentioned engineered guide RNAs are respectively referred to as "pcgRNA-1", "pcgRNA-2", "pcgRNA-3", "pcgRNA-4", "pcgRNA-5", "pcgRNA-6", "pcgRNA-7" and "pcgRNA-8".

[0041] The spacer is also called a targeting segment, which comprises a nucleotide sequence (guide sequence) complementary to (and therefore hybridizing with) a specific sequence (target site) in a target nucleic acid (e.g., target dsDNA, target ssRNA, target ssDNA, complementary strand of double-stranded target DNA, etc.). The first direct repeat sequence and the second direct repeat sequence are also called protein binding segments ("protein binding sequences", Direct Repeat or DR), which interact (bind) with the Cas12i protein provided by the present invention. The site-specific binding of a target nucleic acid (e.g., genomic DNA, dsDNA, RNA, etc.) may occur at a position (e.g., a target sequence of a target locus) determined by base pairing complementarity between an engineered guide RNA (spacer) and a target nucleic acid.

[0042] In some embodiments, the percent complementarity between the guide segment of the engineered guide RNA and the target site of the target nucleic acid is 60% or more (e.g., 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%). In some embodiments, the percent complementarity between the guide segment of the engineered guide RNA and the target site of the target nucleic acid is 80% or more (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%). In some embodiments, the percent complementarity between the guide segment of the engineered guide RNA and the target site of the target nucleic acid is 90% or more (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100%). In some embodiments, the percent complementarity between the guide segment of the engineered guide RNA and the target site of the target nucleic acid is 100%.

[0043] In some embodiments, the spacer sequence of the engineered guide RNA has a length of 15 to 40 nucleotides (e.g., 15 nucleotides (nt) to 20nt, 20nt to 25nt, 25nt to 30nt, 30nt to 35nt, 35nt to 40nt, 40nt to 45nt, or 45nt to 50nt). In some embodiments, the spacer sequence of the engineered guide RNA has a length in the range of 17-30 nucleotides (nt) (e.g., 17-25, 17-22, 17-20, 19-30, 19-25, 19-22, 19-20, 20-30, 20-25, or 20-22 nt). In some embodiments, the spacer sequence of the engineered guide RNA has a length in the range of 17-25 nucleotides (nt) (e.g., 17-22, 17-20, 19-25, 19-22, 19-20, 20-25, or 20-22 nt). In some embodiments, the spacer sequence of the engineered guide RNA has a length of 17 or more nt (e.g., 18 or more, 19 or more, 20 or more, 21 or more, or 22 or more nt; 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, etc.). In some embodiments, the spacer sequence of the engineered guide RNA has a length of 19 or more nt (e.g., 20 or more, 21 or more, or 22 or more nt; 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, etc.). In some embodiments, the spacer sequence of the engineered guide RNA has a length of 17 nt. In some embodiments, the spacer sequence of the engineered guide RNA has a length of 18 nt. In some embodiments, the spacer sequence of the engineered guide RNA has a length of 19 nt. In some embodiments, the spacer sequence of the engineered guide RNA has a length of 20 nt. In some embodiments, the spacer sequence of the engineered guide RNA has a length of 21 nt. In some embodiments, the spacer sequence of the engineered guide RNA has a length of 22 nt. In some embodiments, the spacer sequence of the engineered guide RNA has a length of 23 nt.

[0044] In some embodiments, the 3' end of the engineered guide RNA is further connected to at least three consecutive poly U sequences (polyU), and the 5' end or / and 3' end of the engineered guide RNA contain 2'-O-methyl, 2'-O-methyl 3' phosphorothioate or 2'-O-methyl 3' thio PACE modification; preferably, the 3' end of the engineered guide RNA contains 2'-O-methyl, 2'-O-methyl 3' phosphorothioate or 2'-O-methyl 3' thio PACE modification; preferably, the double end of the engineered guide RNA contains 2'-O-methyl, 2'-O-methyl 3' phosphorothioate or 2'-O-methyl 3' thio PACE modification. Preferably, the poly U sequence is three consecutive U, four consecutive U, five consecutive U or six consecutive U.

[0045] In a preferred embodiment, the 3' end of the engineered guide RNA is also connected to three consecutive poly-U sequences (polyU) and its 3' end contains a 2'-O-methyl 3' phosphorothioate modification, more preferably, wherein the engineered guide RNA is a nucleotide sequence shown in any one of SEQ ID NO.61 to 68, wherein the symbol "*" represents a phosphorothioate modification and "m" represents a 2'-O-methyl group modification. In the following examples, the above-mentioned engineered guide RNAs are respectively referred to as "pcgRNA-1 (3' end modification)", "pcgRNA-2 (3' end modification)", "pcgRNA-3 (3' end modification)", "pcgRNA-4 (3' end modification)", "pcgRNA-5 (3' end modification)", "pcgRNA-6 (3' end modification)", "pcgRNA-7 (3' end modification)" and "pcgRNA-8 (3' end modification)".

[0046] In a preferred embodiment, the 3' end of the engineered guide RNA is also connected to three consecutive poly-U sequences (polyU) and its double ends contain 2'-O-methyl 3' phosphorothioate modification, more preferably, wherein the engineered guide RNA is a nucleotide sequence shown in any one of SEQ ID NO.69 to 76, wherein the symbol "*" represents phosphorothioate modification, and "m" represents 2'-O-methyl group modification. In the following examples, the above-mentioned engineered guide RNAs are respectively referred to as "pcgRNA-1 (double-end modification)", "pcgRNA-2 (double-end modification)", "pcgRNA-3 (double-end modification)", "pcgRNA-4 (double-end modification)", "pcgRNA-5 (double-end modification)", "pcgRNA-6 (double-end modification)", "pcgRNA-7 (double-end modification)" and "pcgRNA-8 (double-end modification)".

[0047] In another aspect of the present invention, a nucleic acid composition is provided, which comprises (i) a ribonucleotide sequence encoding the Cas12i editor; preferably, the ribonucleotide sequence is codon-optimized for expression in prokaryotic or eukaryotic cells; preferably, the ribonucleotide sequence is mRNA; the mRNA further comprises 5'UTR, 3'UTR and a poly A sequence (polyA); preferably, the Cas12i editor comprises or is an amino acid sequence as shown in any one of SEQ ID NO.1, 2, 3, 4, 5, 6, 7, 8, 100, 101 or 102; (ii) an engineered guide RNA, the engineered guide RNA comprising a first direct repeat sequence, a spacer sequence and a second direct repeat sequence connected sequentially from the 5' end to the 3' end; preferably, the engineered guide RNA comprises a nucleotide sequence as shown in any one of SEQ ID NOs.53 to 60 or a nucleotide sequence having 1 to 10 nucleotide substitutions, deletions and / or insertions compared with the nucleotide sequence as shown in any one of SEQ ID NOs.53 to 60; more preferably, wherein the engineered guide RNA is SEQ ID NO. The nucleotide sequence shown in any one of items 53 to 60, wherein N is any nucleotide (A, G, C or U), and n is an integer from 15 to 40, and (N)n is a spacer sequence, and (N)n is specifically complementary to the target nucleic acid.

[0048] In a preferred embodiment, in the nucleic acid composition, the mass ratio of the mRNA of the Cas12i editor to the engineered guide RNA is (0.5-4):1; preferably, the mass ratio of the mRNA of the Cas12i editor to the engineered guide RNA is (0.5-2.5):1.

[0049] In a preferred embodiment, in the nucleic acid composition, the nucleic acid composition contains 1 μg of the mRNA of the Cas12i editor and 2 μg of the engineered guide RNA; preferably, the nucleic acid composition contains 2 μg of the mRNA of the Cas12i editor and 2 μg of the engineered guide RNA; preferably, the nucleic acid composition contains 10 μg of the mRNA of the Cas12i editor and 4 μg of the engineered guide RNA.

[0050] Another aspect of the present invention provides a composition or kit, comprising a ribonucleotide sequence encoding the Cas12i editor and the engineered guide RNA encapsulated in a lipid nanoparticle (LNP); or a ribonucleotide sequence encoding the Cas12i editor and the engineered guide RNA encapsulated in a virus-like particle (VLP). In a preferred embodiment, the composition or kit further comprises a pharmaceutically acceptable carrier.

[0051] The composition or kit of the present invention may further include a pharmaceutically acceptable carrier, such as one or more additional reagents, for example, i) a buffer; ii) a protease inhibitor; iii) a nuclease inhibitor; iv) reagents required for developing or visualizing a detectable label; v) a positive and / or negative control target DNA; vi) a positive and / or negative control Cas12i guide RNA, etc. The composition or kit of the present invention may include: a) components of the CRISPR-Cas gene editing system of the present invention as described above; and b) a therapeutic agent.

[0052] The preparation of the pharmaceutical composition described herein can be prepared by any method known in the art of pharmacology. Generally, such preparation methods include combining the active ingredient with an excipient and / or one or more other auxiliary ingredients, and then, if necessary and / or desired, shaping and / or packaging the product into the desired single-dose or multi-dose unit.

[0053] Another aspect of the present invention provides a method for modifying a target nucleic acid, the method comprising contacting the target nucleic acid with the CRISPR-Cas gene editing system, the nucleic acid composition, the composition or the kit, wherein the contact causes the target nucleic acid to be modified; preferably, the modification comprises increasing or decreasing the expression of the target sequence in the target nucleic acid. In a preferred embodiment, the target nucleic acid is selected from: double-stranded DNA, single-stranded DNA, RNA, genomic DNA and extrachromosomal DNA. In a preferred embodiment, the contact occurs outside the cell in vitro, inside the cultured cell, or inside the cell in vivo. In a preferred embodiment, the cell is a eukaryotic cell, more preferably a human cell.

[0054] In some embodiments, the modification comprises increasing or decreasing the expression of a target sequence in the target nucleic acid, or the modification comprises deaminating a target adenine or a target cytosine in the target nucleic acid to achieve a base pair conversion.

[0055] The CRISPR-Cas gene editing system of the present invention can be used in a variety of methods. The method for modifying a target nucleic acid of the present invention includes contacting the target nucleic acid with the following substances: a) a ribonucleotide sequence encoding a Cas12i editor of the present invention; and b) one or more (e.g., two) engineered guide RNAs. In some embodiments, the contacting step is performed in in vitro cells. In some embodiments, the contacting step is performed in in vivo cells. In some embodiments, the contacting step is performed in ex vivo cells.

[0056] In various embodiments, the disclosed editing methods produce at least about 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98% or 99% on-target DNA base editing efficiency at the target nuclear base pair. The contacting step may result in at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74% or 75% DNA base editing efficiency. In particular, the step of contacting results in a target-based editing efficiency greater than 75%. In certain embodiments, a base editing efficiency of 99% can be achieved.

[0057] In some embodiments, the editing methods of the disclosed base editors result in an actual or average off-target DNA editing frequency of about 2.0% or less, 1.75% or less, 1.5% or less, 1.2% or less, 1% or less, 0.9% or less, 0.8% or less, 0.75% or less, 0.7% or less, 0.65% or less, or 0.6% or less in some embodiments, and the disclosed editing methods produce an actual or average off-target DNA editing frequency of 0.5%, less than 0.5%, less than 0.4%, less than 0.35%, less than 0.3%, less than 0.25%, less than 0.2%, or less than 0.1%.

[0058] Another aspect of the present invention provides that the CRISPR-Cas gene editing system provided herein can be used to treat various rare diseases, tumors, cancers, inflammation, viral infections, genetic diseases, central nervous system diseases, aging and various autoimmune diseases and common and chronic diseases. More specifically, the disease treated can be hypertension, hyperlipidemia, hepatitis B virus (HBV), hepatocellular carcinoma (HCC), scapulohumeral muscular dystrophy (FSHD), heterozygous familial hypercholesterolemia (HeFH), alpha-1 antitrypsin deficiency (A1AD), non-arteritic anterior ischemic optic neuropathy (NAION), Duchenne muscular dystrophy (DMD) or retinitis pigmentosa. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 Schematic diagram showing the structures of conventional gRNA (crRNA) and the engineered guide RNA (pcgRNA) of the present invention.

[0060] Figure 2 A map showing the nucleic acid expression vector of enCas12i-001-N229R mRNA and enCas12i-001-N229R-T5 exomRNA of the present invention.

[0061] Figure 3Shown are the sequencing results of enCas12i-001-N229R-T5 exo mRNA and the engineered guide RNA (pcgRNA) of the present invention.

[0062] Figure 4 The editing efficiency of enCas12i-001-N229R-T5 exo mRNA and the engineered guide RNA (pcgRNA) of the present invention at endogenous targets is shown.

[0063] Figure 5 Shown are the sequencing results of different forms of Cas12i editors (enCas12i-001-N229R mRNA, enCas12i-001-N229R-T5 exo mRNA, and enCas12i-001-N229R-T5 exo fusion protein) and the engineered guide RNA (pcgRNA) of the present invention.

[0064] Figure 6 The editing efficiency of different forms of Cas12i editors (enCas12i-001-N229R mRNA, enCas12i-001-N229R-T5 exo mRNA, and enCas12i-001-N229R-T5 exo fusion protein) and the engineered guide RNA (pcgRNA) of the present invention at endogenous targets is shown.

[0065] Figure 7 A graph showing the sequencing results of different targets using different amounts of Cas12i editors in the form of mRNA (enCas12i-001-N229R mRNA and enCas12i-001-N229R-T5 exo mRNA) and the engineered guide RNA (pcgRNA) of the present invention.

[0066] Figure 8 The editing efficiency of Cas12i editors in mRNA form (enCas12i-001-N229R mRNA and enCas12i-001-N229R-T5 exo mRNA) and the engineered guide RNA (pcgRNA) of the present invention at different endogenous targets is shown.

[0067] Fig. 9 A summary diagram showing the editing efficiency of Cas12i editors (enCas12i-001-N229R mRNA and enCas12i-001-N229R-T5 exo mRNA) in the form of mRNA with different dosages and the engineered guide RNA (pcgRNA) of the present invention at different endogenous targets. DETAILED DESCRIPTION

[0068] Sequence Listing Example

[0069] Example 1. Testing the modification of engineered guide RNA

[0070] like Figure 1 As shown, three modified engineered guide RNAs (pcgRNAs) were synthesized by conventional chemical synthesis methods. The spacer sequence of the engineered guide RNA targets the CCR5 gene, and the sequence of the spacer sequence (CCR5) is SEQ ID NO.77; the sequence of the engineered guide RNA (CCR5 pcgRNA (5' end modification)) with 3 bases at the 5' end modified by 2'-O-methyl 3' phosphorothioate is SEQ ID NO.78; the sequence of the engineered guide RNA (CCR5 pcgRNA (3' end modification)) with 3 bases at the 3' end modified by 2'-O-methyl 3' phosphorothioate is SEQ ID NO.79; the sequence of the engineered guide RNA (CCR5 pcgRNA (double-end modification)) with 3 bases at both ends modified by 2'-O-methyl 3' phosphorothioate is SEQ ID NO.80.

[0071] like Figure 2As shown in A, an enCas12i-001-N229R-T5 exo mRNA nucleic acid expression vector (promoter-5'UTR-kozak-BPSV40NLS-linker-3×FLAG-linker-enCas12i-001-N229R-linker-T5 exo-BPSV40NLS-3'UTR-polyA) is constructed, wherein the nucleotide sequence of 5'UTR is SEQ ID NO.97, the amino acid sequence of BPSV40 NLS is SEQ ID NO.9, the amino acid sequence of 3×FLAG is SEQ ID NO.44, the amino acid sequence of enCas12i-001-N229R is SEQ ID NO.7, the amino acid sequence of T5 exo is SEQ ID NO.8, the nucleotide sequence of 3'UTR is SEQID NO.98, the nucleotide sequence of polyA is SEQ ID NO.99, linker is a conventional glycine-serine polymer, and enCas12i-001-N229R-T5 The nucleotide sequence of the exo mRNA nucleic acid expression vector is SEQ ID NO. 103. The linearized recombinant plasmid (enCas12i-001-N229R-T5 exo mRNA nucleic acid expression vector) was transcribed using an in vitro transcription kit to obtain enCas12i-001-N229R-T5 exo mRNA.

[0072] Then, the 293T cells were grouped according to Table 1, and enCas12i-001-N229R-T5 exomRNA and CCR5pcgRNA corresponding to the three modified methods were electrotransferred into the corresponding 293T cells according to the dosage in Table 1 to knock out the CCR5 gene of the 293T cells; the electrotransferred 293T cells were cultured to obtain 293T cells with CCR5 knocked out. Next, primers were designed around the genomic region of the spacer sequence (CCR5, SEQ ID NO.77) to perform PCR on each 293T cell sample with CCR5 knocked out to PCR amplify the spacer sequence fragment of the CCR5 gene from the genomic DNA; the resulting products were then run on gel for verification, purified, and subjected to Sanger sequencing and TIDE analysis. The results are shown in Table 1, and the sequencing results are shown in Figure 3 As shown in Figure 2, the TIDE analysis results are as follows: Figure 4 As shown. These results show that the engineered guide RNA (pcgRNA) with 3' end and double end modification has higher editing efficiency. Subsequent examples are tested using engineered guide RNA (pcgRNA) with double end modification.

[0073] Table 1

[0074] Example 2. Testing the editing efficiency of different forms of Cas12i editors (enCas12i-001-N229R mRNA, enCas12i-001-N229R-T5 exo mRNA and enCas12i-001-N229R-T5 exo fusion protein)

[0075] In order to obtain a fusion protein of enCas12i-001-N229R and T5 exo, the N-terminus of the fusion protein is fused with a His tag and BPSV40 NLS, and the C-terminus of the fusion protein is fused with BPSV40 NLS; the recombinant plasmid pET-enCas12i encoding the enCas12i-001-N229R-T5exo fusion protein was transformed into Escherichia coli (Arctic Express (DE3)) for expression, and the strain was grown in LB broth supplemented with antibiotics. The bacteria were grown at 37°C to OD 600 The growth temperature was reduced to 16°C and the expression was induced for 20 h. The cells were pelleted and resuspended in loading buffer (20 mM KH2PO4, pH 7.0, 0.5 M NaCl, 10 mM imidazole, 5% glycerol) and disrupted by sonication. Cell debris was removed by centrifugation. The supernatant was loaded onto Ni 2+ Charged 5ml HiTrap chelating HP column (GE Healthcare), and eluted with a linear increasing concentration gradient of imidazole. The fractions containing enCas12i-001-N229R-T5 exo fusion protein were combined and subsequently loaded onto a HiTrap SP HP (Cytiva) column, using a linear increasing concentration gradient of NaCl (from 0.15M to 1MNaCl) for elution. The fractions containing enCas12i-001-N229R-T5 exo fusion protein were combined and dialyzed against 30mMTris pH 8.0, 350mM NaCl, 10% Glycerol, 0.1mM EDTA, 1mM DTT, and stored at -20°C. The enCas12i-001-N229R-T5 exo fusion protein was successfully expressed. The amino acid sequence of the enCas12i-001-N229R-T5 exo fusion protein of this embodiment is SEQ ID NO.101.

[0076] According to the method of Example 1, the linearized recombinant plasmid (enCas12i-001-N229R-T5 exo mRNA nucleic acid expression vector) was transcribed using an in vitro transcription kit to obtain enCas12i-001-N229R-T5 exo mRNA, wherein the nucleotide sequence of the enCas12i-001-N229R-T5 exo mRNA nucleic acid expression vector is SEQ ID NO.103.

[0077] like Figure 2 As shown in B, an enCas12i-001-N229R mRNA nucleic acid expression vector (promoter-5'UTR-kozak-BPSV40NLS-linker-3×FLAG-linker-enCas12i-001-N229R-linker-BPSV40 NLS-3'UTR-polyA) was constructed, wherein the nucleotide sequence of 5'UTR was SEQ ID NO.97, the amino acid sequence of BPSV40 NLS was SEQID NO.9, the amino acid sequence of 3×FLAG was SEQ ID NO.44, the amino acid sequence of enCas12i-001-N229R was SEQID NO.7, the nucleotide sequence of 3'UTR was SEQ ID NO.98, the nucleotide sequence of polyA was SEQ ID NO.99, linker was a conventional glycine-serine polymer, and the nucleotide sequence of enCas12i-001-N229R mRNA nucleic acid expression vector was SEQ ID NO.104. The linearized recombinant plasmid (enCas12i-001-N229RmRNA nucleic acid expression vector) was transcribed using an in vitro transcription kit to obtain enCas12i-001-N229R mRNA.

[0078] According to the method of Example 1, Figure 1 As shown, conventional chemical synthesis methods were used to synthesize double-end modified conventional gRNA and double-end modified engineered guide RNA (pcgRNA); the double-end modification was 2'-O-methyl 3' phosphorothioate modification of the three bases at the 5' end and the 3' end, the spacer sequences of the two gRNAs targeted the RNF2 gene, and the sequence of the spacer sequence (RNF2) was SEQ ID NO.81; the sequence of the double-end modified conventional gRNA (RNF2 crRNA (double-end modification)) was SEQ ID NO.82, the sequence of the unmodified RNF2 pcgRNA (RNF2 pcgRNA) was SEQ ID NO.83, and the sequence of the double-end modified RNF2 pcgRNA (RNF2pcgRNA (double-end modification)) was SEQ ID NO.84.

[0079] Then, the 293T cells were grouped according to Table 2, and the enCas12i-001-N229R-T5 exo fusion protein, enCas12i-001-N229R-T5exo mRNA, enCas12i-001-N229R mRNA and corresponding different RNF2 gRNA were electrotransferred into the corresponding 293T cells according to the dosage of Table 2 to knock out the RNF2 gene of the 293T cells; the electrotransferred 293T cells were cultured to obtain 293T cells with RNF2 knocked out. Next, primers were designed around the genomic region of the spacer sequence (RNF2, SEQ ID NO.81) to perform PCR on each 293T cell sample with RNF2 knocked out to PCR amplify the spacer sequence fragment of the RNF2 gene from the genomic DNA; the resulting product was then run for gel verification, PCR purified, and Sanger sequencing and TIDE analysis were performed. The results are shown in Table 2, and the sequencing results are as follows Figure 5 As shown in Figure 2, the TIDE analysis results are as follows: Figure 6 As shown. These results show that, compared to the Cas12i editor (enCas12i-001-N229R-T5 exo fusion protein) in protein form, the gene editing system formed by the engineered guide RNA (pcgRNA) disclosed in the present invention and the Cas12i editor (enCas12i-001-N229R mRNA) in the form of mRNA has a higher editing efficiency, that is, pcgRNA cannot improve the editing efficiency of enCas12i-001-N229R-T5 exo fusion protein; In addition, the engineered guide RNA (pcgRNA) of the present invention can effectively improve the editing efficiency of the Cas12i editor (enCas12i-001-N229R-T5 exo mRNA and enCas12i-001-N229R mRNA) in the form of mRNA in the RNF2 target gene, and compared to conventional gRNA, the editing system formed by the engineered guide RNA (pcgRNA) and enCas12i-001-N229R-T5 exo mRNA has a higher editing efficiency. Subsequent examples were tested using a double-ended modified engineered guide RNA (pcgRNA) and a Cas12i editor in mRNA form (enCas12i-001-N229R-T5 exo mRNA and enCas12i-001-N229R mRNA).

[0080] Table 2

[0081] Example 3. Testing the editing efficiency of Cas12i editors (enCas12i-001-N229R mRNA and enCas12i-001-N229R-T5 exo mRNA) in different target sites with different amounts of mRNA

[0082] like Figure 1 As shown, conventional chemical synthesis methods are used to synthesize double-end modified conventional gRNA and double-end modified engineered guide RNA (pcgRNA); the double-end modification is 2'-O-methyl 3' phosphorothioate modification of the three bases at the 5' end and the 3' end, and the spacer sequences of the two gRNAs are targeted TRAC, CD70-1 and CD70-2 genes; wherein the sequence of the spacer sequence (TRAC) is SEQ ID NO.85, the sequence of the spacer sequence (CD70-1) is SEQ ID NO.89, and the sequence of the spacer sequence (CD70-2) is SEQ ID NO.93. The sequence of the double-end modified conventional gRNA (TRAC crRNA (double-end modification)) is SEQ ID NO.86, the sequence of the unmodified TRAC pcgRNA (TRAC pcgRNA) is SEQ ID NO.87, and the sequence of the double-end modified TRAC pcgRNA (TRACpcgRNA (double-end modification)) is SEQ ID NO.88. The sequence of the conventional gRNA with double-end modification (CD70-1 crRNA (double-end modification)) is SEQ ID NO.90, the sequence of the unmodified CD70-1 pcgRNA (CD70-1 pcgRNA) is SEQ ID NO.91, and the sequence of the double-end modified CD70-1 pcgRNA (CD70-1 pcgRNA (double-end modification)) is SEQ ID NO.92. The sequence of the conventional gRNA with double-end modification (CD70-2 crRNA (double-end modification)) is SEQ ID NO.94, the sequence of the unmodified CD70-2pcgRNA (CD70-2 pcgRNA) is SEQ ID NO.95, and the sequence of the double-end modified CD70-2pcgRNA (CD70-2pcgRNA (double-end modification)) is SEQ ID NO.96.

[0083] With reference to Example 1 and Example 2, as Figure 2As shown, enCas12i-001-N229R-T5 exo mRNA nucleic acid expression vector (promoter-5'UTR-kozak-BPSV40 NLS-linker-3×FLAG-linker-enCas12i-001-N229R-linker-T5 exo-BPSV40 NLS-3'UTR-polyA) and enCas12i-001-N229R mRNA nucleic acid expression vector (promoter-5'UTR-kozak-BPSV40NLS-linker-3×FLAG-linker-enCas12i-001-N229R-linker-BPSV40 NLS-3'UTR-polyA) were constructed, the nucleotide sequence of enCas12i-001-N229R-T5 exo mRNA nucleic acid expression vector is SEQ ID NO.103, enCas12i-001-N229R The nucleotide sequence of the mRNA nucleic acid expression vector is SEQ ID NO. 104. The linearized recombinant plasmid (enCas12i-001-N229R-T5 exo mRNA nucleic acid expression vector and enCas12i-001-N229R mRNA nucleic acid expression vector) was transcribed using an in vitro transcription kit to obtain enCas12i-001-N229R-T5 exo mRNA and enCas12i-001-N229R mRNA.

[0084] Then, the 293T cells were grouped according to Table 3, and enCas12i-001-N229R-T5 exomRNA, enCas12i-001-N229R mRNA and corresponding different gRNAs were electrotransferred into the corresponding 293T cells according to the dosage in Table 3 to knock out the corresponding genes of the 293T cells; the electrotransferred 293T cells were cultured to obtain 293T cells with corresponding genes knocked out. Next, primers were designed around the genomic region of the spacer sequence (TRAC (SEQ ID NO.85), CD70-1 (SEQ ID NO.89), CD70-2 (SEQ ID NO.93)) to perform PCR on each 293T cell sample with the corresponding gene knocked out, so as to PCR amplify the spacer sequence fragment of the corresponding gene from the genomic DNA; the resulting product was then run on gel for verification, PCR purified, and subjected to Sanger sequencing and TIDE analysis. The results are shown in Table 3, and the sequencing results are shown in Table 3. Figure 7 As shown in Figure 2, the TIDE analysis results are as follows: Figure 8 The TIDE results of Example 2 and Example 3 are summarized as Fig. 9These results show that the engineered guide RNA (pcgRNA) of the present invention can effectively improve the editing efficiency of Cas12i editors in the form of mRNA (enCas12i-001-N229R-T5 exo mRNA and enCas12i-001-N229RmRNA) in multiple target genes, and the editing system formed by the engineered guide RNA (pcgRNA) and enCas12i-001-N229R-T5exo mRNA has a higher editing efficiency. In addition, Fig. 9 The results show that in the CRISPR-Cas gene editing system of the present invention, the mass ratio of the Cas12i editor in the form of mRNA (enCas12i-001-N229R-T5 exo mRNA or enCas12i-001-N229R mRNA) to the engineered guide RNA (pcgRNA) of the present invention is (0.5-4): 1, which can produce effective editing efficiency, especially the gene editing system composed of 1 μg of Cas12i editor in the form of mRNA (enCas12i-001-N229R-T5exo mRNA or enCas12i-001-N229R mRNA) and 2 μg of pcgRNA, and 2 μg of Cas12i editor in the form of mRNA (enCas12i-001-N229R-T5 exo mRNA or enCas12i-001-N229R The gene editing system consists of 10 μg of Cas12i editor in mRNA form (enCas12i-001-N229R-T5 exo mRNA or enCas12i-001-N229R mRNA) and 4 μg of pcgRNA.

[0085] Table 3

Claims

1. A CRISPR-Cas gene editing system, comprising: (a) a ribonucleotide sequence encoding a Cas12i editor, wherein the Cas12i editor comprises a Cas12i protein; the Cas12i protein comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence shown in any one of SEQ ID NO.1 to 6; and (b) an engineered guide RNA, wherein the engineered guide RNA comprises a first direct repeat sequence, a spacer sequence, and a second direct repeat sequence connected in sequence from the 5' end to the 3' end; the spacer sequence is specifically complementary to the target nucleic acid, and the engineered guide RNA is complexed with the Cas12i editor to guide the Cas12i protein to bind to the target nucleic acid.

2. The CRISPR-Cas gene editing system according to claim 1, wherein the Cas12i protein is mutated so that it has enhanced nucleic acid cleavage activity and enhanced nucleic acid binding activity; preferably, according to the sequence number shown in SEQ ID NO.1, the Cas12i protein has an amino acid substitution at position N229, preferably substituted with lysine, arginine or histidine, more preferably substituted with arginine; more preferably, the Cas12i protein comprises the amino acid sequence shown in SEQ ID NO.

7.

3. The CRISPR-Cas gene editing system according to claim 1 or 2, wherein the Cas12i editor further comprises a 5'-3' exonuclease domain, and the 5'-3' exonuclease domain is fused to the N-terminus and / or C-terminus of the Cas12i protein; preferably, the 5'-3' exonuclease domain is from T5 phage; more preferably, the 5'-3' exonuclease domain comprises an amino acid sequence having at least 99% sequence identity compared to the amino acid sequence shown in SEQ ID NO.

8.

4. The CRISPR-Cas gene editing system according to any one of claims 1 to 3, wherein the Cas12i editor further comprises one or more heterologous polypeptides, and the one or more heterologous polypeptides are independently epitope tags, nuclear localization signals, reporter gene sequences or DNA binding domains; preferably, the one or more heterologous polypeptides have one or more of the following enzymatic activities: reverse transcriptase activity, nuclease activity, methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitination activity, adenylation activity, deadenylation activity, SUMOylation activity, deSUMOylation activity, ribosylation activity, deribosylation activity, myristoylation activity, desmildomyristoylation activity, Cordyceps activity, glycosylation activity (e.g., from O-GlcNAc transferase) and deglycosylation activity, DNA repair activity, DNA damage activity, deaminase activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity and glycosylase activity; more preferably, the enzymatic activity domain has one or more of the following enzymatic activities: deaminase activity, methyltransferase activity, demethylase activity, acetyltransferase activity and deacetylase activity; preferably, the one or more heterologous polypeptides are independently a transcription repression domain, a transcription activation domain, an adenosine deaminase domain, a cytidine deaminase domain.

5. The CRISPR-Cas gene editing system according to claim 4, wherein the structure of the Cas12i editor is selected from: NH2-[Cas12i protein]-COOH; NH2-[nuclear localization signal]-[Cas12i protein]-COOH; NH2-[Cas12i protein]-[nuclear localization signal]-COOH; NH2-[nuclear localization signal]-[Cas12i protein]-[nuclear localization signal]-COOH; NH2-[epitope tag]-[Cas12i protein]-COOH; NH2-[Cas12i protein]-[epitope tag]-COOH; NH2-[epitope tag]-[Cas12i protein]-[epitope tag]-COOH; NH2-[nuclear localization signal]-[epitope tag]-[Cas12i protein]-COOH; NH2-[nuclear localization signal]-[Cas12i protein]-[epitope tag]-COOH; NH2-[nuclear localization signal]-[epitope tag]-[Cas12i protein]-[epitope tag]-COOH; NH2-[epitope tag]-[Cas12i protein]-[nuclear localization signal]-COOH; NH2-[Cas12i protein]-[epitope tag]-[nuclear localization signal]-COOH; NH2-[epitope tag]-[Cas12i protein]-[epitope tag]-[nuclear localization signal]-COOH; NH2-[nuclear localization signal]-[epitope tag]-[Cas12i protein]-[nuclear localization signal]-COOH; NH2-[nuclear localization signal]-[Cas12i protein]-[epitope tag]-[nuclear localization signal]-COOH; NH2-[nuclear localization signal]-[epitope tag]-[Cas12i protein]-[epitope tag]-[nuclear localization signal]-COOH; NH2-[Cas12i protein]-[reporter gene sequence]-COOH; NH2-[reporter gene sequence]-[Cas12i protein]-COOH; NH2-[reporter gene sequence]-[Cas12i protein]-[reporter gene sequence]-COOH; NH2-[nuclear localization signal]-[Cas12i protein]-[reporter gene sequence]-COOH; NH2-[nuclear localization signal]-[reporter gene sequence]-[Cas12i protein]-COOH; NH2-[nuclear localization signal]-[reporter gene sequence]-[Cas12i protein]-[reporter gene sequence]-COOH; NH2-[Cas12i protein]-[reporter gene sequence]-[nuclear localization signal]-COOH; NH2-[reporter gene sequence]-[Cas12i protein]-[nuclear localization signal]-COOH; NH2-[reporter gene sequence]-[Cas12i protein]-[reporter gene sequence]-[nuclear localization signal]-COOH; NH2-[nuclear localization signal]-[Cas12i protein]-[reporter gene sequence]-[nuclear localization signal]-COOH; NH2-[nuclear localization signal]-[reporter gene sequence]-[Cas12i protein]-[nuclear localization signal]-COOH; NH2-[nuclear localization signal]-[reporter gene sequence]-[Cas12i protein]-[reporter gene sequence]-[nuclear localization signal]-COOH; NH2-[Cas12i protein]-[DNA binding domain]-COOH; NH2-[DNA binding domain]-[Cas12i protein]-COOH; NH2-[DNA binding domain]-[Cas12i protein]-[DNA binding domain]-COOH; NH2-[nuclear localization signal]-[Cas12i protein]-[DNA binding domain]-COOH; NH2-[nuclear localization signal]-[DNA binding domain]-[Cas12i protein]-COOH; NH2-[nuclear localization signal]-[DNA binding domain]-[Cas12i protein]-[DNA binding domain]-COOH; NH2-[Cas12i protein]-[DNA binding domain]-[nuclear localization signal]-COOH; NH2-[DNA binding domain]-[Cas12i protein]-[nuclear localization signal]-COOH; NH2-[DNA binding domain]-[Cas12i protein]-[DNA binding domain]-[nuclear localization signal]-COOH; NH2-[nuclear localization signal]-[Cas12i protein]-[DNA binding domain]-[nuclear localization signal]-COOH; NH2-[nuclear localization signal]-[DNA binding domain]-[Cas12i protein]-[nuclear localization signal]-COOH; NH2-[nuclear localization signal]-[DNA binding domain]-[Cas12i protein]-[DNA binding domain]-[nuclear localization signal]-COOH; NH2-[Cas12i protein]-[5'-3' exonuclease domain]-COOH; NH2-[nuclear localization signal]-[Cas12i protein]-[5'-3' exonuclease functional domain]-COOH; NH2-[Cas12i protein]-[5'-3' exonuclease domain]-[nuclear localization signal]-COOH; NH2-[nuclear localization signal]-[Cas12i protein]-[5'-3' exonuclease functional domain]-[nuclear localization signal]-COOH; NH2-[epitope tag]-[Cas12i protein]-[5'-3' exonuclease domain]-COOH; NH2-[Cas12i protein]-[5'-3' exonuclease domain]-[epitope tag]-COOH; NH2-[epitope tag]-[Cas12i protein]-[5'-3' exonuclease domain]-[epitope tag]-COOH; NH2-[nuclear localization signal]-[epitope tag]-[Cas12i protein]-[5'-3' exonuclease domain]-COOH; NH2-[nuclear localization signal]-[Cas12i protein]-[5'-3' exonuclease domain]-[epitope tag]-COOH; NH2-[nuclear localization signal]-[epitope tag]-[Cas12i protein]-[5'-3' exonuclease domain]-[epitope tag]-COOH; NH2-[epitope tag]-[Cas12i protein]-[5'-3' exonuclease domain]-[nuclear localization signal]-COOH; NH2-[Cas12i protein]-[5'-3' exonuclease domain]-[epitope tag]-[nuclear localization signal]-COOH; NH2-[epitope tag]-[Cas12i protein]-[5'-3' exonuclease domain]-[epitope tag]-[nuclear localization signal]-COOH; NH2-[nuclear localization signal]-[epitope tag]-[Cas12i protein]-[5'-3' exonuclease domain]-[nuclear localization signal]-COOH; NH2-[nuclear localization signal]-[Cas12i protein]-[5'-3' exonuclease domain]-[epitope tag]-[nuclear localization signal]-COOH; NH2-[nuclear localization signal]-[epitope tag]-[Cas12i protein]-[5'-3' exonuclease functional domain]-[epitope tag]-[nuclear localization signal]-COOH; NH2-[Cas12i protein]-[5'-3' exonuclease functional domain]-[reporter gene sequence]-COOH; NH2-[reporter gene sequence]-[Cas12i protein]-[5'-3' exonuclease functional domain]-COOH; NH2-[reporter gene sequence]-[Cas12i protein]-[5'-3' exonuclease functional domain]-[reporter gene sequence]-COOH; NH2-[nuclear localization signal]-[Cas12i protein]-[5'-3' exonuclease functional domain]-[reporter gene sequence]-COOH; NH2-[nuclear localization signal]-[reporter gene sequence]-[Cas12i protein]-[5'-3' exonuclease functional domain]-COOH; NH2-[nuclear localization signal]-[reporter gene sequence]-[Cas12i protein]-[5'-3' exonuclease functional domain]-[reporter gene sequence]-COOH; NH2-[Cas12i protein]-[5'-3' exonuclease domain]-[reporter gene sequence]-[nuclear localization signal]-COOH; NH2-[reporter gene sequence]-[Cas12i protein]-[5'-3' exonuclease functional domain]-[nuclear localization signal]-COOH; NH2-[reporter gene sequence]-[Cas12i protein]-[5'-3' exonuclease functional domain]-[reporter gene sequence]-[nuclear localization signal]-COOH; NH2-[nuclear localization signal]-[Cas12i protein]-[5'-3' exonuclease functional domain]-[reporter gene sequence]-[nuclear localization signal]-COOH; NH2-[nuclear localization signal]-[reporter gene sequence]-[Cas12i protein]-[5'-3' exonuclease functional domain]-[nuclear localization signal]-COOH; NH2-[nuclear localization signal]-[reporter gene sequence]-[Cas12i protein]-[5'-3' exonuclease functional domain]-[reporter gene sequence]-[nuclear localization signal]-COOH; NH2-[Cas12i protein]-[5'-3' exonuclease domain]-[DNA binding domain]-COOH; NH2-[DNA binding domain]-[Cas12i protein]-[5'-3' exonuclease domain]-COOH; NH2-[DNA binding domain]-[Cas12i protein]-[5'-3' exonuclease functional domain]-[DNA binding domain]-COOH; NH2-[nuclear localization signal]-[Cas12i protein]-[5'-3' exonuclease domain]-[DNA binding domain]-COOH; NH2-[nuclear localization signal]-[DNA binding domain]-[Cas12i protein]-[5'-3' exonuclease functional domain]-COOH; NH2-[nuclear localization signal]-[DNA binding domain]-[Cas12i protein]-[5'-3' exonuclease functional domain]-[DNA binding domain]-COOH; NH2-[Cas12i protein]-[5'-3' exonuclease domain]-[DNA binding domain]-[nuclear localization signal]-COOH; NH2-[DNA binding domain]-[Cas12i protein]-[5'-3' exonuclease domain]-[nuclear localization signal]-COOH; NH2-[DNA binding domain]-[Cas12i protein]-[5'-3' exonuclease functional domain]-[DNA binding domain]-[nuclear localization signal]-COOH; NH2-[nuclear localization signal]-[Cas12i protein]-[5'-3' exonuclease functional domain]-[DNA binding domain]-[nuclear localization signal]-COOH; NH2-[nuclear localization signal]-[DNA binding domain]-[Cas12i protein]-[5'-3' exonuclease functional domain]-[nuclear localization signal]-COOH; NH2-[nuclear localization signal]-[DNA binding domain]-[Cas12i protein]-[5'-3' exonuclease functional domain]-[DNA binding domain]-[nuclear localization signal]-COOH.

6. The CRISPR-Cas gene editing system according to claim 1, wherein the first direct repeat sequence and the second direct repeat sequence are the same sequence or different sequences; the first direct repeat sequence comprises the nucleotide sequence shown in any one of SEQ ID NOs. 45 to 52 or a nucleotide sequence having 1 to 10 nucleotide substitutions, deletions and / or insertions compared with the nucleotide sequence shown in any one of SEQ ID NOs. 45 to 52; preferably, the first direct repeat sequence is a nucleotide sequence shown in any one of SEQ ID NOs. 45 to 52; the second direct repeat sequence comprises a nucleotide sequence shown in any one of SEQ ID NOs. 45 to 52 or a nucleotide sequence having 1 to 10 nucleotide substitutions, deletions and / or insertions compared with the nucleotide sequence shown in any one of SEQ ID NOs. 45 to 52; preferably, the second direct repeat sequence is a nucleotide sequence shown in any one of SEQ ID NOs. 45 to 52.

7. The CRISPR-Cas gene editing system according to claim 1, wherein the 3' end of the engineered guide RNA is also connected to at least three consecutive poly-U sequences (polyU), and the 5' end or / and 3' end of the engineered guide RNA contains 2'-O-methyl, 2'-O-methyl 3' phosphorothioate or 2'-O-methyl 3' thio PACE modification; preferably, the 3' end of the engineered guide RNA contains 2'-O-methyl, 2'-O-methyl 3' phosphorothioate or 2'-O-methyl 3' thio PACE modification; preferably, the double ends of the engineered guide RNA contain 2'-O-methyl, 2'-O-methyl 3' phosphorothioate or 2'-O-methyl 3' thio PACE modification.

8. A nucleic acid composition comprising (i) a ribonucleotide sequence encoding a Cas12i editor as described in any one of claims 1 to 7; preferably, the ribonucleotide sequence is codon-optimized for expression in prokaryotic or eukaryotic cells; preferably, the ribonucleotide sequence is mRNA; the mRNA further comprises a 5'UTR, a 3'UTR and a poly A sequence (polyA); preferably, the Cas12i editor comprises or is an amino acid sequence as shown in any one of SEQ ID NOs. 1 to 7, 100 to 102; (ii) an engineered guide RNA, wherein the engineered guide RNA comprises a first direct repeat sequence, a spacer sequence, and a second direct repeat sequence connected in sequence from the 5' end to the 3' end; preferably, the engineered guide RNA comprises a nucleotide sequence as shown in any one of SEQ ID NOs. 53 to 60, or a nucleotide sequence having 1 to 10 nucleotide substitutions, deletions and / or insertions compared with the nucleotide sequence as shown in any one of SEQ ID NOs. 53 to 60; more preferably, the engineered guide RNA is a nucleotide sequence as shown in any one of SEQ ID NOs. 53 to 60, wherein, N is any nucleotide (A, G, C or U), and n is an integer from 15 to 40, said (N) n is a spacer sequence, said (N) n specifically complementary to the target nucleic acid; Preferably, the mass ratio of the mRNA of the Cas12i editor to the engineered guide RNA is (0.5-4):1; Preferably, the nucleic acid composition contains 1 μg of the mRNA of the Cas12i editor and 2 μg of the engineered guide RNA; preferably, the nucleic acid composition contains 2 μg of the mRNA of the Cas12i editor and 2 μg of the engineered guide RNA; preferably, the nucleic acid composition contains 10 μg of the mRNA of the Cas12i editor and 4 μg of the engineered guide RNA.

9. A composition or kit comprising A ribonucleotide sequence encoding a Cas12i editor as described in any one of claims 1 to 7 and an engineered guide RNA as described in any one of claims 1 to 7 encapsulated in a lipid nanoparticle (LNP); or A ribonucleotide sequence encoding a Cas12i editor as described in any one of claims 1 to 7 and an engineered guide RNA as described in any one of claims 1 to 7 encapsulated in a virus-like particle (VLP).

10. A method for modifying a target nucleic acid, the method comprising contacting the target nucleic acid with the CRISPR-Cas gene editing system according to any one of claims 1 to 7, the nucleic acid composition according to claim 8, the composition or the kit according to claim 9, wherein the contact results in the target nucleic acid being modified; preferably, the modification comprises increasing or decreasing the expression of a target sequence in the target nucleic acid.

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