CRISPR-Cas gene editing system for preparing universal CAR-T

Abstract

The invention discloses a CRISPR (clustered regularly interspaced short palindromic repeats)-Cas gene editing system for preparing a universal CAR-T. The invention discloses a CRISPR-Cas gene editing system for preparing a universal CAR-T. The CRISPR-Cas gene editing system comprises a Cas12i editor and an engineered guide RNA, a nucleic acid composition for encoding the Cas12i editor 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 cell therapy technology, in particular to the field of CAR-T cell therapy technology. Specifically, the present invention relates to a CRISPR-Cas gene editing system for preparing a universal CAR-T, which comprises RNA encoding a Cas12i editor and an engineered guide RNA, as well as a nucleic acid composition encoding them. The present invention also relates to a CRISPR-Cas gene editing composition or kit for preparing a universal CAR-T, which comprises the CRISPR-Cas12i gene editing system of the present invention encapsulated in lipid nanoparticles (LNP), or the CRISPR-Cas12i gene editing system of the present invention encapsulated in virus-like particles (VLP). The present invention also relates to a method for preparing a universal CAR-T, which comprises obtaining T cells from a healthy donor and preparing them into CAR-T cells through the CRISPR-Cas12i gene editing system of the present invention. Background Art

[0002] Tumor immunotherapy has received extensive attention and application in recent years, especially the emergence of chimeric antigen receptor T cells (CAR-T) technology, which has made a milestone in human control of tumors. Currently, the U.S. Food and Drug Administration (FDA) and the China Center for Drug Evaluation (CDE) have approved a number of CAR-T cell drugs for marketing. However, all the approved ones are autologous CAR-T, and their clinical application still has certain limitations, such as complex preparation process, long time and high cost. In addition, some patients' own T cells do not meet the requirements for preparing CAR-T cells.

[0003] Based on the defects of these autologous CAR-T cells, scientists have proposed the concept of universal CAR-T (UCART) cells. Universal CAR-T cells refer to T cells obtained from healthy donors and prepared into CAR-T cells through genetic engineering technology. Its advantages can well make up for the shortcomings of autologous CAR-T cell therapy. However, universal CAR-T needs to solve its attack on the receptor or host (GVHD) and the immune rejection of universal CAR-T cells by the receptor or host (HvGR). To solve these two major problems, it is necessary to further transform T cells from healthy donors through gene editing or non-gene editing methods on the basis of inserting chimeric antigen receptor (CAR) genes to avoid GVHD and HvGR, so that they can survive in patients and effectively identify and kill tumor cells. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a CRISPR-Cas gene editing system for preparing universal CAR-T. The universal CAR-T cells prepared by the Cas12i editor provided by the present invention and its corresponding engineered guide RNA can efficiently knock out one or more of the target nucleic acid sequences targeting CD7 gene, CD70 gene, B2M gene, TRAC gene and CIITA gene, thereby obtaining a universal CAR-T with better efficacy and higher safety.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] One aspect of the present invention provides a CRISPR-Cas gene editing system for preparing a universal CAR-T, comprising: (a) a Cas12i editor; wherein the Cas12i editor is an amino acid sequence of a Cas12i editor or a ribonucleotide sequence encoding the Cas12i editor, and the Cas12i editor comprises a Cas12i protein; the Cas12i protein comprises or has an amino acid sequence having at least 99% sequence identity compared 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 and a target nucleic acid sequence connected in sequence from the 5' end to the 3' end; the target nucleic acid sequence is specifically complementary to the target gene, and the engineered guide RNA is complexed with the Cas12i editor to guide the Cas12i protein to bind to the target nucleic acid; wherein the target nucleic acid sequence targets one or more of the CD7 gene, CD70 gene, B2M gene, TRAC gene and CIITA gene to knock out the corresponding gene; preferably, the target nucleic acid sequence targeting the CD7 gene comprises or has a sequence identity with SEQ ID NO. NO.61 to 70; the target nucleic acid sequence targeting the CD70 gene comprises or has a sequence having at least 90% sequence identity compared to the sequence shown in SEQ ID NO.83 or 84; the target nucleic acid sequence targeting the B2M gene comprises or has a sequence having at least 90% sequence identity compared to the sequence shown in SEQ ID NO.89 to 98; the target nucleic acid sequence targeting the TRAC gene comprises or has a sequence having at least 90% sequence identity compared to the sequence shown in SEQ ID NO.112 to 130; the target nucleic acid sequence targeting the CIITA gene comprises or has a sequence having at least 90% sequence identity compared to the sequence shown in SEQ ID NO.153 to 160.

[0007] Preferably, the Cas12i protein 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.

[0008] In some embodiments, the ribonucleotide sequence encoding the Cas12i editor of the present invention is codon optimized for expression in a target cell. 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 encoding the same protein at the same time. Therefore, the codon may change, but the encoded protein remains unchanged; preferably, the ribonucleotide sequence is codon optimized for expression in eukaryotic cells. For example, if the expected target cell is a human cell, a human codon-optimized ribonucleotide sequence encoding the Cas12i editor can be used. As another non-limiting example, if the expected host cell is a mammalian T cell, a mammalian codon-optimized ribonucleotide sequence encoding the Cas12i editor can be generated, such as a human T cell. If the expected host cell is an animal cell (such as a mammalian cell), a corresponding animal codon-optimized ribonucleotide sequence encoding the Cas12i editor can be generated.

[0009] 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.).

[0010] In some embodiments, the CRISPR-Cas gene editing system can be the RNP of the Cas12i editor and the engineered guide RNA, or the mRNA encoding the Cas12i editor and the engineered guide RNA.

[0011] In other embodiments, the nucleic acid composition molecules of the CRISPR-Cas gene editing system of the present invention 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.

[0012] 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.

[0013] 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).

[0014] 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 Cas12i protein, a 3'UTR (such as a 3'UTR from a human β-globin) and a poly A sequence (polyA). Specifically, the 5'UTR comprises or is a nucleotide sequence as shown in SEQ ID NO.169, the 3'UTR comprises or is a nucleotide sequence as shown in SEQ ID NO.170, the length of the poly A sequence (polyA) is 50-200nt, and polyA comprises or is a nucleotide sequence as shown in SEQ ID NO.171.

[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 Cas12i protein, 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.104.

[0016] 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).

[0017] 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.175 or 176.

[0018] 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.

[0019] 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.

[0020] In other embodiments, the Cas12i protein is mutated so that it has (1) partial or complete inactivation of nucleic acid cleavage activity; (2) enhanced nucleic acid cleavage activity; (3) enhanced nucleic acid binding activity.

[0021] In other preferred embodiments, the Cas12i protein is mutated to enhance its nucleic acid binding activity, partially or completely inactivate nucleic acid cleavage activity, or enhance nucleic acid cleavage activity. In such an embodiment, 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 a reduction in the nucleic acid cleavage activity of the Cas12i protein, for example, compared to the Cas12i protein, the nucleic acid cleavage activity is reduced 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. 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.

[0022] 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". Specifically, enCas12i-001 to enCas12i-006 each form an editor for knocking out a target gene.

[0023] 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.

[0024] In other embodiments, the Cas12i protein is mutated to completely inactivate its nucleic acid cleavage activity, and according to the sequence numbering shown in SEQ ID NO.1, the Cas12i protein has an amino acid substitution at position D1009, preferably substituted with alanine; referred to as "denCas12i-001-D1009A". Specifically, denCas12i-001-D1009A to denCas12i-006-D1009A are each fused with a transcriptional repression domain to form an epigenetic suppressor, denCas12i-001-D1009A to denCas12i-006-D1009A are each fused with a transcriptional activation domain to form an epigenetic activator, and denCas12i-001-D1009A to denCas12i-006-D1009A are each fused with a transcriptional activation domain to form an epigenetic activator. Each of them is fused with an adenosine deaminase domain to form an adenine base editor, denCas12i-001-D1009A to denCas12i-006-D1009A are each fused with a cytidine deaminase domain to form a cytosine base editor, and denCas12i-001-D1009A to denCas12i-006-D1009A are each fused with a cytidine deaminase domain and a uracil DNA glycosylase inhibitor domain to form a cytosine base editor.

[0025] 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.

[0026] 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.

[0027] In some embodiments, the structure of the Cas12i editor is selected from: NH2-[Mutant protein of Cas12i]-[5'-3' exonuclease functional domain]-COOH; NH2-[5'-3' exonuclease functional domain]-[Mutant protein of Cas12i]-COOH; NH2-[5'-3' exonuclease functional domain]-[Cas12i protein]-COOH; NH2-[Cas12i protein]-[5'-3' exonuclease functional domain]-COOH.

[0028] 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).

[0029] 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.172.

[0030] In some embodiments, 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: transcriptional regulatory activity, deaminase activity and inhibition of uracil DNA glycosylase activity; more preferably, the one or more heterologous polypeptides are independently transcription repression domains, transcriptional activation domains, adenosine deaminase domains, cytidine deaminase domains and uracil DNA glycosylase inhibitor domains.

[0031] 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).

[0032] In a preferred embodiment, the epitope tag is 3×FLAG, and the 3×FLAG comprises the amino acid sequence shown in SEQ ID NO.34.

[0033] 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), and more specifically, the amino acid sequence of 2 NLS (2×NLS) includes the amino acid sequence shown in SEQ ID NO.15. 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 order can be: NH2-[Cas12i editor]-[NLS]-COOH; NH2-[NLS]-[Cas12i editor]-COOH; NH2-[NLS]-[Cas12i editor]-[NLS]-COOH; wherein]-[represents a connecting peptide that may optionally exist according to the definition below (the same below).

[0034] 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).

[0035] 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 15.

[0036] 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.

[0037] In other embodiments, the Cas12i editor fused with a heterologous polypeptide (BPSV40NLS and His tag) comprises or is the amino acid sequence shown in SEQ ID NO.173 or 174.

[0038] 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.

[0039] In some embodiments, the heterologous polypeptide is selected from one or more of the following enzymatic activities: transcriptional regulatory activity, deaminase activity and inhibition of uracil DNA glycosylase activity; more preferably, the one or more heterologous polypeptides are independently a transcription repression domain, a transcriptional activation domain, an adenosine deaminase domain, a cytidine deaminase domain and a uracil DNA glycosylase inhibitor domain.

[0040] In a preferred embodiment, the Cas12i editor is an editor for knocking out the target gene, and the Cas12i editor is an epigenetic editor. In a preferred embodiment, the Cas12i editor is a base editor, such as a cytosine base editor or an adenine base editor. The editor, epigenetic editor, and base editor provided by the present invention can be used to modify the T cell target gene from a healthy donor to transform T cells to avoid GVHD and avoid HvGR.

[0041] In some preferred embodiments, the transcription repression domain is selected from KRAB catalytic domain (such as ZIM3 or KOX1), DNA methyltransferase (such as DNMT3A, DNMT3B, DNMT1, or DNMT3L) or a combination thereof; wherein the deaminase domain includes an adenosine deaminase domain, a cytidine deaminase domain or a combination thereof; preferably, the cytidine deaminase is selected from activation-induced cytidine deaminase (AID), apolipoprotein B mRNA editing complex (APOBEC) and PmCDA1; preferably, the adenosine deaminase domain is TadA, ecTadA, saTadA, ecTadA7.10, TadA-8e, TadA8.17, TadA8.20, TadA9 or a combination thereof; preferably, the uracil DNA glycosylase inhibitor domain is a uracil glycosylase inhibitor (UGI).

[0042] In some embodiments, the structure of the Cas12i editor is selected from: NH2-[one or more heterologous polypeptides]-[Cas12i protein]-COOH; NH2-[Cas12i protein]-[one or more heterologous polypeptides]-COOH; NH2-[one or more heterologous polypeptides]-[Cas12i protein]-[one or more heterologous polypeptides]-COOH; NH2-[one or more heterologous polypeptides]-[Cas12i protein]-[one or more heterologous polypeptides]-COOH; NH2-[one or more heterologous polypeptides]-[mutant protein of Cas12i]-[one or more heterologous polypeptides]-COOH; NH2-[one or more heterologous polypeptides]-[mutant protein of Cas12i]-[one or more heterologous polypeptides]-COOH; NH2-[one or more heterologous polypeptides]-[mutant protein of Cas12i]-[one or more heterologous polypeptides]-COOH; NH2-[one or more heterologous polypeptides]-[mutant protein of Cas12i]-[5'- 3' exonuclease functional domain]-COOH; NH2-[Mutant protein of Cas12i]-[5'-3' exonuclease functional domain]-[one or more heterologous polypeptides]-COOH; NH2-[one or more heterologous polypeptides]-[Mutant protein of Cas12i]-[5'-3' exonuclease functional domain]-[one or more heterologous polypeptides]-COOH; NH2-[one or more heterologous polypeptides]-[5'-3' exonuclease functional domain]-[Cas12i protein]-COOH; NH2-[5'-3' exonuclease functional domain]-[Cas12i protein]-[one or more heterologous polypeptides]-COOH; or NH2-[one or more heterologous polypeptides]-[5'-3' exonuclease functional domain]-[Cas12i protein]-[one or more heterologous polypeptides]-COOH.

[0043] In some embodiments, the structure of the Cas12i editor is selected from: NH2-[KRAB catalytic domain]-[Cas12i protein]-[DNA methyltransferase]-COOH; NH2-[DNA methyltransferase]-[Cas12i protein]-[KRAB catalytic domain]-COOH; NH2-[Cas12i protein]-[adenosine deaminase domain]-COOH; NH2-[adenosine deaminase domain]-[Cas12i protein]-COOH; NH2-[cytidine deaminase ]-[Cas12i protein]-COOH; NH2-[Cas12i protein]-[cytidine deaminase]-COOH; NH2-[cytidine deaminase]-[Cas12i protein]-[UGI]-COOH; NH2-[UGI]-[Cas12i protein]-[cytidine deaminase]-COOH; NH2-[cytidine deaminase]-[Cas12i protein]-[2×UGI]-COOH; NH2-[2×UGI]-[Cas12i protein]-[cytidine deaminase]-COOH.

[0044] In some embodiments, the structure of the Cas12i editor is selected from: NH2-[KRAB catalytic domain]-[one or more heterologous polypeptides]-[Cas12i protein]-[DNA methyltransferase]-COOH; NH2-[KRAB catalytic domain]-[Cas12i protein]-[one or more heterologous polypeptides]-[DNA methyltransferase]-COOH; NH2-[KRAB catalytic domain]-[one or more heterologous polypeptides]-[Cas12i protein]-[one or more heterologous polypeptides]-[DNA methyltransferase]-COOH; NH2-[DNA methyltransferase]-[one or more heterologous polypeptides]-[Cas12i protein]- [KRAB catalytic domain]-COOH; NH2-[DNA methyltransferase]-[Cas12i protein]-[one or more heterologous polypeptides]-[KRAB catalytic domain]-COOH; NH2-[DNA methyltransferase]-[one or more heterologous polypeptides]-[Cas12i protein]-[one or more heterologous polypeptides]-[KRAB catalytic domain]-COOH; NH2-[one or more heterologous polypeptides]-[Cas12i protein]-[adenosine deaminase domain]-COOH; NH2-[Cas12i protein]-[one or more heterologous polypeptides]-[adenosine deaminase domain]-COOH; NH2-[one or more heterologous polypeptides]-[Ca s12i protein]-[one or more heterologous polypeptides]-[adenosine deaminase domain]-COOH; NH2-[adenosine deaminase domain]-[one or more heterologous polypeptides]-[Cas12i protein]-COOH; NH2-[adenosine deaminase domain]-[Cas12i protein]-[one or more heterologous polypeptides]-COOH; NH2-[adenosine deaminase domain]-[Cas12i protein]-[one or more heterologous polypeptides]-COOH; NH2-[adenosine deaminase domain]-[one or more heterologous polypeptides]-[Cas12i protein]-[one or more heterologous polypeptides]-COOH; NH2-[cytidine deaminase]-[one or more heterologous polypeptides]-[Cas12i protein]-COOH; NH2-[Cas12i protein]-[one or more heterologous polypeptides] -[Cytidine deaminase]-COOH; NH2-[Cytidine deaminase]-[one or more heterologous polypeptides]-[Cas12i protein]-[one or more heterologous polypeptides]-COOH; NH2-[one or more heterologous polypeptides]-[Cas12i protein]-[Cytidine deaminase]-COOH; NH2-[Cytidine deaminase]-[Cas12i protein]-[one or more heterologous polypeptides]-COOH; NH2-[one or more heterologous polypeptides]-[Cas12i protein]-[one or more heterologous polypeptides]-[Cytidine deaminase]-COOH; NH2-[Cytidine deaminase]-[one or more heterologous polypeptides]-[Cas12i protein]-[UGI]-COOH;NH2-[Cytidine deaminase]-[Cas12i protein]-[one or more heterologous polypeptides]-[UGI]-COOH; NH2-[Cytidine deaminase]-[one or more heterologous polypeptides]-[Cas12i protein]-[one or more heterologous polypeptides]-[UGI]-COOH; NH2-[UGI]-[one or more heterologous polypeptides]-[Cas12i protein]-[Cytidine deaminase]-COOH; NH2-[UGI]-[Cas12i protein]-[one or more heterologous polypeptides]-[Cytidine deaminase]-COOH; NH2-[UGI]-[one or more heterologous polypeptides]-[Cas12i protein]- [One or more heterologous polypeptides]-[Cytidine deaminase]-COOH; NH2-[Cytidine deaminase]-[One or more heterologous polypeptides]-[Cas12i protein]-[2×UGI]-COOH; NH2-[Cytidine deaminase]-[Cas12i protein]-[One or more heterologous polypeptides]-[2×UGI]-COOH; NH2-[Cytidine deaminase]-[One or more heterologous polypeptides]-[Cas12i protein]-[One or more heterologous polypeptides]-[2×UGI]-COOH; NH2-[2×UGI]-[One or more heterologous polypeptides]-[Cas12i protein]-[Cytidine deaminase]-COOH; NH2-[2×UGI]-[Cas12i protein]-[one or more heterologous polypeptides]-[cytidine deaminase]-COOH; NH2-[2×UGI]-[one or more heterologous polypeptides]-[Cas12i protein]-[one or more heterologous polypeptides]-[cytidine deaminase]-COOH.

[0045] 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.

[0046] 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. 16 to 33. 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.

[0047] In some embodiments, the engineered guide RNA further comprises a second direct repeat sequence; the engineered guide RNA comprises a first direct repeat sequence, a target nucleic acid sequence, and a second direct repeat sequence connected in sequence from the 5' end to the 3' end; 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.35 to 42, 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.35 to 42; preferably, the first direct repeat sequence is the nucleotide sequence shown in any one of SEQ ID NOs.35 to 42; the second direct repeat sequence comprises the nucleotide sequence shown in any one of SEQ ID NOs.35 to 42, 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.35 to 42; preferably, the second direct repeat sequence is the nucleotide sequence shown in any one of SEQ ID NOs.35 to 42. 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".

[0048] In some preferred embodiments, the target nucleic acid sequence of the engineered guide RNA can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; preferably, the target nucleic acid sequence of the engineered guide RNA is 2; the two target nucleic acid sequences are separated by a first direct repeat sequence or a second direct repeat sequence; for example, when the target nucleic acid sequence of the engineered guide RNA is 2, the engineered guide RNA comprises the first direct repeat sequence, the first target nucleic acid sequence, the first direct repeat sequence and the second target nucleic acid sequence connected in sequence from the 5' end to the 3' end, or the engineered guide RNA comprises the first direct repeat sequence, the first target nucleic acid sequence, the second direct repeat sequence and the second target nucleic acid sequence connected in sequence from the 5' end to the 3' end.

[0049] In some embodiments, the engineered guide RNA comprises or is a nucleotide sequence as shown in any one of SEQ ID NO. 43 to 50 or a reverse complementary sequence thereof, 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. 43 or a reverse complementary sequence thereof, 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.44 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.45 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.46 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.47 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.48 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.49 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.50 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 "cgRNA-1", "cgRNA-2", "cgRNA-3", "cgRNA-4", "cgRNA-5", "cgRNA-6", "cgRNA-7" and "cgRNA-8".

[0050] In some embodiments, the engineered guide RNA comprises or is a nucleotide sequence as shown in any one of SEQ ID NO.51 to 58 or a reverse complementary sequence thereof, 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, 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.51 or a reverse complementary sequence thereof, 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.52 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.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 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 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 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".

[0051] In some embodiments, the engineered guide RNA (cgRNA and pcgRNA), i.e., SEQ ID NO.71 to SEQ ID NO.82, SEQ ID NO.85 to SEQ ID NO.88, SEQ ID NO.99 to SEQ ID NO.111, SEQ ID NO.131 to SEQ ID NO.152, and SEQ ID NO.161 to SEQ ID NO.168, is modified at both ends: three consecutive poly-U sequences (polyU) are connected to the 3' end of the gRNA (cgRNA and pcgRNA), and 3 consecutive bases at the 5' and 3' ends of the gRNA are modified with 2'-O-methyl 3' phosphorothioate. For example, the engineered guide RNA with double-end modification is the nucleotide sequence shown in SEQ ID NO.59 or 60, and the symbol "*" in the sequence indicates phosphorothioate modification, and "m" indicates 2'-O-methyl group modification.

[0052] The target nucleic acid sequence is also called a spacer sequence, a targeting section, which comprises a nucleotide sequence (guide sequence) complementary to (and therefore hybridizing with) a specific sequence (target site) in a target gene (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 sections ("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 (target nucleic acid sequence) and a target nucleic acid.

[0053] In some embodiments, the complementarity percentage between the target nucleic acid sequence of the engineered guide RNA and the target site of the target gene 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 complementarity percentage between the target nucleic acid sequence of the engineered guide RNA and the target site of the target gene 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 complementarity percentage between the target nucleic acid sequence of the engineered guide RNA and the target site of the target gene 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 target nucleic acid sequence of the engineered guide RNA and the target site of the target gene is 100%.

[0054] In some embodiments, the target nucleic acid 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 target nucleic acid 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 target nucleic acid 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 target nucleic acid 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 target nucleic acid 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 target nucleic acid sequence of the engineered guide RNA has a length of 17 nt. In some embodiments, the target nucleic acid sequence of the engineered guide RNA has a length of 18 nt. In some embodiments, the target nucleic acid sequence of the engineered guide RNA has a length of 19 nt. In some embodiments, the target nucleic acid sequence of the engineered guide RNA has a length of 20 nt. In some embodiments, the target nucleic acid sequence of the engineered guide RNA has a length of 21 nt. In some embodiments, the target nucleic acid sequence of the engineered guide RNA has a length of 22 nt. In some embodiments, the target nucleic acid sequence of the engineered guide RNA has a length of 23 nt.

[0055] 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.

[0056] In a preferred embodiment, the 3' end of the engineered guide RNA is also connected to three consecutive poly-U sequences (polyU) and its 5' end or / and 3' end contain 2'-O-methyl 3' phosphorothioate modification, more preferably, wherein the engineered guide RNA is a nucleotide sequence shown in SEQ ID NO.59 or 60, wherein the symbol "*" in the sequence below 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 "cgRNA-1 (double-end modification)" and "pcgRNA-1 (double-end modification)".

[0057] In another aspect of the present invention, a nucleic acid composition is provided, which comprises (i) a Cas12i editor; wherein the Cas12i editor is an amino acid sequence of a Cas12i editor or a ribonucleotide sequence encoding the Cas12i editor, and the Cas12i editor comprises a Cas12i protein; the Cas12i protein comprises or has an amino acid sequence having at least 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO.1 to 6 or 7; preferably, the ribonucleotide sequence is codon-optimized for expression in prokaryotes 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 shown in any one of SEQ ID NO.1, 2, 3, 4, 5, 6 or 7; (ii) an engineered guide RNA, wherein the engineered guide RNA comprises a first direct repeat sequence and a target nucleic acid sequence connected sequentially from the 5' end to the 3' end; preferably, the engineered guide RNA comprises SEQ ID NO.43 to 58 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 NO.43 to 58; more preferably, the engineered guide RNA is a nucleotide sequence shown in any one of SEQ ID NO.43 to 58, wherein N is any nucleotide (A, G, C or U), and n is an integer from 15 to 40, and the (N)n is a target nucleic acid sequence; preferably, the target nucleic acid sequence targeting the CD7 gene comprises or has a sequence having at least 90% sequence identity compared with the sequence shown in any one of SEQ ID NO.61 to 70; the target nucleic acid sequence targeting the CD70 gene comprises or has a sequence having at least 90% sequence identity compared with the sequence shown in SEQ ID NO.83 or 84; the target nucleic acid sequence targeting the B2M gene comprises or has a sequence having at least 90% sequence identity compared with the sequence shown in any one of SEQ ID NO.89 to 98; the target nucleic acid sequence targeting the TRAC gene comprises or has a sequence having at least 90% sequence identity compared with the sequence shown in SEQ ID NO. A sequence having at least 90% sequence identity compared to the sequence shown in any one of SEQ ID NOs. 112 to 130; the target nucleic acid sequence targeting the CIITA gene comprises or has at least 90% sequence identity compared to the sequence shown in any one of SEQ ID NOs. 153 to 160.

[0058] In a preferred embodiment, the nucleic acid composition comprises: (1) mRNA encoding the Cas12i editor, and an engineered guide RNA of a sequence shown in any one of SEQ ID NO.71 to SEQ ID NO.82; (2) mRNA encoding the Cas12i editor, and an engineered guide RNA of a sequence shown in any one of SEQ ID NO.85 to SEQ ID NO.88 and SEQ ID NO.177; (3) mRNA encoding the Cas12i editor, and an engineered guide RNA of a sequence shown in any one of SEQ ID NO.99 to SEQ ID NO.111; (4) mRNA encoding the Cas12i editor, and an engineered guide RNA of a sequence shown in any one of SEQ ID NO.131 to SEQ ID NO.152; (5) mRNA encoding the Cas12i editor, and an engineered guide RNA of a sequence shown in any one of SEQ ID NO.161 to SEQ ID NO.168; (6) protein encoding the Cas12i editor, and SEQ ID NO.71 to SEQ ID NO.82; (7) a protein encoding the Cas12i editor, and an engineered guide RNA of any one of SEQ ID NO.85 to SEQ ID NO.88 and SEQ ID NO.177; (8) a protein encoding the Cas12i editor, and an engineered guide RNA of any one of SEQ ID NO.99 to SEQ ID NO.111; (9) a protein encoding the Cas12i editor, and an engineered guide RNA of any one of SEQ ID NO.131 to SEQ ID NO.152; (10) a protein encoding the Cas12i editor, and an engineered guide RNA of any one of SEQ ID NO.161 to SEQ ID NO.168; preferably, the Cas12i editor is a Cas12i protein and a mutant thereof, a Cas12i protein and a mutant thereof with a 5'-3' exonuclease functional domain; more preferably, the Cas12i editor comprises or is SEQ ID The amino acid sequence shown in any one of NO.1, 2, 3, 4, 5, 6, 7, 172, 173 or 174, and the vector encoding the mRNA of the Cas12i editor contains or is the sequence shown in SEQ ID NO.175 or 176.

[0059] Another aspect of the present invention provides a composition or kit comprising a CRISPR-Cas gene editing system of the present invention encapsulated in a lipid nanoparticle (LNP); or a CRISPR-Cas gene editing system of the present invention encapsulated in a virus-like particle (VLP). In a preferred embodiment, the composition or kit further comprises a pharmaceutically acceptable carrier.

[0060] 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.

[0061] 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.

[0062] Another aspect of the present invention provides a method for preparing a universal CAR-T, the method comprising obtaining T cells from a healthy donor; then contacting the target nucleic acid of the T cell with the CRISPR-Cas gene editing system of the present invention, the nucleic acid composition of the present invention, the composition or the kit of the present invention, wherein the contact causes the target gene to be modified and the CAR gene to be inserted into the genome of the T cell; preferably, the modification comprises increasing or decreasing the expression of the target gene.

[0063] Specifically, the chimeric antigen receptor (CAR) gene is a CAR gene in the prior art that can enable T cells to target multiple tumor antigens. The present invention does not impose any limitations or elaborations on the CAR gene.

[0064] In a preferred embodiment, the target gene is selected from the group consisting of 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 human T cell.

[0065] 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, or the modification comprises repressing or activating the transcription of the target gene to silence or activate the expression of the target gene in the cell.

[0066] In various embodiments, the editing method of the disclosed CRISPR-Cas gene editing system produces at least about 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98% or 99% on-target DNA base editing efficiency (such as knocking out target genes or silencing target genes) 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.

[0067] In some embodiments, the editing methods of the disclosed CRISPR-Cas gene editing systems 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%.

[0068] Another aspect of the present invention provides a universal CAR-T prepared using the CRISPR-Cas gene editing system provided herein, which can be used to treat various cancers; preferably, the cancer is a hematological malignancy or some solid tumors.

[0069] Specifically, the CRISPR-Cas gene editing system provided by the present invention can modify one or more target sequences in the CD7 gene, CD70 gene, B2M gene, TRAC gene or CIITA gene of mammalian T cells, such as cutting the target sequence, or performing base conversion on a specific site of the target sequence, or methylating or other epigenetic modifications to the target sequence to knock out the target gene or regulate (such as silencing) the expression of the target gene, so that the modified T cells can avoid the occurrence of GVHD and HvGR, and at the same time, the chimeric antigen receptor (CAR) gene is inserted, and ultimately universal CAR-T cells can be produced. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 A schematic diagram showing the structure of the double-end modified engineered guide RNA (cgRNA and pcgRNA) provided by the present invention.

[0071] 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.

[0072] Figure 3 A schematic diagram showing the structure of the enCas12i-001-N229R-T5 exo fusion protein and the enCas12i-001-N229R protein of the present invention.

[0073] Figure 4 A schematic diagram showing the structure of a double-end modified gRNA containing two target nucleic acid sequences provided by the present invention. DETAILED DESCRIPTION

[0074] Sequence Listing Example

[0075] Example 1. Testing the editing efficiency of different forms of Cas12i editors (mRNA form and protein form) in the CD7 gene

[0076] First, the editing efficiency of the editors (enCas12i-001-N229R-T5 exo mRNA and enCas12i-001-N229R mRNA) at CD7-target nucleic acid sequences 1 to 9 was tested. Figure 1As shown, a conventional chemical synthesis method is used to synthesize a double-end modified (including connecting three consecutive poly-U sequences (polyU) at the 3' end of the gRNA, and performing 2'-O-methyl 3' phosphorothioate modification on three consecutive bases at the 5' end and 3' end of the gRNA), and the sequence of the double-end modified cgRNA is SEQ ID NO.59, and (N)n of SEQ ID NO.59 is the target nucleic acid sequence of the CD7 gene. The target nucleic acid sequence of the CD7 gene is SEQ ID NO.61 to SEQ ID NO.70, and the sequence of the obtained engineered guide RNA (cgRNA) is SEQ ID NO.71 to SEQ ID NO.80.

[0077] like Figure 2 As shown in A, an 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) was constructed, wherein the nucleotide sequence of 5'UTR was SEQ ID NO.169, the amino acid sequence of BPSV40 NLS was SEQ ID NO.13, the amino acid sequence of 3×FLAG was SEQ ID NO.34, the amino acid sequence of enCas12i-001-N229R was SEQ ID NO.7, the amino acid sequence of T5 exo was SEQ ID NO.8, the nucleotide sequence of 3'UTR was SEQ ID NO.170, and the nucleotide sequence of polyA was SEQ ID NO.171, linker is a conventional glycine-serine polymer, the nucleotide sequence of enCas12i-001-N229R-T5 exo mRNA nucleic acid expression vector is SEQ ID NO.175, and the nucleotide sequence of enCas12i-001-N229R mRNA nucleic acid expression vector is SEQ ID NO.176. The linearized recombinant plasmids (enCas12i-001-N229R-T5 exo mRNA nucleic acid expression vector and enCas12i-001-N229RmRNA nucleic acid expression vector) were transcribed using an in vitro transcription kit to obtain enCas12i-001-N229R-T5 exo mRNA and enCas12i-001-N229RmRNA.

[0078] Then, the 293T cells were grouped according to Table 1, and the enCas12i-001-N229R-T5 exomRNA and the corresponding CD7 cgRNA were electrotransfected into the corresponding 293T cells according to the dosage of Table 1 to knock out the CD7 gene of the 293T cells; the electrotransfected 293T cells were cultured to obtain 293T cells with CD7 knocked out. Next, primers were designed around the genomic region corresponding to the target nucleic acid sequence (SEQ ID NO.61 to SEQ ID NO.69) of the CD7 gene to perform PCR on each 293T cell sample with CD7 knocked out to PCR amplify the target nucleic acid sequence fragment of the CD7 gene from the genomic DNA; the resulting product was then run for gel verification, purified, and subjected to Sanger sequencing and TIDE analysis, and the results are shown in Table 1. These results show that, except for CD7-cgRNA7, the remaining double-end modified CD7-cgRNAs have an editing efficiency of greater than 90% for the CD7 gene. Subsequently, double-end modified CD7-cgRNA3, CD7-cgRNA4, CD7-cgRNA6, CD7-cgRNA9 and CD7-cgRNA10 were used for testing.

[0079] Table 1

[0080] Next, the editing efficiency of the editors prepared above (enCas12i-001-N229R-T5 exo mRNA and enCas12i-001-N229RmRNA) in CD7-target nucleic acid sequences 3, 4, 6, 9 and 10 was tested. The 293T cells were grouped according to Table 2, and enCas12i-001-N229R-T5 exo mRNA and enCas12i-001-N229R mRNA and corresponding CD7 cgRNA (CD7-cgRNA3, CD7-cgRNA4, CD7-cgRNA6, CD7-cgRNA9 and CD7-cgRNA10, respectively) were electroporated into the corresponding 293T cells according to the dosage of Table 2 to knock out the CD7 gene of 293T cells; the electroporated 293T cells were cultured to obtain 293T cells with CD7 knocked out. Next, primers were designed around the genomic region corresponding to the target nucleic acid sequence (SEQ ID NO. 63, 64, 66, 69 and 70) of the CD7 gene to perform PCR on each 293T cell sample with CD7 knocked out, so as to PCR amplify the target nucleic acid sequence fragment of the CD7 gene from the genomic DNA; the resulting product was then run on gel for verification, purified, and subjected to Sanger sequencing and TIDE analysis, and the results are shown in Table 2. These results show that compared with nCas12i-001-N229R mRNA, the editing efficiency of enCas12i-001-N229R-T5 exo mRNA at multiple CD7 targets was significantly improved, and the editing efficiency of CD7-cgRNA4, CD7-cgRNA6 and CD7-cgRNA9 was subsequently tested using protein editors.

[0081] Table 2

[0082] In addition, the editing efficiency of two editors in protein form (enCas12i-001-N229R-T5 exo fusion protein and enCas12i-001-N229R protein) at CD7-target nucleic acid sequences 4, 6 and 9 was tested.

[0083] according to Figure 3The protein structure schematic diagram shown in the figure was used to prepare enCas12i-001-N229R-T5 exo fusion protein and enCas12i-001-N229R protein. The N-termini of these two proteins were fused with a His tag and BPSV40 NLS in sequence, and the C-termini were fused with BPSV40 NLS. The method for preparing the fusion protein is as follows: the recombinant plasmid pET-enCas12i encoding the enCas12i-001-N229R-T5 exo 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 an OD of 1. 600 The growth temperature was reduced to 16°C and 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 1M NaCl) for elution. The fractions containing enCas12i-001-N229R-T5 exo fusion protein were combined and dialyzed against 30mM Tris 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 enCas12i-001-N229R protein was obtained by the same method. The amino acid sequence of the enCas12i-001-N229R-T5 exo fusion protein of the present embodiment is SEQ ID NO.173, and the amino acid sequence of the enCas12i-001-N229R protein is SEQ ID NO.174.

[0084] According to Table 3, 293T cells were grouped, and enCas12i-001-N229R-T5 exo fusion protein and enCas12i-001-N229R protein and corresponding CD7 cgRNA (CD7-cgRNA4, CD7-cgRNA6 and CD7-cgRNA9, respectively) were electrotransferred to corresponding 293T cells according to the dosage of Table 3 to knock out the CD7 gene of 293T cells; the electrotransferred 293T cells were cultured to obtain 293T cells with CD7 knocked out. Then, primers were designed around the genomic region corresponding to the target nucleic acid sequence (SEQ ID NO.64, 66 and 69) of the CD7 gene to perform PCR on each 293T cell sample with CD7 knocked out, so as to PCR amplify the target nucleic acid sequence fragment of the CD7 gene from the genomic DNA; the resulting product was then run on gel for verification, purified, and subjected to Sanger sequencing and TIDE analysis, and the results are shown in Table 3.

[0085] The results in Tables 1 to 3 show that both mRNA and protein forms of Cas12i editors can efficiently knock out the CD7 gene.

[0086] Table 3

[0087] In addition, the editing efficiency of the editor (enCas12i-001-N229R-T5 exo mRNA) and different forms of engineered guide RNA (cgRNA and pcgRNA) at CD7-target nucleic acid sequences 4 and 9 were tested.

[0088] EnCas12i-001-N229R-T5 exo mRNA was obtained according to the above-mentioned mRNA transcription method. The engineered guide RNA (cgRNA and pcgRNA) modified at both ends was synthesized by conventional chemical synthesis method, and the sequence of the cgRNA modified at both ends was SEQ ID NO.59, and the sequence of the pcgRNA modified at both ends was SEQ ID NO.60, wherein (N) n of SEQ ID NO.59 and SEQ ID NO.60 is the target nucleic acid sequence of CD7 gene. The target nucleic acid sequence of CD7 gene is SEQ ID NO.64 and SEQ ID NO.69, and the obtained CD7-cgRNA4 (SEQ ID NO.74), CD7-cgRNA9 (SEQ ID NO.79), CD7-pcgRNA4 (SEQID NO.81) and CD7-pcgRNA9 (SEQ ID NO.82).

[0089] The 293T cells were grouped according to Table 4, and enCas12i-001-N229R-T5 exo mRNA and corresponding CD7 gRNA were electrotransferred into the corresponding 293T cells according to the dosage of Table 4 to knock out the CD7 gene of the 293T cells; the electrotransferred 293T cells were cultured to obtain 293T cells with CD7 knocked out. Next, primers were designed around the genomic region corresponding to the target nucleic acid sequence (SEQ ID NO.64 and SEQ ID NO.69) of the CD7 gene to perform PCR on each 293T cell sample with CD7 knocked out to PCR amplify the target nucleic acid sequence fragment of the CD7 gene from the genomic DNA; the resulting product was then run for gel verification, purified, and subjected to Sanger sequencing and TIDE analysis, and the results are shown in Table 4. These results show that pcgRNA can improve the editing efficiency of the enCas12i-001-N229R-T5exo mRNA editor.

[0090] Table 4

[0091] Finally, the editing efficiency of editors in mRNA and protein forms and engineered guide RNAs (cgRNA and pcgRNA) in human T cells CD7-target nucleic acid sequence 9 was tested. According to the above method and the grouping and dosage of Table 5, different editors and corresponding CD7 gRNAs (CD7-cgRNA9 and CD7-pcgRNA9) were electroporated into T cells. The editing results are shown in Table 5, indicating that the CRISPR-Cas gene editing system provided by the present invention can effectively edit T cells.

[0092] Table 5

[0093] Example 2. Testing the editing efficiency of different Cas12i editors (mRNA form and protein form) in the CD70 gene

[0094] According to the mRNA transcription method of Example 1, enCas12i-001-N229R mRNA and enCas12i-001-N229R-T5 exo mRNA were obtained. According to the chemical synthesis method of Example 1, the double-end modified engineered guide RNA (cgRNA and pcgRNA) was synthesized, and the target nucleic acid sequence of the CD70 gene was SEQ ID NO.83 and SEQ ID NO.84, and the obtained CD70-cgRNA2 (SEQ ID NO.85), CD70-cgRNA5 (SEQ ID NO.86), CD70-pcgRNA2 (SEQ ID NO.87) and CD70-pcgRNA5 (SEQ ID NO.88).

[0095] According to Table 6, 293T cells were grouped, and enCas12i-001-N229R mRNA and enCas12i-001-N229R-T5 exo mRNA and corresponding gRNA were electrotransferred into corresponding 293T cells according to the dosage of Table 6 to knock out the CD70 gene of 293T cells; the electrotransferred 293T cells were cultured to obtain 293T cells with CD70 knocked out. Then, primers were designed around the genomic region corresponding to the target nucleic acid sequence (SEQ ID NO.83 and SEQ ID NO.84) of the CD70 gene to perform PCR on each 293T cell sample with CD70 knocked out, so as to PCR amplify the target nucleic acid sequence fragment of the CD70 gene from the genomic DNA; the resulting product was then run on gel for verification, purified, and subjected to Sanger sequencing and TIDE analysis, the results are shown in Table 6, and the results show that both the Cas12i editor in the form of mRNA and the engineered guide RNA (cgRNA and pcgRNA) can effectively edit the CD70 gene.

[0096] Subsequently, enCas12i-001-N229R-T5 exo fusion protein and enCas12i-001-N229R protein were prepared according to the protein preparation method of Example 1, and enCas12i-001-N229R-T5exo fusion protein and enCas12i-001-N229R protein and corresponding gRNA were electroporated into corresponding 293T cells with reference to the above method and the grouping and dosage of Table 6. The editing results are as shown in Table 6, indicating that the protein form editor and engineered guide RNA (cgRNA) provided by the present invention can effectively edit the target sequence of the cell.

[0097] Table 6

[0098] Example 3. Testing the editing efficiency of different Cas12i editors (mRNA form and protein form) in the B2M gene

[0099] According to the mRNA transcription method of Example 1, enCas12i-001-N229R mRNA and enCas12i-001-N229R-T5 exo mRNA were obtained. According to the chemical synthesis method of Example 1, the double-end modified engineered guide RNA (cgRNA) was synthesized, and the target nucleic acid sequence of the B2M gene was SEQ ID NO.89 to SEQ ID NO.97, corresponding to B2M-cgRNA1 to B2M-cgRNA11, and the sequences of these cgRNAs were SEQ ID NO.99 to SEQ ID NO.107, respectively.

[0100] The 293T cells were grouped according to Table 7, and enCas12i-001-N229R-T5 exo mRNA and the corresponding B2M-cgRNA were electroporated into the corresponding 293T cells according to the dosage in Table 7 to knock out the B2M gene of the 293T cells; the electroporated 293T cells were cultured to obtain 293T cells with B2M knocked out. Next, primers were designed around the genomic region corresponding to the target nucleic acid sequence (SEQ ID NO.89 to SEQ ID NO.97) of the B2M gene, and PCR was performed on each 293T cell sample with B2M knocked out to PCR amplify the target nucleic acid sequence fragment of the B2M gene from the genomic DNA; the resulting product was then run on gel for verification, purified, and subjected to Sanger sequencing and TIDE analysis. The results are shown in Table 7. The target editing efficiency of all B2M-cgRNAs was higher than 75%. The results showed that the Cas12i editor in the form of mRNA and the engineered guide RNA (cgRNA) can effectively edit the B2M gene, and the optimal gRNA is B2M-cgRNA1 with an efficiency of 96.1%.

[0101] Table 7

[0102] According to the chemical synthesis method of Example 1, the double-end modified engineered guide RNA (pcgRNA) was synthesized, and the target nucleic acid sequence of the B2M gene was SEQ ID NO.89, 90, 97 and 98, corresponding to B2M-pcgRNA1 (SEQ ID NO.108), B2M-pcgRNA3 (SEQ ID NO.109), B2M-pcgRNA11 (SEQ ID NO.110) and B2M-pcgRNA12 (SEQ ID NO.111). 293T cells were grouped according to Table 8, and enCas12i-001-N229R-T5exo mRNA and corresponding B2M-pcgRNA were electrotransferred into corresponding 293T cells according to the dosage of Table 8 to knock out the B2M gene of 293T cells; the electrotransferred 293T cells were cultured to obtain 293T cells with B2M knocked out. Next, primers were designed around the genomic region corresponding to the target nucleic acid sequence (SEQ ID NO.89, 90, 97 and 98) of the B2M gene, and PCR was performed on each 293T cell sample with B2M knocked out to PCR amplify the target nucleic acid sequence fragment of the B2M gene from the genomic DNA; the resulting product was then run on gel for verification, purified, and subjected to Sanger sequencing and TIDE analysis. The results are shown in Table 8. The Cas12i editor and engineered guide RNA (pcgRNA) provided by the present invention have high editing efficiency for the B2M gene.

[0103] Table 8

[0104] Finally, the editing efficiency of editors in the form of mRNA and protein and engineered guide RNA (pcgRNA and cgRNA) in B2M-target nucleic acid sequence 1 and B2M-target nucleic acid sequence 11 of T cells was tested. According to the protein preparation method of Example 1, enCas12i-001-N229R-T5 exo fusion protein and enCas12i-001-N229R protein were prepared, and different editors and corresponding B2M-pcgRNA were electrotransferred into T cells with reference to the above method and the grouping and dosage of Table 9. The editing results are shown in Table 9, indicating that the editors in the form of mRNA and protein and engineered guide RNA (pcgRNA and cgRNA) provided by the present invention can effectively edit T cells.

[0105] Table 9

[0106] Example 4. Testing the editing efficiency of the Cas12i editor in the form of mRNA in the TRAC gene

[0107] According to the mRNA transcription method of Example 1, enCas12i-001-N229R mRNA and enCas12i-001-N229R-T5 exo mRNA were obtained. According to the chemical synthesis method of Example 1, the engineered guide RNA (cgRNA) with double-end modification was synthesized, and the target nucleic acid sequence of the TRAC gene was SEQ ID NO.112 to SEQ ID NO.127, corresponding to TRAC-cgRNA1 to TRAC-cgRNA10, TRAC-TAC-cgRNA1 to TRAC-TAC-cgRNA6. The sequences of TRAC-cgRNA1 to TRAC-cgRNA10 are SEQ ID NO.131 to SEQ ID NO.140, and the sequences of TRAC-TAC-cgRNA1 to TRAC-TAC-cgRNA6 are SEQ ID NO.141 to SEQ ID NO.146.

[0108] According to Table 10, 293T cells are grouped, and enCas12i-001-N229R-T5 exomRNA and corresponding gRNA are electrotransferred to corresponding 293T cells according to the dosage of Table 10 to knock out the TRAC gene of 293T cells; The electrotransferred 293T cells are cultured to obtain 293T cells knocking out TRAC. Then, primers are designed around the genomic region corresponding to the target nucleic acid sequence (SEQ ID NO.112 to 127) of the TRAC gene to perform PCR on each 293T cell sample knocking out TRAC, so as to PCR amplify the target nucleic acid sequence fragment of the TRAC gene from genomic DNA; The resulting product is then run on gel for verification, purified, and subjected to Sanger sequencing and TIDE analysis, and the results are shown in Table 10. The Cas12i editor provided by the present invention and the engineered guide RNA (cgRNA) have a high editing efficiency for the TRAC gene, especially TRAC-cgRNA2 and TRAC-TAC-cgRNA2.

[0109] Table 10

[0110] Then TRAC-TAC-cgRNA2 was selected for testing, and the two mRNA editors and TRAC-TAC-cgRNA2 were electroporated into 293T cells according to the above method and the grouping and dosage in Table 11. The editing results are shown in Table 11, indicating that the enCas12i-001-N229R-T5 exo mRNA provided by the present invention has a higher editing efficiency at the same target site.

[0111] Table 11

[0112] Next, the double-ended modified engineered guide RNA (cgRNA and pcgRNA) was synthesized according to the chemical synthesis method of Example 1. The target nucleic acid sequence of the TRAC gene was SEQ ID NO.128 to SEQ ID NO.130, and the corresponding TRAC-cgRNA-YH (SEQ ID NO.147), TRAC-cgRNA-LW02 (SEQ ID NO.148), TRAC-cgRNA-LW04 (SEQ IDNO.149), TRAC-pcgRNA-YH (SEQ ID NO.150), TRAC-pcgRNA-LW02 (SEQ ID NO.151), and TRAC-pcgRNA-LW04 (SEQ ID NO.152) were obtained. According to Table 12, 293T cells were grouped, and enCas12i-001-N229R-T5 exo mRNA, enCas12i-001-N229R mRNA and corresponding gRNA were electrotransferred into corresponding 293T cells according to the dosage of Table 12 to knock out the TRAC gene of 293T cells; the electrotransferred 293T cells were cultured to obtain 293T cells with TRAC knocked out. Next, primers were designed around the genomic region corresponding to the target nucleic acid sequence (SEQ ID NO.128 to 130) of the TRAC gene to perform PCR on each 293T cell sample with TRAC knocked out, so as to PCR amplify the target nucleic acid sequence fragment of the TRAC gene from the genomic DNA; the resulting product was then run on gel for verification, purified, and subjected to Sanger sequencing and TIDE analysis, and the results are shown in Table 12. It shows that the different engineered guide RNAs (cgRNA and pcgRNA) provided by the present invention can guide the editor to the target site for efficient cutting, especially TRAC-cgRNA-LW04 and TRAC-pcgRNA-LW04.

[0113] Table 12

[0114] Example 5. Testing the editing efficiency of different forms of Cas12i editors (mRNA form and protein form) in the CIITA gene

[0115] According to the mRNA transcription method of Example 1, enCas12i-001-N229R mRNA and enCas12i-001-N229R-T5 exo mRNA were obtained. According to the chemical synthesis method of Example 1, the engineered guide RNA (cgRNA) modified at both ends was synthesized, and the target nucleic acid sequence of the CIITA gene was SEQ ID NO.153 to SEQ ID NO.160, corresponding to CIITA-cgRNA1, CIITA-cgRNA6 to CIITA-cgRNA12. The sequences of CIITA-cgRNA1, CIITA-cgRNA6 to CIITA-cgRNA12 are SEQ ID NO.161 to SEQ ID NO.168, respectively. The 293T cells were grouped according to Table 13, and enCas12i-001-N229R-T5exo mRNA and the corresponding gRNA were electroporated into the corresponding 293T cells according to the dosage in Table 13 to knock out the CIITA gene of the 293T cells; the electroporated 293T cells were cultured to obtain 293T cells with CIITA knocked out. Next, primers were designed around the genomic region corresponding to the target nucleic acid sequence (SEQ ID NO. 153 to 160) of the CIITA gene, and PCR was performed on each 293T cell sample with CIITA knocked out to PCR amplify the target nucleic acid sequence fragment of the CIITA gene from the genomic DNA; the resulting product was then run on gel for verification, purified, and subjected to Sanger sequencing and TIDE analysis. The results are shown in Table 13. The target editing efficiency of most CIITA-cgRNAs was higher than 75%. The results showed that the Cas12i editor in the form of mRNA and the engineered guide RNA (cgRNA) can effectively edit the CIITA gene, and the preferred gRNAs are CIITA-cgRNA1, CIITA-cgRNA8 to CIITA-cgRNA12.

[0116] Table 13

[0117] Next, the editing efficiency of enCas12i-001-N229RmRNA and enCas12i-001-N229R-T5 exo mRNA in CIITA-cgRNA1, CIITA-cgRNA8, CIITA-cgRNA10 to CIITA-cgRNA12 was tested. Referring to the test method of Example 1, 293T cells were grouped according to Table 14, and the editors (enCas12i-001-N229RmRNA and enCas12i-001-N229R-T5 exo mRNA) and corresponding gRNA were electrotransferred into the corresponding 293T cells according to the dosage of Table 14 to knock out the CIITA gene of 293T cells; The electrotransferred 293T cells were cultured to obtain 293T cells with CIITA knocked out. Next, primers were designed around the genomic region corresponding to the target nucleic acid sequence (SEQ ID NO. 153, 156, 158, 159 and 160) of the CIITA gene to perform PCR on each 293T cell sample with CIITA knocked out, to PCR amplify the target nucleic acid sequence fragment of the CIITA gene from genomic DNA; the resulting product was then run on gel for verification, purified, and subjected to Sanger sequencing and TIDE analysis. The results are shown in Table 14, and the target editing efficiency of most CIITA-cgRNAs is higher than 75%. The results show that the Cas12i editor in the form of mRNA and the engineered guide RNA (cgRNA) can effectively edit the CIITA gene. CIITA-cgRNA8 and CIITA-cgRNA9 were subsequently selected as gRNAs for testing.

[0118] Table 14

[0119] According to the protein preparation method of Example 1, enCas12i-001-N229R-T5 exo fusion protein and enCas12i-001-N229R protein were prepared, and enCas12i-001-N229R-T5 exo fusion protein and enCas12i-001-N229R protein and corresponding gRNA were electrotransferred into corresponding 293T cells with reference to the above method and the grouping and dosage of Table 15. The editing results are shown in Table 15. The above results show that the editors in the form of mRNA and protein and the engineered guide RNA (cgRNA) provided by the present invention can effectively edit the target sequence of the cell.

[0120] Table 15

[0121] Example 6. Testing the editing efficiency of gRNA containing two target nucleic acid sequences in the CD70 gene

[0122] According to the mRNA transcription method of Example 1, enCas12i-001-N229R-T5 exo mRNA was obtained. Figure 4 As shown, a double-end modified gRNA containing two target nucleic acid sequences (i.e., dual-target-gRNA) was synthesized by a chemical synthesis method. From the 5' end to the 3' end of the dual-target-gRNA, there are the first direct repeat sequence (SEQ ID NO.35), CD70-target nucleic acid sequence 2 (SEQ ID NO.83), the first direct repeat sequence (SEQ ID NO.35) and CD70-target nucleic acid sequence 5 (SEQ ID NO.84), and the sequence of the dual-target-gRNA is SEQ ID NO.177.

[0123] According to Table 16, 293T cells were grouped, and enCas12i-001-N229R-T5 exomRNA and the above-mentioned dual-target-gRNA were electrotransferred into the corresponding 293T cells according to the dosage of Table 16 to knock out the CD70 gene of 293T cells; the electrotransferred 293T cells were cultured to obtain 293T cells with CD70 knocked out. Then, primers were designed around the genomic region corresponding to the target nucleic acid sequence (SEQID NO.83 and SEQ ID NO.84) of the CD70 gene to perform PCR on each 293T cell sample with CD70 knocked out, so as to PCR amplify the target nucleic acid sequence fragment of the CD70 gene from the genomic DNA; the resulting product was then run on gel for verification, purified, and subjected to Sanger sequencing and TIDE analysis. The results are shown in Table 16. The use of gRNA containing two target nucleic acid sequences can simultaneously and efficiently knock out the two target sequences of the CD70 gene.

[0124] Table 16

Claims

1. A CRISPR-Cas gene editing system for preparing universal CAR-T, comprising: (a) Cas12i editor; wherein The Cas12i editor is an amino acid sequence of a Cas12i editor or a ribonucleotide sequence encoding the Cas12i editor, and the Cas12i editor comprises a Cas12i protein; the Cas12i protein comprises or has an amino acid sequence having at least 99% sequence identity with 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 and a target nucleic acid sequence connected sequentially from the 5' end to the 3' end; the target nucleic acid sequence is specifically complementary to the target gene, and the engineered guide RNA is complexed with the Cas12i editor to guide the Cas12i protein to bind to the target nucleic acid; Wherein, the target nucleic acid sequence targets one or more of the CD7 gene, the CD70 gene, the B2M gene, the TRAC gene and the CIITA gene to knock out the corresponding gene; Preferably, the target nucleic acid sequence targeting the CD7 gene comprises or has a sequence with at least 90% sequence identity compared with the sequence shown in any one of SEQ ID NO.61 to 70; the target nucleic acid sequence targeting the CD70 gene comprises or has a sequence with at least 90% sequence identity compared with the sequence shown in any one of SEQ ID NO.83 or 84; the target nucleic acid sequence targeting the B2M gene comprises or has a sequence with at least 90% sequence identity compared with the sequence shown in any one of SEQ ID NO.89 to 98; the target nucleic acid sequence targeting the TRAC gene comprises or has a sequence with at least 90% sequence identity compared with the sequence shown in any one of SEQ ID NO.112 to 130; the target nucleic acid sequence targeting the CIITA gene comprises or has a sequence with at least 90% sequence identity compared with the sequence shown in any one of SEQ ID NO.153 to 160.

2. The CRISPR-Cas gene editing system according to claim 1, wherein the Cas12i protein is mutated so that it has (1) partial or complete inactivation of nucleic acid cleavage activity; (2) enhanced nucleic acid cleavage activity; (3) enhanced nucleic acid binding activity; Preferably, according to the sequence numbering 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 mutant protein of Cas12i 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: transcriptional regulatory activity, deaminase activity and inhibition of uracil DNA glycosylase activity; more preferably, the one or more heterologous polypeptides are independently transcription repression domains, transcription activation domains, adenosine deaminase domains, cytidine deaminase domains and uracil DNA glycosylase inhibitor domains.

5. The CRISPR-Cas gene editing system according to claim 4, wherein the structure of the Cas12i editor is selected from: NH2-[one or more heterologous polypeptides]-[Cas12i protein]-COOH; NH2-[Cas12i protein]-[one or more heterologous polypeptides]-COOH; NH2-[one or more heterologous polypeptides]-[Cas12i protein]-[one or more heterologous polypeptides]-COOH; NH2-[one or more heterologous polypeptides]-[Mutated proteins of Cas12i]-COOH; NH2-[one or more heterologous polypeptides]-[Mutated proteins of Cas12i]-[one or more heterologous polypeptides]-COOH; NH2-[one or more heterologous polypeptides]-[Mutated proteins of Cas12i]-[one or more heterologous polypeptides]-COOH; NH2-[one or more heterologous polypeptides]-[Mutated proteins of Cas12i]-[one or more heterologous polypeptides]-COOH; NH2-[mutant protein of Cas12i]-[5'-3' exonuclease functional domain]-COOH; NH2-[mutant protein of Cas12i]-[5'-3' exonuclease functional domain]-[one or more heterologous polypeptides]-COOH; NH2-[one or more heterologous polypeptides]-[mutant protein of Cas12i]-[5'-3' exonuclease functional domain]-[one or more heterologous polypeptides]-COOH; NH2-[one or more heterologous polypeptides]-[5'-3' exonuclease functional domain]-[Cas12i protein]-COOH; NH2-[5'-3' exonuclease functional domain]-[Cas12i protein]-[one or more heterologous polypeptides]-COOH; or NH2-[one or more heterologous polypeptides]-[5'-3' exonuclease functional domain]-[Cas12i protein]-[one or more heterologous polypeptides]-COOH.

6. The CRISPR-Cas gene editing system according to claim 1, wherein the engineered guide RNA further comprises a second direct repeat sequence; the engineered guide RNA comprises a first direct repeat sequence, a target nucleic acid sequence and a second direct repeat sequence connected in sequence from the 5' end to the 3' end; the first direct repeat sequence and the second direct repeat sequence are the same sequence or different sequences; the first direct repeat sequence comprises a nucleotide sequence as shown in any one of SEQ ID NOs. 35 to 42 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. 35 to 42; preferably, wherein the first direct repeat sequence is a nucleotide sequence as shown in any one of SEQ ID NOs. 35 to 42; the second direct repeat sequence comprises a nucleotide sequence as shown in any one of SEQ ID NOs. 35 to 42 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. 35 to 42; preferably, wherein the second direct repeat sequence is a nucleotide sequence as shown in any one of SEQ ID NOs. 35 to 42.

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) Cas12i editor; wherein The Cas12i editor is an amino acid sequence of a Cas12i editor or a ribonucleotide sequence encoding the Cas12i editor, and the Cas12i editor comprises a Cas12i protein; the Cas12i protein comprises or has an amino acid sequence having at least 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO.1 to 6; 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, 172 to 174; (ii) an engineered guide RNA, wherein the engineered guide RNA comprises a first direct repeat sequence and a target nucleic acid 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. 43 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. 43 to 60; more preferably, the engineered guide RNA is a nucleotide sequence as shown in any one of SEQ ID NOs. 43 to 60, wherein N is any nucleotide (A, G, C or U), and n is an integer from 15 to 40, and the (N) n is the target nucleic acid sequence; Preferably, the target nucleic acid sequence targeting the CD7 gene comprises or has a sequence with at least 90% sequence identity compared with the sequence shown in any one of SEQ ID NO.61 to 70; the target nucleic acid sequence targeting the CD70 gene comprises or has a sequence with at least 90% sequence identity compared with the sequence shown in any one of SEQ ID NO.83 or 84; the target nucleic acid sequence targeting the B2M gene comprises or has a sequence with at least 90% sequence identity compared with the sequence shown in any one of SEQ ID NO.89 to 98; the target nucleic acid sequence targeting the TRAC gene comprises or has a sequence with at least 90% sequence identity compared with the sequence shown in any one of SEQ ID NO.112 to 130; the target nucleic acid sequence targeting the CIITA gene comprises or has a sequence with at least 90% sequence identity compared with the sequence shown in any one of SEQ ID NO.153 to 160.

9. A composition or kit comprising The CRISPR-Cas gene editing system of any one of claims 1 to 7 encapsulated in lipid nanoparticles (LNPs); or The CRISPR-Cas gene editing system of any one of claims 1 to 7 encapsulated in a virus-like particle (VLP).

10. A method for preparing a universal CAR-T, the method comprising obtaining T cells from a healthy donor; then contacting the target nucleic acid of the T cells 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 causes the target gene to be modified and the CAR gene to be inserted into the genome of the T cells; Preferably, the modification comprises increasing or decreasing the expression of the target gene.

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