WO2023241669A1

WO2023241669A1 - Crispr-cas effector protein, gene editing system therefor, and application

Info

Publication number: WO2023241669A1
Application number: PCT/CN2023/100529
Authority: WO
Inventors: 张红玲; 任文丹
Original assignee: 尧唐(上海)生物科技有限公司
Priority date: 2022-06-16
Filing date: 2023-06-15
Publication date: 2023-12-21
Also published as: CN114921439A; CN114921439B

Abstract

Provided are a CRISPR-Cas effector protein, a gene editing system therefor, and an application. The CRISPR-Cas effector protein comprises a protein which is at least 70% identical to the amino acid sequence of any one of SEQ ID NOs: 1 to 5. The present application can solve the problem in the prior art of low CRISPR/Cas system cutting efficiency, and is suitable for the field of gene editing.

Description

CRISPR-Cas effector proteins, their gene editing systems and applications

This application is based on the Chinese application with CN application number 202210681597.5 and the filing date is June 16, 2022, and claims its priority. The disclosure content of the CN application is incorporated into this application as a whole.

Technical field

The present invention relates to the field of gene editing, specifically to a CRISPR-Cas effector protein, its gene editing system and applications.

Background technique

The Clustered regularly interspaced short palindromic repeats (CRISPR) system was formed by bacteria and archaea to defend against the DNA of invading phages. The immune interference process of the CRISPR system mainly includes three stages: adaptation, expression and interference. In the adaptation phase, the CRISPR system integrates short DNA fragments from phages or plasmids between the leader sequence and the first repeat sequence. Each integration is accompanied by the replication of the repeat sequence, thereby forming a new repeat-spacer sequence unit. During the expression stage, the CRISPR locus is transcribed into a CRISPR RNA (crRNA) precursor (pre-crRNA), which is further processed into a small crRNA at the repetitive sequence in the presence of Cas protein and tracrRNA. Mature crRNA and Cas protein form Cas/crRNA complex. In the interference stage, crRNA guides the Cas/crRNA complex to find the target through its region complementary to the target sequence, and causes a double-stranded DNA break at the target position through the nuclease activity of the Cas protein, thereby causing the target DNA to lose its original shape. There are functions.

CRISPR systems are divided into three families: I, II, and III. Among them, the most common type II system is the CRISPR/Cas9 system. The Cas9 protein can convert pre -crRNA is processed into mature crRNA that binds to tracrRNA. Later, it was discovered that by artificially constructing a single-stranded chimera guide RNA (guide RNA) that simulates the crRNA:tracrRNA complex, it can effectively mediate the recognition and cleavage of the target by the Cas9 protein. The three bases immediately adjacent to the 3' end of the target must be in the form of 5'-NGG-3', thus forming the PAM (protospacer adjacent motif) structure required for the Cas/crRNA complex to recognize the target. However, the different CRISPR/Cas currently existing have different advantages and disadvantages. For example, Cas9, C2c1 and CasX all require two RNAs for guide RNA. Common Cas9, C2c1, CasY and Cpf1 are usually around 1300 amino acids in size. In addition, the PAM sequences of Cas9, Cpf1, CasX, and CasY are complex and diverse. And the existing CRISPR/Cas systems have problems such as serious off-target effects and low cleavage efficiency. Therefore, it is of great significance to develop new CRISPR/Cas systems with low off-target effects and high cleavage efficiency.

Contents of the invention

The main purpose of the present invention is to provide a CRISPR-Cas effector protein, its gene editing system and application, so as to solve the problem of low cutting efficiency of the CRISPR/Cas system in the existing technology.

In order to achieve the above object, according to the first aspect of the present invention, a CRISPR-Cas effector protein is provided, the CRISPR-Cas effector protein includes any one of SEQ ID NO: 2, 1, 3-5 Proteins with at least 70% amino acid sequence identity.

Further, the CRISPR-Cas effector protein includes more than 80%, preferably more than 90%, more preferably more than 95%, further preferably more than 99% of the amino acid sequence of any one of SEQ ID NO: 2, 1, 3-5. Identity protein; preferably, the CRISPR-Cas effector protein includes a RuvC domain.

Further, the CRISPR-Cas effector protein includes: a) the protein shown in any one of SEQ ID NO: 2, 1, 3-5; or b) based on the amino acid sequence shown in SEQ ID NO: 1, Proteins with one or more point mutations as follows: N21X, N23X, R25X, K26X, Q482X, S484X, R486X, S489X, R493X, H511X, C513X, H515X, N516X, R518X, R540X, K558X, Y560X, K562X, K565X, T600X, T672X, D676X, Q680X, Y683X, L686X, D693X, Y731X, G767X, R772X, K832X, K833X, Q836X, M896X; or c) Based on the amino acid sequence shown in SEQ ID NO: 2, perform one of the following or Proteins with multiple point mutations: R19X, R28X, R32X, K512X, N527X, W531X, R553X, K581X, K589X, I590X, R605X, K611X, R612X, R615X, Y777X, E877X, R931X; or d) SEQ ID NO: 3 Based on the amino acid sequence shown, the protein has one or more point mutations as follows: K8X, F15X, N17X, K20X, K471X, W483X, H502X, R505X, K557X, K556X, R560X, Y673X, L676X, Y723X, N822X, K823X, E826X, K827X, K830X, K880X, L887X; or e) A protein with one or more point mutations based on the amino acid sequence shown in SEQ ID NO: 4: K317X, W330X, Y351X, K354X, D392X , F395X, N399X, Y509X, V512X, Y568X, N662X, K663X, E666X, R667X, K670X, K719X, L726X; or f) Based on the amino acid sequence shown in SEQ ID NO: 5, perform one or more of the following points Mutated proteins: M9X, V16X, D18X, K21X, K518X, W531X, F550X, K553X, R609X, Y612X, R616X, Y730X, L733X, Y781X, N879X, K880X, E883X, K884X, K887X, K936X, F943 X; where X is any Amino acids.

In order to achieve the above object, according to the second aspect of the present invention, a CRISPR-Cas effector fusion protein is provided, which includes the above-mentioned CRISPR-Cas effector protein, or CRISPR-Cas effector protein Derivatives or functional fragments of CRISPR-Cas effector proteins, as well as heterologous functional domains.

Further, the heterologous functional domain is located at the N-terminal, C-terminal or internal part of the CRISPR-Cas effector fusion protein; preferably, the heterologous functional domain includes a positioning signal, a reporter protein, a CRISPR-Cas effector protein targeting part, One of DNA binding domain, epitope tag, transcription activation domain, transcription repression domain, nuclease, deamination domain, methylase, demethylase, transcription release factor, HDAC, cleavage active polypeptide, ligase or more; preferably, the localization signal includes a nuclear localization signal and/or a nuclear export signal; preferably, the nuclear export signal includes human protein tyrosine kinase 2; preferably, the reporter protein includes glutathione-S-transferase , one or more of horseradish peroxidase, chloramphenicol acetyltransferase, β-galactosidase, β-glucuronidase or autofluorescent protein; preferably, the autofluorescent protein includes green One or more of fluorescent protein, HcRed, DsRed, cyan fluorescent protein, yellow fluorescent protein or blue fluorescent protein; preferably, the DNA binding domain includes one or more of methylation binding protein, LexADBD or Gal4DBD ; Preferably, the epitope tag includes one or more of histidine tag, V5 tag, FLAG tag, influenza virus hemagglutinin tag, Myc tag, VSV-G tag or thioredoxin tag; Preferably, The transcriptional activation domain includes VP64 and/or VPR; preferably, the transcriptional repression domain includes KRAB and/or SID; Preferably, the nuclease includes FokI; Preferably, the deamination domain includes one or more of ADAR1, ADAR2, APOBEC, AID or TAD; Preferably, the cleavage active polypeptide includes single-stranded RNA A polypeptide with cleavage activity, a polypeptide with double-stranded RNA cleavage activity, a polypeptide with single-stranded DNA cleavage activity or a polypeptide with double-stranded DNA cleavage activity; preferably, the ligase includes DNA ligase and/or RNA ligase.

In order to achieve the above object, according to the third aspect of the present invention, a DNA molecule encoding the above-mentioned CRISPR-Cas effector protein or CRISPR-Cas effector fusion protein is provided.

Further, the DNA molecule is a DNA molecule that is codon-optimized according to the codon preference of the host cell; preferably, the host cell includes a prokaryotic cell or a eukaryotic cell; preferably, the DNA molecule includes those in SEQ ID NO: 6 to 10 Any nucleotide sequence has 70% or more, preferably 90% or more, more preferably 95% or more, still more preferably 99%, even more preferably 100% nucleotide identity.

In order to achieve the above object, according to the fourth aspect of the present invention, a recombinant vector is provided, which contains the above-mentioned DNA molecule.

Further, the DNA molecule is connected to a promoter; preferably, the promoter includes one or more of an inducible promoter, a constitutive promoter or a tissue-specific promoter; preferably, the promoter includes T7, SP6, T3 , CMV, EF1a, SV40, PGK1, human β-actin, CAG, U6, H1, T7, T7lac, one or more of araBAD, trp, lac or Ptac; preferably, the recombinant vector includes retroviral vector, lentivirus vector, Viral vector, adenovirus vector, adeno-associated virus vector, herpes simplex vector, plasmid vector or phagemid vector; preferably, the recombinant vector includes a plasmid vector.

In order to achieve the above object, according to the fifth aspect of the present invention, a host cell is provided, which host cell is transformed with the above recombinant vector.

In order to achieve the above objects, according to the sixth aspect of the present invention, a gene editing system is provided. The gene editing system includes: a) an RNA guide or a nucleic acid encoding an RNA guide. The RNA guide includes a direct repeat sequence and a spacer. sub-sequence, spacer sequence for hybridization with the target nucleic acid; b) the above-mentioned CRISPR-Cas effector protein, or CRISPR-Cas effector fusion protein, or DNA molecule, or recombinant vector, or host cell; DNA molecule, recombinant vector or The host cell can express CRISPR-Cas effector protein or CRISPR-Cas effector fusion protein. In the gene editing system, the CRISPR-Cas effector protein or CRISPR-Cas effector fusion protein combines with the RNA guide to perform the targeting function. The hybridization sequence formed by hybridization between the spacer sequence and the target nucleic acid.

Further, the gene editing system does not contain tracrRNA.

Further, the RNA guide includes one or more types.

Further, the target nucleic acid includes DNA; preferably, DNA includes DNA derived from eukaryotes or DNA derived from prokaryotes; preferably, eukaryotes include animals or plants; preferably, DNA includes non-human mammalian DNA, Human DNA, insect DNA, avian DNA, reptile DNA, amphibian DNA, rodent DNA, fish DNA, worm DNA, nematode DNA or yeast DNA; preferably, non-human mammalian DNA includes non-human primate DNA .

Further, the 3' end of the direct repeat sequence includes a stem-loop structure. The stem-loop structure includes a first stem nucleotide chain, a cyclic nucleotide chain and a second stem nucleotide chain connected in sequence. The first stem nucleotide The cyclic nucleotide chain and the second stem nucleotide chain hybridize to each other to form the stem of the stem-loop structure, and the cyclic nucleotide chain forms the ring of the stem-loop structure; preferably, the length of the first stem nucleotide chain is 5 or 6 nucleotides ; Preferably, the length of the second stem nucleotide chain is 5 nucleotides; Preferably, the length of the cyclic nucleotide chain is 6, 7 or 8 nucleotides.

Further, the stem-loop structure includes the nucleotide sequence of SEQ ID NO: 25, 28, 31, 34 or 37.

Further, the direct repeat sequence includes a nucleotide sequence having at least 80% identity with the nucleotide sequence of SEQ ID NO: 24, 27, 30, 33 or 36; preferably, the direct repeat sequence includes a nucleotide sequence with SEQ ID NO: The nucleotide sequence of 24, 27, 30, 33 or 36 has at least 85% or more, more preferably more than 90%, further preferably more than 95% identity; preferably, the direct repeat sequence includes SEQ ID NO: 24, 27, 30, 33 or 36 nucleotide sequence.

Further, more than 80% of the spacer sequence is complementary to the target nucleic acid; preferably, more than 90% of the spacer sequence is more than 95%, more preferably more than 99%, and even more preferably 100% is complementary to the target nucleic acid; preferably , the length of the spacer sequence is 18-41nt; preferably, the length of the spacer sequence is 18-37nt; preferably, the length of the spacer sequence is 18-26 or 34-36nt; preferably, the length of the spacer sequence is 20nt.

Further, the direct repeat sequence includes a first direct repeat sequence and a second direct repeat sequence; preferably, the RNA guide includes a first direct repeat sequence, a spacer sequence and a second direct repeat sequence connected in sequence; preferably, The first direct repeat sequence is the same as the second direct repeat sequence.

Further, the target nucleic acid includes a pre-spacer adjacent motif, the CRISPR-Cas effector protein or CRISPR-Cas effector fusion protein can recognize the pre-spacer adjacent motif, and the pre-spacer adjacent motif includes the nucleic acid sequence 5' -TTN-3', where N is any nucleotide; preferably, N is A, C or T.

Further, the CRISPR-Cas effector protein or CRISPR-Cas effector fusion protein is combined with the RNA guide to form a protein-nucleic acid complex; preferably, the protein-nucleic acid complex is non-naturally occurring or modified; preferably , at least one component of the protein-nucleic acid complex is non-naturally occurring or modified.

Further, the target nucleic acid is modified through the targeting effect of the CRISPR-Cas effector protein or CRISPR-Cas effector fusion protein and the RNA guide on the target nucleic acid; preferably, the modification includes cutting or nicking; preferably, the modification results in (1) the cell contains an alteration in the expression of at least one gene product; or (2) the cell contains an alteration in the expression of at least one gene product, wherein the expression of at least one gene product is increased; or (3) the cell contains at least one Alteration in the expression of gene products, wherein the expression of at least one gene product is reduced; or (4) the cell contains an edited genome; preferably, the modification results in cytotoxicity; preferably, the above modification results in inhibition of gene expression, reduction in gene expression, or Enhance gene expression.

Further, the gene editing system includes a target nucleic acid or a nucleic acid encoding the target nucleic acid. The target nucleic acid includes a homology arm fragment and a donor template nucleic acid; preferably, the target nucleic acid includes a sequence capable of hybridizing with the spacer sequence; preferably, the homology arm fragment It includes a 5' homology arm and a 3' homology arm. The target nucleic acid is composed of a 5' homology arm, a donor template nucleic acid and a 3' homology arm sequentially connected.

Further, the gene editing system exists in a deliverable form, and the delivery system is used to bring the gene editing system into contact with the target nucleic acid; preferably, the delivery system delivers the gene editing system into cells containing the target nucleic acid; preferably, the delivery system is in a deliverable form. Formulas include nanoparticles, liposomes, exosomes, microvesicles, protein capsids or particles used in gene guns.

In order to achieve the above object, according to the seventh aspect of the present invention, a gene editing vector is provided, which contains the above-mentioned nucleic acid encoding an RNA guide.

Further, the gene editing vector also contains the above-mentioned DNA molecule; preferably, the DNA molecule and the nucleic acid encoding the RNA guide are located on the same or different vectors; preferably, the DNA molecule is connected to the first regulatory element; preferably, the nucleic acid encoding the RNA guide The nucleic acid of the object is connected to the second regulatory element; preferably, the first regulatory element and the second regulatory element are independently selected from one or more of inducible promoters, constitutive promoters or tissue-specific promoters; preferably Ground, the first regulatory element and the second regulatory element are independently selected from T7, SP6, T3, CMV, EF1a, SV40, PGK1, human β-actin, CAG, U6, H1, T7, T7lac, araBAD, trp, lac or Ptac one or more of them.

In order to achieve the above object, according to the eighth aspect of the present invention, a method for combining the above gene editing system with a target nucleic acid in a cell is provided. The method includes: delivering the gene editing system to the cell, and the cell includes the target nucleic acid; CRISPR-Cas effector proteins or CRISPR-Cas effector fusion proteins combine with the RNA guide to allow the spacer sequence to bind to the target nucleic acid.

Further, the target nucleic acid is double-stranded DNA or single-stranded DNA; preferably, the combination of the gene editing system and the target nucleic acid in the cell results in a change in the expression state of the target nucleic acid; preferably, the combination of the gene editing system and the target nucleic acid in the cell, Cause the target nucleic acid to be cleaved; preferably, the target nucleic acid is cleaved to cause the destruction of the target nucleic acid, or the replacement of a specific site of the target nucleic acid, or the removal of the target nucleic acid site, or the change of the function of the target nucleic acid region, or two positions on the target nucleic acid. The sequence between points is inverted.

In order to achieve the above object, according to the ninth aspect of the present invention, a cell containing a gene editing system is provided. The cell containing a gene editing system includes the above gene editing system or a gene editing vector.

Further, the cell containing the gene editing system contains a modified target target locus, and the target target locus is a locus modified by the gene editing system; preferably, the modification of the target target locus results in: (1) containing the gene editing system The cell contains an alteration in the expression of at least one gene product; or (2) the cell containing the gene editing system contains an alteration in the expression of at least one gene product, wherein the expression of at least one gene product is increased; or (3) the cell containing the gene The cell containing the editing system contains an altered expression of at least one gene product, wherein the expression of at least one gene product is reduced; or (4) the cell containing the gene editing system contains an edited genome; preferably, the cell containing the gene editing system It includes eukaryotic cells or prokaryotic cells; preferably, eukaryotic cells include animal cells, plant cells or human cells; preferably, animal cells include mammalian cells.

In order to achieve the above object, according to the tenth aspect of the present invention, a method for targeting and editing a target nucleic acid is provided, which method includes contacting the target nucleic acid with the above-mentioned gene editing system.

In order to achieve the above object, according to an eleventh aspect of the present invention, a method for non-specific degradation of single-stranded DNA after identifying a target nucleic acid is provided, which method includes contacting the target nucleic acid with the above-mentioned gene editing system.

In order to achieve the above object, according to a twelfth aspect of the present invention, there is provided a method of targeting and nicking the non-spacer complementary strand of the double-stranded target DNA after identifying the spacer complementary strand of the double-stranded target DNA. , the method includes contacting double-stranded target DNA with the above-mentioned gene editing system.

In order to achieve the above object, according to the thirteenth aspect of the present invention, a method for targeting and cutting double-stranded target DNA is provided, which method includes contacting the double-stranded target DNA with the above-mentioned gene editing system.

Further, before nicking the spacer complementary strand of the double-stranded DNA, the non-spacer complementary strand of the double-stranded target DNA is nicked.

In order to achieve the above object, according to the fourteenth aspect of the present invention, there is provided a method for specifically editing double-stranded nucleic acids, which method includes contacting the following under sufficient conditions for a sufficient amount of time, (1) the above-mentioned CRISPR - a Cas effector protein, or a CRISPR-Cas effector fusion protein, another enzyme with sequence-specific nicking activity, and an RNA guide that directs the CRISPR-Cas effector protein or CRISPR-Cas effector fusion protein, The activity of a sequence-specific nicking enzyme relative to another nicks the opposing strand; and (2) a double-stranded nucleic acid; the method results in the formation of a double-stranded break.

In order to achieve the above object, according to the fifteenth aspect of the present invention, a method for editing double-stranded nucleic acid is provided, which method includes contacting the following under sufficient conditions for a sufficient amount of time: (1) the above-mentioned CRISPR-Cas An effector protein, or a CRISPR-Cas effector fusion protein, and a fusion protein of a protein domain with DNA modification activity, and an RNA guide targeting double-stranded nucleic acid; and (2) double-stranded nucleic acid; CRISPR-Cas fusion protein Cas effectors are modified to nick the non-target strand of a double-stranded nucleic acid.

Further, the two strands of the double-stranded nucleic acid are cleaved at different sites, resulting in staggered cleavage; preferably, the two strands of the double-stranded nucleic acid are cleaved at the same site, resulting in a flat double-stranded break.

In order to achieve the above object, according to the sixteenth aspect of the present invention, a method for targeting and cutting single-stranded target DNA is provided, which method includes contacting the target nucleic acid with the above-mentioned gene editing system.

In order to achieve the above object, according to the seventeenth aspect of the present invention, a method for inducing cell state changes is provided, which method includes contacting the above gene editing system with a target nucleic acid in the cell.

Further, the cell state includes apoptosis or dormancy; preferably, the cells include eukaryotic cells or prokaryotic cells; preferably, the cells include mammalian cells or plant disease cells; preferably, the cells include cancer cells; preferably, the cells include infection Sexual cells or cells infected by an infectious agent; preferably, the cells include virus-infected cells, prion-infected cells; preferably, the cells include fungal cells, protozoa or parasite cells.

In order to achieve the above objects, according to the eighteenth aspect of the present invention, there is provided an application of the above-mentioned gene editing system in preparing drugs for treating conditions or diseases in subjects.

Further, the application includes administering the gene editing system to a subject or ex vivo cells of a subject; preferably, the spacer sequence is complementary to at least 15 nucleotides of the target nucleic acid associated with the disorder or disease, the CRISPR-Cas effect The daughter protein or CRISPR-Cas effector fusion protein cleaves the target nucleic acid; Preferably, the disorder or disease includes cancer, infectious disease, metabolic disease or genetic disease; Preferably, the cancer includes Wilms tumor, Ewing sarcoma, neuroendocrine disease tumor, glioblastoma, neuroblastoma, melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, kidney cancer, pancreatic cancer, lung cancer, biliary tract cancer, cervical cancer, endometrium Cancer, esophageal cancer, gastric cancer, head and neck cancer, medullary thyroid cancer, ovarian cancer, glioma, lymphoma, leukemia, myeloma, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia Leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or urinary bladder One or more cancers; preferably, the metabolic disease includes familial hypercholesterolemia (FH); preferably, the genetic disease includes transthyretin amyloidosis (ATTR), primary hyperoxaluric acid syndrome (PH1), hereditary angioedema (HAE); preferably, the disorder or disease includes cystic fibrosis, progressive Duchenne muscular dystrophy, Becker muscular dystrophy, alpha-1-antitrypsin deficiency, Pandora's disease Bayi disease, myotonic dystrophy, Huntington's disease, fragile X syndrome, Friedreich's ataxia, amyotrophic lateral sclerosis, frontotemporal dementia, hereditary chronic kidney disease, hyperlipidemia, high cholesterol One or more of Leber's congenital amaurosis, sickle cell disease or beta thalassemia; preferably, the infectious agent of the infectious disease includes human immunodeficiency virus, herpes simplex virus-1, type B One or more of hepatitis (HEPATITIS B) or herpes simplex virus-2.

In order to achieve the above object, according to the nineteenth aspect of the present invention, a eukaryotic cell line is provided, which eukaryotic cell line contains the above-mentioned cells containing the gene editing system, or is the progeny of the cells containing the gene editing system.

In order to achieve the above object, according to the twentieth aspect of the present invention, a multicellular organism is provided, which includes the above-mentioned cell containing the gene editing system.

Further, multicellular organisms include model animals or model plants.

In order to achieve the above object, according to the twenty-first aspect of the present invention, a method for obtaining plants with target traits is provided, using the above gene editing system to contact plant cells, modifying the genes of the plant cells or introducing the target genes, The modified or target gene can express the target traits, obtain modified plant cells, and use the modified plant cells for regeneration to obtain plants with the target traits.

In order to achieve the above object, according to the twenty-second aspect of the present invention, a method for identifying target traits in plants is provided. The target genes in plant cells can express the target traits, and the above gene editing system is used to contact the plant cells, thereby Identify the gene of interest.

In order to achieve the above object, according to the twenty-third aspect of the present invention, a kit is provided, which kit includes one or more components selected from the following: the above-mentioned CRISPR-Cas effector protein, DNA molecules, Recombinant vector, host cell, gene editing system, gene editing vector, cell containing the gene editing system, eukaryotic cell line, or multicellular organism; the components of the kit are in the same or different containers.

In order to achieve the above object, according to the twenty-fourth aspect of the present invention, a container is provided, which container contains the above-mentioned kit.

Further, the container includes a sterile container; preferably, the container includes a syringe.

In order to achieve the above object, according to the twenty-fifth aspect of the present invention, an implantable device is provided, which includes the above gene editing system.

Further, the gene editing system is in the matrix; preferably, the gene editing system is in the reservoir.

According to a twenty-sixth aspect of the present invention, there is provided a method of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject any one or more of the following:

The above-mentioned CRISPR-Cas effector protein, or a derivative of the above-mentioned CRISPR-Cas effector protein or a functional fragment of the above-mentioned CRISPR-Cas effector protein, as well as a heterologous functional domain, and an RNA guide or an RNA guide encoding the RNA guide nucleic acid;

The above-mentioned CRISPR-Cas effector fusion protein, and an RNA guide or a nucleic acid encoding the RNA guide;

The above-mentioned DNA molecules;

The above recombinant vector;

The above gene editing system;

The above gene editing vector;

The above-mentioned cells containing gene editing systems;

The above eukaryotic cell lines;

The above-mentioned multicellular organisms;

The above-mentioned kit;

The above-mentioned container; or the above-mentioned implantable device.

Further, the subject includes a subject suffering from a condition or disease, including cancer, metabolic disease or genetic disease or infectious disease;

Further, the cancer includes Wilms tumor, Ewing sarcoma, neuroendocrine tumor, glioblastoma, neuroblastoma, melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, Kidney cancer, pancreatic cancer, lung cancer, biliary tract cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, medullary thyroid cancer, ovarian cancer, glioma, lymphoma, leukemia, myeloma, acute lymphocyte One or more of leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or urinary bladder cancer;

Further, metabolic diseases include familial hypercholesterolemia (FH);

Further, genetic diseases include transthyretin amyloidosis (ATTR), primary hyperoxaluric acidosis (PH1), and hereditary angioedema (HAE);

Further, the infectious agent of the infectious disease includes one or more of human immunodeficiency virus, herpes simplex virus-1, hepatitis B (HEPATITIS B) or herpes simplex virus-2;

Further, the conditions or diseases include cystic fibrosis, progressive Duchenne muscular dystrophy, Baker muscular dystrophy, alpha-1-antitrypsin deficiency, Pompe disease, myotonic dystrophy, Huntington's disease, Fragile One or more of syncytosis or beta thalassemia.

According to a twenty-seventh aspect of the present invention, there is provided the use of the above gene editing system in treating disorders or diseases.

Further, the conditions or diseases include cancer, infectious diseases, metabolic diseases or genetic diseases;

The cancers include Wilms tumor, Ewing sarcoma, neuroendocrine tumors, glioblastoma, neuroblastoma, melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, kidney cancer, Pancreatic cancer, lung cancer, biliary tract cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, medullary thyroid cancer, ovarian cancer, glioma, lymphoma, leukemia, myeloma, acute lymphoblastic leukemia, acute One or more of myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or urinary bladder cancer;

Further, the metabolic disease includes familial hypercholesterolemia (FH);

Further, the genetic diseases include transthyretin amyloidosis (ATTR), primary hyperoxaluric acidosis (PH1), and hereditary angioedema (HAE);

Applying the technical solution of the present invention, the above-mentioned CasY1, CasY2, CasY3, CasY4 and CasY5 can all exert the cleavage activity of Cas protein, and the cleavage efficiency is higher than that of the existing CRISPR/Cas system.

Description of the drawings

The description and drawings that constitute a part of this application are used to provide a further understanding of the present invention. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the attached picture:

Figure 1 shows a schematic diagram of the protein domain of CasY1-CasY5 according to Embodiment 1 of the present invention, in which D, E, and D represent the catalytic residues of the three conserved motifs I, II, and III of the RuvC domain, and h represents the bridge. spiral structure.

Figure 2 shows a schematic diagram of the secondary structure analysis results of the direct repeat sequences of CasY1, CasY2, CasY3, CasY4 and CasY5 according to Embodiment 1 of the present invention.

Figure 3 shows a schematic diagram of in vivo screening of effectors and library plasmid design according to Example 2 of the present invention.

Figure 4 shows a schematic diagram of the negative selection screening workflow according to Embodiment 2 of the present invention.

Figure 5 shows the PAM domain analysis results of CasY1, CasY2, CasY3, CasY4, and CasY5 according to Embodiment 2 of the present invention.

Figure 6 shows a schematic diagram of CasY1, CasY2, CasY3, CasY4 and CasY5 targeted cutting plasmids according to Embodiment 3 of the present invention.

Figure 7 shows the bacterial cleavage results of CasY1, CasY2, CasY3, CasY4 and CasY5 according to Example 3 of the present invention.

Figure 8 shows the in vitro cleavage results of CasY1, CasY2, CasY3, CasY4, and CasY5 according to Embodiment 4 of the present invention. Figure a shows the in vitro cleavage results of CasY1, and figure b shows the in vitro cleavage of CasY2. Figure c shows the in vitro cleavage results of CasY3, figure d shows the in vitro cleavage results of CasY4, and figure e shows the in vitro cleavage results of CasY5.

Figure 9 shows a statistical graph of the cleavage activities of CasY1, CasY2, CasY3, CasY4, CasY5 and Lbcpf1 on different target genes in 293T cells according to Example 5 of the present invention.

Detailed ways

It should be noted that, as long as there is no conflict, the embodiments and features in the embodiments of this application can be combined with each other. The present invention will be described in detail below with reference to examples.

Terminology explanation:

The term "cleavage" as used herein refers to a DNA break in a target nucleic acid produced by the nuclease of the CRISPR system described herein. In some embodiments, the cleavage event is a double-stranded DNA break. In some embodiments, the cleavage is a single-stranded DNA break. In some embodiments, the nick is a break in one of the DNA strands of the double-stranded DNA.

The term "CRISPR-Cas system" as used in the present invention refers to type V-I CRISPR-Cas effector proteins (i.e., Cas 12i effector proteins) and one or more RNA guides, and/or encodes the CRISPR-Cas effectors described above A nucleic acid containing a daughter protein or one or more RNA guides, and optionally a promoter operably linked to expression of a CRISPR-Cas effector or to the RNA guide or both.

The term "direct repeat" or "direct repeat" as used in the present invention refers to multiple short direct repeats that show very little or no sequence change in a CRISPR array. Appropriately, the direct repeats of Cas12i can form a stem-loop structure.

The term "stem-loop structure" used in the present invention refers to a nucleic acid having a secondary structure that includes a nucleotide region known or predicted to form a double-stranded (stem portion). ) are connected on one side by a region (loop portion) of mostly single-stranded nucleotides. The terms "hairpin" and "turn-back" structures are also used herein to refer to stem-loop structures. Such structures are well known in the art, and these terms are used consistent with their commonly known meanings in the art. As is known in the art, stem-loop structures do not require precise base pairing. Thus, a stem may include one or more base mismatches.

Alternatively, base pairing may be exact, ie, not include any mismatches. Direct repeat sequences have a stem-loop structure. The directly repeated stems included in the RNA guides consist of five complementary nucleobases that hybridize to each other, and the loop length is 6, 7, or 9 nucleotides.

The terms "CRISPR RNA", "crRNA" or "crRNA" as used in the invention refer to RNA molecules containing guide sequences used by CRISPR effectors to target specific nucleic acid sequences. Typically, crRNA contains spacer sequences that mediate target recognition and direct repeat sequences (also known as direct repeat or "DR" sequences) that form complexes with CRISPR-Cas effector proteins.

The term "donor template nucleic acid" as used herein refers to a nucleic acid molecule that can be used by one or more cellular proteins to alter the structure of the target nucleic acid after the CRISPR enzyme described herein has altered the target nucleic acid.

In some embodiments, the donor template nucleic acid is a double-stranded nucleic acid.

In some embodiments, the donor template nucleic acid is a single-stranded nucleic acid.

In some embodiments, the donor template nucleic acid is linear.

In some embodiments, the donor template nucleic acid is circular (eg, a circular plasmid). In some embodiments, the donor template nucleic acid is an exogenous nucleic acid molecule. In some embodiments, the donor template nucleic acid is an endogenous nucleic acid molecule (eg, a chromosome).

As used in the present invention, the terms "CRISPR-Cas effector", "CRISPR effector", "effector", "CRISPR-related protein" or "CRISPR enzyme", "CRISPR-Cas effector protein", or "Cas effector" "Protein" refers to a protein that performs an enzymatic activity or binds to a target site on a nucleic acid specified by an RNA guide.

The CRISPR-Cas effector protein associated within the CRISPR-Cas system may also be referred to as "Cas" or "Cas enzyme" or "Cas protein" in the present invention. Cas enzymes can recognize short motifs associated near target DNA, called prespacer adjacent motifs (PAMs).

CasY1, CasY2, CasY3, CasY4 and CasY5 proteins in the present invention can recognize PAMs containing or consisting of TTN, where N represents any nucleotide.

For example, PAM can be TTA, TTC, TTT or TTG.

In some embodiments, the CRISPR-Cas effector protein has endonuclease activity, nickase activity, and/or exonuclease activity.

The term "RNA guide" as used herein refers to any RNA molecule that facilitates the targeting of a protein described in the invention to a target nucleic acid. Exemplary "RNA guides" include, but are not limited to, crRNA, pre-crRNA (eg, DR-spacer-DR), and mature crRNA (eg, mature DR-spacer, mature DR-spacer-mature DR).

The term "targeting" as used in the present invention refers to the preferential or specific binding of a complex including a CRISPR-associated protein and an RNA guide (such as crRNA) to other nucleic acids that do not have the same or similar sequence as the target nucleic acid. For example, the ability to hybridize to a specific target nucleic acid.

The term "target nucleic acid" as used in the present invention refers to a specific nucleic acid substrate that contains a nucleic acid sequence complementary to all or part of the spacer in the RNA guide. In some embodiments, the target nucleic acid comprises a gene or a sequence within a gene. In some embodiments, the target nucleic acid includes a non-coding region (eg, a promoter). In some embodiments, the target nucleic acid is single-stranded or double-stranded.

In this specification, the numerical range represented by "numeric value A to numerical value B" refers to the range including the endpoint values A and B.

In the present invention, the numerical value represented by "at least numerical value A" means a range including greater than or equal to numerical value A.

In the present invention, the use of "substantially" or "substantially" means that the standard deviation from the theoretical model or theoretical data is within the range of 5%, preferably 3%, and more preferably 1%.

In the present invention, the meaning expressed by "can" includes both the meaning of performing a certain process and not performing a certain process.

In the present invention, "optional" or "optionally" means that the next described event or situation may or may not occur, and the description includes situations where the event occurs and situations where the event does not occur.

In this specification, references to "some specific/preferred embodiments", "other specific/preferred embodiments", "implementations", etc. refer to the specific elements described related to the embodiment (for example, Features, structures, properties and/or characteristics) are included in at least one embodiment described herein and may or may not be present in other embodiments. Additionally, it is to be understood that the described elements may be combined in various embodiments in any suitable manner.

As used herein, the terms "nucleic acid" and "nucleic acid molecule" refer to a compound containing a nucleobase and an acidic moiety, such as a nucleoside, a nucleotide, or a polymer of nucleotides. Typically, polymeric nucleic acids, such as nucleic acid molecules containing three or more nucleotides, are linear molecules in which adjacent nucleotides are linked to each other by phosphodiester bonds. In some embodiments, "nucleic acid" refers to a single nucleic acid residue (eg, a nucleotide and/or nucleoside). In some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising three or more individual nucleotide residues. As used herein, the terms "oligonucleotide" and "polynucleotide" are used interchangeably to refer to a polymer of nucleotides (eg, a string of at least three nucleotides). In some embodiments, "nucleic acid" includes RNA as well as single- and/or double-stranded DNA. Nucleic acids may be naturally occurring, for example, in the context of genomes, transcripts, mRNA, tRNA, rRNA, siRNA, snRNA, plasmids, cosmids, chromosomes, chromatids or other naturally occurring nucleic acid molecules. On the other hand, a nucleic acid molecule may be a non-naturally occurring molecule, such as a recombinant DNA or RNA, an artificial chromosome, an engineered genome or a fragment thereof, or a synthetic DNA, RNA, DNA/RNA hybrid, or include non-naturally occurring nucleotides or nucleosides. Furthermore, the terms "nucleic acid," "DNA," "RNA," and/or similar terms include nucleic acid analogs, for example, analogs having backbones other than a phosphodiester backbone. Nucleic acids can be purified from natural sources, produced and optionally purified using recombinant expression systems, chemically synthesized, and the like. Where appropriate, for example in the case of chemically synthesized molecules, the nucleic acid may comprise nucleoside analogs, for example analogs with chemically modified bases or sugars and backbone modifications.

As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably herein and refer to a polymer of amino acids of any length. The polymer can be linear or branched, it can contain modified amino acids, and it can be interrupted by non-amino acids. The term also includes amino acid polymers that have been modified (eg, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component).

As used herein, "CRISPR-Cas effector fusion protein" refers to a hybrid polypeptide comprising protein domains from at least two different proteins. A protein can be located at the amino-terminal (N-terminal, N-terminal) portion or the carboxyl-terminal (C-terminal, C-terminal) portion of a CRISPR-Cas effector fusion protein, thus forming an "amino-terminal CRISPR-Cas" respectively Effector fusion protein" or "carboxy-terminal CRISPR-Cas effector fusion protein". Any protein provided herein can be produced by any method known in the art. For example, the proteins provided herein can be produced via recombinant protein expression and purification, which is particularly suitable for CRISPR-Cas effector fusion proteins containing peptide linkers. Methods used for recombinant protein expression and purification are Well known and include those described below, see, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)).

It is clear to those skilled in the art that the structure of a protein can be changed without adversely affecting its activity and functionality. For example, one or more conservative amino acid substitutions can be introduced in the amino acid sequence of the protein without affecting the activity and/or functionality of the protein molecule. Or adversely affect the three-dimensional structure. Examples and implementations of conservative amino acid substitutions will be apparent to those skilled in the art. Specifically, the amino acid residue can be replaced with another amino acid residue belonging to the same group as the site to be replaced, that is, a non-polar amino acid residue can be substituted for another non-polar amino acid residue, and a polar uncharged amino acid residue can be substituted. An amino acid residue is substituted for another polar uncharged amino acid residue, a basic amino acid residue is substituted for another basic amino acid residue, and an acidic amino acid residue is substituted for another acidic amino acid residue. Such substituted amino acid residues may or may not be encoded by the genetic code. Conservative substitutions in which one amino acid is replaced by another amino acid belonging to the same group fall within the scope of the invention as long as the substitution does not result in inactivation of the biological activity of the protein. Therefore, the protein involved in the present invention may contain one or more conservative substitutions in the amino acid sequence, as long as the non-conservative substitution does not significantly affect the desired function and biological activity of the protein of the invention.

Conservative amino acid substitutions can be made at one or more predicted non-essential amino acid residues. "Non-essential" amino acid residues are amino acid residues that can be altered (deletion, substitution or replacement) without altering biological activity, whereas "essential" amino acid residues are required for biological activity. A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Amino acid substitutions can be made in non-conserved regions of the above-mentioned Cas proteins. Generally, such substitutions are not made to conserved amino acid residues, or to amino acid residues located within conserved motifs where such residues are required for protein activity. However, those skilled in the art will appreciate that functional variants may have fewer conservative or non-conservative changes in conserved regions.

As used herein, the term "CRISPR" refers to clustered regularly interspaced short palindromic repeats (Clustered regularly interspaced short palindromic repeats), which are derived from the immune system of microorganisms.

As used in the present invention, the term "target sequence" refers to the nucleotide sequence in the target nucleic acid that is complementary or at least partially complementary to crRNA. After the Cas protein, crRNA and the target sequence form a ternary complex, the Cas protein exerts its influence on the target nucleic acid. Specific cleavage activity on target nucleic acid strands and/or non-nucleotide strands. In this disclosure, "target sequence" is used interchangeably with "target nucleic acid," "target polynucleotide," "target sequence," and "target nucleic acid sequence."

As used in the present invention, the term "target strand" refers to the nucleotide strand in the target nucleic acid that hybridizes to crRNA; the term "non-target strand" refers to the nucleotide strand in the target nucleic acid that does not hybridize with crRNA. The nucleotide chains in which hybridization pairing occurs.

As used herein, the term "deaminase" or "deaminase domain" refers to a protein or enzyme that catalyzes a deamination reaction. In some embodiments, the deaminase is an adenosine deaminase, which catalyzes the hydrolytic deamination of adenine to hypoxanthine. In some embodiments, the deaminase is an adenosine deaminase, which catalyzes the hydrolytic deamination of adenosine or adenine (A) to inosine (I).

As used in this disclosure, "Base Editor (BE)" or "nucleobase editor" refers to a reagent that binds to a polynucleotide and has nucleobase modifying activity. In various embodiments, a base editor comprises a nucleobase modifying polypeptide (e.g., a deaminase) and a nucleic acid programmable nucleotide binding domain (e.g., a guide RNA) that binds to a guide polynucleotide (e.g., a guide RNA) For example, nucleic acid-programmable DNA-binding proteins). In various embodiments, the agent is a biomolecular complex comprising a protein domain having base editing activity, i.e., capable of modifying bases (e.g., A, T, C, G or U). In some embodiments, the polynucleotide programmable DNA binding domain is fused or linked to a deaminase domain. In one embodiment, the agent is a CRISPR-Cas effector fusion protein comprising a domain with base editing activity. In some embodiments, a domain with base editing activity is capable of deaminating bases within a nucleic acid molecule. In some embodiments, the base editor is capable of deaminating one or more bases within a DNA molecule. In some embodiments, the base editor is an adenosine base editor (ABE).

The terms "coding sequence" or "protein coding sequence" as used interchangeably herein refer to a polynucleotide fragment that encodes a protein. This region or sequence has a start codon near the 5' end and a stop codon near the 3' end. The coding sequence may also be called an open reading frame.

The terms "nuclear localization sequence" and "nuclear localization signal (NuclearLocalization Signal, NLS)" refer to the amino acid sequence that promotes protein import into the nucleus. Nuclear localization sequences are known in the art and are described, for example, in Plank et al., International PCT application, PCT/EP2000/011690, filed on November 23, 2000, published as WO/2001/038547 on May 31, 2001, which The contents are incorporated herein by reference for their disclosure of exemplary nuclear localization sequences. In some embodiments, the NLS is an optimized NLS, such as described by Koblan et al., Nature Biotech. 2018 doi: 10.1038/nbt.4172.

As used in this disclosure, the terms "complementary" or "hybrid" are used to refer to "polynucleotide" and "oligonucleotide" (which are interchangeable terms referring to is the nucleotide sequence). For example, the sequence "CAGT" is complementary to the sequence "GTCA". Complementarity can be "partial" or "total." "Partial" complementarity means that one or more nucleic acid bases are mismatched according to the base pairing rules. "Total" or "complete" complementarity between nucleic acids means that each nucleic acid base is matched with another base under base pairing. Base matching rules. The degree of complementarity between nucleic acid strands has an important impact on the efficiency and strength of hybridization between nucleic acid strands. This is particularly important in amplification reactions and detection methods that depend on binding between nucleic acids.

As used herein, the term "hybridization" refers to any process that uses a nucleic acid strand to combine with a complementary strand through base pairing to form a hybridization complex to pair complementary nucleic acids.

As used in this disclosure, the terms "nucleic acid sequence" and "nucleotide sequence" refer to oligonucleotides or polynucleotides, fragments or portions thereof, and refer to genomic or synthetic sources that may be single- or double-stranded of DNA or RNA, and represents the sense or antisense strand.

As used in this disclosure, the terms "sequence identity" and "percent identity" refer to the percentage of nucleotides or amino acids that are identical (i.e., identical) between two or more polynucleotides or polypeptides. Sequence identity between two or more polynucleotides or polypeptides can be determined by aligning the nucleotide or amino acid sequences of the polynucleotides or polypeptides and the aligned polynucleotides or polypeptides containing The number of positions with identical nucleotides or amino acid residues is scored and compared to the number of positions in the aligned polynucleotide or polypeptide that contain different nucleotides or amino acid residues. Polynucleotides may differ at one position, for example, by containing different nucleotides (ie, substitutions or mutations) or missing nucleotides (ie, nucleotide insertions or nucleotide deletions in one or both polynucleotides). Polypeptides may differ at one position, for example, by containing a different amino acid (ie, a substitution or mutation) or a missing amino acid (ie, an amino acid insertion or amino acid deletion in one or both polypeptides). Passable sequence identity Calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of amino acid residues in the polynucleotide or polypeptide. For example, percent identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of nucleotides or amino acid residues in the polynucleotide or polypeptide and multiplying by 100.

Illustratively, two or more sequences or subsequences have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleotides "sequence identity" or "percent identity". In certain embodiments, the sequences are substantially identical throughout the entire length of either or both compared biopolymers (eg, polynucleotides).

The term "vector" refers to a means of introducing a nucleic acid sequence into a cell thereby producing a transformed cell. Vectors include plasmids, transposons, phages, viruses, liposomes, and episomes. A "recombinant vector" or "expression vector" is a nucleic acid sequence containing a nucleotide sequence to be expressed in a recipient cell. Expression vectors may include additional nucleic acid sequences to facilitate and/or facilitate expression of introduced sequences, such as initiation, termination, enhancers, promoters, and secretion sequences.

As used in this disclosure, the terms "individual" and "subject" are used interchangeably and refer to mammals. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). mouse). In particular, individuals are people.

The methods disclosed herein may be performed in vitro, ex vivo, or in vivo, or the products may be present in in vitro, ex vivo, or in vivo forms. The term "in vitro" refers to experiments using materials, biological substances, cells and/or tissues in laboratory conditions or culture media; whereas the term "in vivo" refers to experiments and procedures using intact multicellular organisms. In some embodiments, methods performed in vivo can be performed on non-human animals. "Ex vivo" refers to an event that exists or occurs outside an organism, such as outside a human or animal body, such as an event that may exist or occur on tissue (eg, a whole organ) or cells taken from an organism.

As used in this disclosure, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g. , lubricants, talc, magnesium stearate, calcium or zinc or stearic acid) or solvent encapsulated materials involving the delivery or transport of compounds from one site of the body (e.g., delivery site) to another site (e.g., organ, tissue or body part). A pharmaceutically acceptable carrier is "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the tissues of the subject (eg, physiologically compatible, sterile, physiological pH, etc.). Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as Sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricant, Such as magnesium stearate, sodium lauryl sulfate and talc; (8) Excipients, such as cocoa butter and suppository wax; (9) Oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, Corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol, and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and laurel Ethyl acid; (13) Agar; (14) Buffers, such as magnesium hydroxide and aluminum hydroxide; (15) Alginic acid; (16) Pyrogen-free water; (17) Isotonic saline; (18) Ringer's solution; (19) ethanol; (20) pH buffer solution; (21) polyester, polycarbonate and/or polyanhydride; (22) bulking agent, such as peptides and amino acids (23) serum components, Such as serum albumin, high density lipoprotein (HDL) and low density lipoprotein (LDL); (22) C2-C12 alcohols, such as ethanol; and (23) others used in pharmaceutical preparations Non-toxic and compatible substances. Wetting agents, colorants, release agents, coating agents, sweeteners, flavorings, aroma Agents, preservatives and antioxidants may also be present in the formulation. Terms such as "excipient,""pharmaceutically acceptable carrier," and the like are used interchangeably herein.

As used herein, the term "effective amount" refers to an amount of biologically active agent sufficient to elicit the desired biological response. For example, in some embodiments, an effective amount of a base editor may refer to an amount of base editor sufficient to induce mutation of a target site that is specifically bound by the mutation of the base editor. As those skilled in the art will appreciate, effective amounts of reagents, such as base editor CRISPR-Cas effector fusion proteins, deaminases, polynucleotides, etc., can vary depending on various factors, such as the desired biological response. , vary, for example, with the specific allele, genome or target site to be edited, with the cells or tissues targeted and the reagents used.

The terms "treatment" and "treatment" mean, as described herein, intended to reverse, alleviate a disease or condition or one or more symptoms thereof, delay the onset of a disease or condition or one or more symptoms thereof, or inhibit a disease or condition or clinical intervention for the progression of one or more of its symptoms. As used herein, the terms "treatment" and "treatment" mean, as described herein, intended to reverse, alleviate a disease or condition or one or more symptoms thereof, delay the onset of a disease or condition or one or more symptoms thereof, or Clinical intervention that inhibits the progression of a disease or condition or one or more of its symptoms. In some embodiments, treatment may be administered after one or more symptoms have developed and/or the disease has been diagnosed. In other embodiments, treatment may be administered in the absence of symptoms, for example, to prevent or delay the onset of symptoms or to inhibit the onset or progression of a disease. For example, the treatment may be administered to a susceptible individual prior to the onset of symptoms (eg, in view of a history of symptoms and/or in view of genetic or other susceptibility factors). Treatment can also be continued after symptoms have subsided, for example to prevent or delay their recurrence.

The term "subject" includes humans and non-human animals. Non-human animals include vertebrates, such as mammals, and non-mammals, such as non-human primates, sheep, cats, horses, cattle, chickens, dogs, mice, rats, goats, rabbits, and pigs. Preferably, the subject is human. Unless otherwise indicated, the terms "patient" or "subject" are used interchangeably herein.

As mentioned in the background, the cutting efficiency of CRISPR/Cas systems in the prior art is low. Therefore, in this application, the inventor attempts to develop new Cas proteins and CRISPR/Cas systems to enrich the CRISPR/Cas system and improve cutting efficiency and targeting to meet the needs of actual use. Therefore, a series of protection schemes of this application are proposed.

In a first typical embodiment of the present application, a CRISPR-Cas effector protein is provided, which CRISPR-Cas effector protein includes at least 70% of the amino acid sequence of any one of SEQ ID NO: 1 to 5. Identity proteins (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84 %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) .

In a preferred embodiment, the CRISPR-Cas effector protein includes more than 80%, preferably more than 90%, more preferably more than 95%, further preferably more than 99% of the amino acid sequence of any one of SEQ ID NO: 1 to 5. % or above identity; preferably, the CRISPR-Cas effector protein includes a RuvC domain.

In a preferred embodiment, the CRISPR-Cas effector protein includes:

a) The protein shown in any one of SEQ ID NO: 1 to 5; or

b) Based on the amino acid sequence shown in SEQ ID NO: 1, a protein with one or more point mutations as follows: N21X, N23X, R25X, K26X, Q482X, S484X, R486X, S489X, R493X, H511X, C513X, H515X, N516X, R518X, R540X, K558X, Y560X, K562X, K565X, T600X, T672X, D676X, Q 680X, Y683X, L686X, D693X, Y731X, G767X, R772X, K832X, K833X, Q836X, M896X; or

c) A protein based on the amino acid sequence shown in SEQ ID NO: 2 with one or more of the following point mutations: R19X, R28X, R32X, K512X, N527X, W531X, R553X, K581X, K589X, I590X, R605X, K611X, R612X, R615X, Y777X, E877X, R931X; or

d) Based on the amino acid sequence shown in SEQ ID NO: 3, a protein with one or more point mutations as follows: K8X, F15X, N17X, K20X, K471X, W483X, H502X, R505X, K557X, K556X, R560X, Y673X, L676X, Y723X, N822X, K823X, E826X, K827X, K830X, K880X, L887X; or

e) Based on the amino acid sequence shown in SEQ ID NO: 4, a protein with one or more point mutations as follows: K317X, W330X, Y351X, K354X, D392X, F395X, N399X, Y509X, V512X, Y568X, N662X, K663X, E666X, R667X, K670X, K719X, L726X; or

f) Based on the amino acid sequence shown in SEQ ID NO: 5, a protein with one or more point mutations as follows: M9X, V16X, D18X, K21X, K518X, W531X, F550X, K553X, R609X, Y612X, R616X, Y730X, L733X, Y781X, N879X, K880X, E883X, K884X, K887X, K936X, F943X; where X is any amino acid.

The above-mentioned CRISPR-Cas effector protein has cutting activity and can specifically or non-specifically cut nucleotide chains to achieve the activity of the CRISPR-Cas system. For homologous proteins and point mutant proteins within the above-mentioned identity limits, compared to the protein shown in any one of SEQ ID NO: 1 to 5, amino acid changes can occur at the active site or inactive site of the protein. , including within or outside the RuvC domain. The protein obtained by changing the amino acid still has the cleavage activity of CRISPR-Cas effector protein.

In the second typical embodiment of the present application, a CRISPR-Cas effector fusion protein is provided. The CRISPR-Cas effector fusion protein includes the above-mentioned CRISPR-Cas effector protein, or a derivative of the CRISPR-Cas effector protein. or functional fragments of CRISPR-Cas effector proteins, as well as heterologous functional domains.

The sequence of the functional fragment is less than the full-length sequence but retains the cleavage function of the above-mentioned CRISPR-Cas effector protein. The missing residues in the functional fragment can be at the N-terminal, C-terminal and/or internal. Derivatives refer to at least about 80% sequence identity with the above-mentioned CRISPR-Cas effector protein, and possess at least one of the same functions, such as the ability to bind to and form a complex with a crRNA containing at least one DR sequence. Reasons for derivative formation include, but are not limited to, conservative amino acid residue substitutions.

CRISPR-Cas effectors are obtained by fusing heterologous functional domains to CRISPR-Cas effector proteins with cleavage activity, or derivatives of CRISPR-Cas effector proteins, or functional fragments of CRISPR-Cas effector proteins. Fusion proteins can have the activity of heterologous functional domains on the basis of normal cleavage activity. In specific use, heterologous functional domains in the existing technology can be flexibly selected to increase the function of the fusion protein. The connection of heterologous functional domains to CRISPR-Cas effector proteins (including derivatives and functional fragments) can be flexibly selected using different lengths in existing technologies The fusion protein linker (Linker) can also be directly connected without using a Linker, which will not affect the activity of CRISPR-Cas effector proteins (including derivatives and functional fragments) and heterologous functional domains.

In a preferred embodiment, the heterologous functional domain is located at the N-terminal, C-terminal or internal part of the CRISPR-Cas effector fusion protein; preferably, the heterologous functional domain includes but is not limited to positioning signal, reporter protein, CRISPR -Cas effector protein targeting portion, DNA binding domain, epitope tag, transcription activation domain, transcription repression domain, nuclease, deamination domain, methylase, demethylase, transcription release factor, HDAC (group One or more of protein deacetylase), cleavage active polypeptide, ligase; Preferably, the localization signal includes but is not limited to nuclear localization signal and/or nuclear export signal; Preferably, the nuclear export signal includes but is not limited to Human protein tyrosine kinase 2; preferably, the reporter protein includes but is not limited to glutathione-S-transferase, horseradish peroxidase, chloramphenicol acetyltransferase, β-galactosidase, β- Glucuronidase, or one or more of fluorescent proteins; preferably, the fluorescent proteins include but are not limited to, but are not limited to, green fluorescent protein, HcRed, DsRed, cyan fluorescent protein, yellow fluorescent protein or blue fluorescent protein One or more of them; preferably, the DNA binding domain includes but is not limited to one of methylation binding proteins, LexA DBD (DNA binding domain of LexA protein) or Gal4DBD (DNA binding domain of GAL4 protein) Or more; preferably, the epitope tag includes but is not limited to one of histidine tag, V5 tag, FLAG tag, influenza virus hemagglutinin tag, Myc tag, VSV-G tag or thioredoxin tag or A variety of; Preferably, the transcription activation domain includes but is not limited to VP64 and/or VPR; Preferably, the transcription repression domain includes but is not limited to KRAB and/or SID; Preferably, the nuclease includes but is not limited to FokI; Preferably, De The amino domain includes but is not limited to one or more of ADAR1, ADAR2, APOBEC, AID or TAD; preferably, the cleavage active polypeptide includes but is not limited to polypeptides with single-stranded RNA cleavage activity, polypeptides with double-stranded RNA cleavage activity polypeptide, a polypeptide with single-stranded DNA cleavage activity or a polypeptide with double-stranded DNA cleavage activity; preferably, the ligase includes but is not limited to DNA ligase and/or RNA ligase.

Flexible selection of the above heterologous functional domains can achieve multiple functions and activities of the fusion protein. The above-mentioned deamination domain includes a deaminase or a functional fragment of a deaminase. The effect of a base editor can be achieved by fusing the above-mentioned deamination domain with the above-mentioned heterologous functional domain and CRISPR-Cas effector protein. Fusion proteins containing nuclear localization signals can interact with nuclear import carriers so that proteins can be transported into the nucleus. The DNA binding domain (DBD) can recognize specific DNA sequences, thereby improving the targeting of the fusion protein.

In the third typical embodiment of the present application, a DNA molecule encoding the above-mentioned CRISPR-Cas effector protein or CRISPR-Cas effector fusion protein is provided.

In a preferred embodiment, the DNA molecule is a DNA molecule that is codon-optimized according to the codon preference of the host cell; preferably, the host cell includes a prokaryotic cell or a eukaryotic cell; preferably, the DNA molecule includes SEQ ID The nucleotide sequence of any one of NO: 6 to 10 has more than 70% (for example, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% , 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, 99% or 100%), preferably 90% or more, more preferably 95% or more, further preferably 99%, and still more preferably 100% identical nucleotides.

The above-mentioned DNA molecules include isolated DNA molecules. It can encode the above-mentioned CRISPR-Cas effector protein or CRISPR-Cas effector fusion protein through transcription and translation. The above-mentioned DNA molecules include single-stranded or double-stranded DNA, which can carry genetic information and thus achieve the function of encoding proteins. According to the host cell in which the DNA molecule is located or where the encoding occurs Codon preference and flexible codon optimization can achieve efficient expression of DNA molecules. The above-mentioned nucleotides shown in SEQ ID NO: 6 to 10 can respectively encode the proteins shown in SEQ ID NO: 1 to 5.

In the fourth typical embodiment of the present application, a recombinant vector is provided, which includes the above-mentioned DNA molecule.

In a preferred embodiment, the DNA molecule is connected to a promoter; preferably, the promoter includes but is not limited to one or more of an inducible promoter, a constitutive promoter or a tissue-specific promoter; preferably , promoters include but are not limited to one or more of T7, SP6, T3, CMV, EF1a, SV40, PGK1, humanβ-actin, CAG, U6, H1, T7, T7lac, araBAD, trp, lac or Ptac; Preferably, the recombinant vector includes, but is not limited to, a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated virus vector, a herpes simplex vector or a phagemid vector; preferably, the recombinant vector includes a plasmid vector.

In the fifth typical embodiment of the present application, a host cell is provided, and the host cell is transformed with the above-mentioned recombinant vector.

Integrate the above-mentioned DNA molecules into the above-mentioned recombinant vector to obtain a recombinant vector capable of replication and expression in host cells, thereby achieving large-scale replication and expression of DNA molecules, thereby realizing the above-mentioned CRISPR-Cas effector protein or CRISPR-Cas effector Expression, purification, or protein activity of fusion proteins in host cells.

In a sixth typical embodiment of the present application, a gene editing system is provided. The gene editing system includes: a) an RNA guide or a nucleic acid encoding an RNA guide. The RNA guide includes a direct repeat sequence and a spacer sequence. , the spacer sequence is used to hybridize with the target nucleic acid; b) the above-mentioned CRISPR-Cas effector protein, or CRISPR-Cas effector fusion protein, or DNA molecule, or recombinant vector, or host cell; DNA molecule, recombinant vector or host cell Able to express CRISPR-Cas effector protein or CRISPR-Cas effector fusion protein. In the gene editing system, the CRISPR-Cas effector protein or CRISPR-Cas effector fusion protein is combined with the RNA guide to target the spacer. The hybridization sequence formed by hybridization of the subsequence with the target nucleic acid.

The above-mentioned gene editing system includes RNA guides or nucleic acids encoding RNA guides, and CRISPR-Cas effector proteins, or CRISPR-Cas effector fusion proteins, or DNA molecules, recombinant vectors or host cells capable of expressing the above-mentioned proteins. This gene editing system hybridizes and combines the RNA guide with the target nucleic acid to form a hybrid sequence; the RNA guide on the hybrid sequence can combine with the above-mentioned protein to form a complex, thereby positioning the protein close to the target nucleic acid, and the protein can exert activity and target. The above-mentioned target nucleic acid may be modified in a targeted or non-targeted manner by cutting, nicking or the like.

In a preferred embodiment, the gene editing system does not contain tracrRNA.

TracrRNA is the abbreviation of trans-activating crRNA. It is transcribed separately in the gene editing system involving Cas9 protein and combines with crRNA to form gRNA (guide RNA), which combines with Cas9 protein to guide protein positioning. If the above gene editing system does not contain tracrRNA, the length and molecular weight of the gRNA that guides protein positioning will be smaller, and the binding domain between gRNA and Cas protein will be smaller, thereby reducing the molecular weight and size of the Cas protein and enriching the applications of the CRISPR-Cas system. Scenes.

In a preferred embodiment, the RNA guide includes one or more types.

The above-mentioned RNA guides include 1 type, 2 types, 3 types, 4 types, 5 types or even more types. The gene editing system includes a variety of RNA guides and can hybridize with multiple target nucleic acids at the same time, thereby exerting the activity of the gene editing system at multiple target nucleic acid sites, which can greatly improve the efficiency of gene editing and reduce the cost of gene editing and cell The time required for experiments such as passaging and verification of editing results. In the existing technology, it is also common to use multiple gRNAs at the same time in actual experimental processes. For example, when performing gene knockout or detection applications, multiple gRNAs may be used.

In a preferred embodiment, the target nucleic acid includes DNA; preferably, DNA includes DNA derived from eukaryotic organisms or DNA derived from prokaryotes; preferably, eukaryotic organisms include but are not limited to animals or plants; preferably, eukaryotic organisms include but are not limited to animals or plants; , DNA includes but is not limited to non-human mammal DNA, human DNA, insect DNA, avian DNA, reptile DNA, amphibian DNA, rodent DNA, fish DNA, worm DNA, nematode DNA or yeast DNA; Preferably, Non-human mammalian DNA includes, but is not limited to, non-human primate DNA.

Using the above gene editing system, RNA guides can be hybridized with DNA from different sources to achieve gene editing of multiple species.

In a preferred embodiment, the 3' end of the direct repeat sequence includes a stem-loop structure, and the stem-loop structure includes a first stem nucleotide chain, a cyclic nucleotide chain, and a second stem nucleotide chain connected in sequence. , the first stem nucleotide chain and the second stem nucleotide chain hybridize with each other to form a stem of a stem-loop structure, and the cyclic nucleotide chain forms a ring of a stem-loop structure; preferably, the length of the first stem nucleotide chain is 5 or 6 nucleotides; preferably, the length of the second stem nucleotide chain is 5 nucleotides; preferably, the length of the cyclic nucleotide chain is 6, 7 or 8 nucleotides.

In a preferred embodiment, the stem-loop structure includes the nucleotide sequence of SEQ ID NO: 25, 28, 31, 34 or 37.

The above-mentioned RNA guide includes direct repeat sequences and spacer sequences, wherein the 3' end of the direct repeat sequence is a stem-loop structure with secondary structure. The stem-loop structure includes a first stem nucleotide chain and a second stem nucleotide chain that can hybridize to each other to form a double strand, and the cyclic nucleotide chain forms a ring structure. This stem-loop structure can bind to CRISPR-Cas effector proteins or CRISPR-Cas effector fusion proteins to guide protein positioning. The N in the sequence indicates all bases.

In the embodiments of this application, the stem-loop structure corresponding to the nucleotide described in SEQ ID NO: 25 can bind to CasY1 protein; the stem-loop structure corresponding to the nucleotide described in SEQ ID NO: 28 can bind to CasY2 Protein binding; the stem-loop structure corresponding to the nucleotide described in SEQ ID NO: 31 can bind to the CasY3 protein; the stem-loop structure corresponding to the nucleotide described in SEQ ID NO: 34 can bind to the CasY4 protein; SEQ The stem-loop structure corresponding to the nucleotide described in ID NO: 37 can bind to the CasY5 protein. Regarding the selection of the stem-loop structure sequence, the protein can be freely combined with the above-mentioned stem-loop structure, or can be flexibly selected according to the above-mentioned identity without affecting the binding of the protein to the RNA guide.

In a preferred embodiment, the direct repeat sequence includes at least 80% (e.g., 80%, 81%, 82%, 83%) with the nucleotide sequence of SEQ ID NO: 24, 27, 30, 33 or 36 , 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100 %) nucleotide sequence identity; preferably, the direct repeat sequence includes at least 85% or more, more preferably 90% or more, and further preferably more than 90% identity with the nucleotide sequence of SEQ ID NO: 24, 27, 30, 33 or 36 A nucleotide sequence with more than 95% identity; preferably, the direct repeat sequence includes the nucleotide sequence of SEQ ID NO: 24, 27, 30, 33 or 36.

In a preferred embodiment, the spacer sequence includes 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) or more complementary to the target nucleic acid; preferably, the spacer sequence includes more than 90%, more preferably More than 95%, more preferably more than 99%, even more preferably 100% complementary to the target nucleic acid; preferably, the length of the spacer sequence is 18-41nt; preferably, the length of the spacer sequence is 18-37nt; preferably, the spacer sequence The length of the subsequence is 18-26nt or 34-36nt; preferably, the length of the spacer subsequence is 20nt.

The above-mentioned spacer sequence includes more than 80% fragments that are complementary to the target nucleic acid. Through at least 80% complementary base pairing, the RNA guide with the spacer sequence can bind firmly to the target nucleic acid, thereby realizing the gene editing system's targeting of the target nucleic acid. modification. The length of the spacer sequence can be flexibly selected within a certain range. If the length is too short, the complementary chain formed will be shorter, have poor binding force, and have weak binding specificity, affecting gene editing efficiency and off-target rate; if the length is too long, The increased length of the RNA guide and the difficulty of complementary pairing will also affect the gene editing efficiency.

In a preferred embodiment, the direct repeat sequence includes a first direct repeat sequence and a second direct repeat sequence; preferably, the RNA guide includes a first direct repeat sequence, a spacer sequence and a second direct repeat sequence connected in sequence. Repeating sequence; preferably, the first direct repeating sequence and the second direct repeating sequence are identical.

An RNA guide in this application refers to any RNA molecule that facilitates targeting of a protein of the invention to a target nucleic acid, including but not limited to crRNA, pre-crRNA (e.g., DR-spacer-DR), and mature crRNA (e.g., mature DR-spacer). sub, mature DR-spacer-mature DR). The RNA guide used in the examples of this application is pre-crRNA. The direct repeat sequence includes a first direct repeat sequence and a second direct repeat sequence located at both ends of the spacer sequence. The stem-loop structure is between the first and second direct repeat sequence. all exist. In the subsequent processing of RNA guides by cells, active RNA guides can be further obtained. For example, for the pre-crRNA composed of DR-spacer-DR, one of the direct repeat sequences is deleted after processing.

In a preferred embodiment, the target nucleic acid contains a pre-spacer adjacent motif, and the CRISPR-Cas effector protein or CRISPR-Cas effector fusion protein is capable of recognizing the pre-spacer adjacent motif (PAM). The adjacent motif comprises the nucleic acid sequence 5'-TTN-3', where N is any nucleotide; preferably, N is A, C or T.

In the CRISPR-Cas system, the Cas enzyme can recognize short motifs associated with the target nucleic acid, thereby completing the cutting and modification of specific sites. The PAM that can be specifically recognized by the CRISPR-Cas effector protein or CRISPR-Cas effector fusion protein in the above gene editing system is 5’-TTN-3’, where N is any nucleotide. And it has the highest recognition efficiency for 5’-TTA-3’, 5’-TTC-3’ and 5’-TTT-3’.

In a preferred embodiment, the CRISPR-Cas effector protein or CRISPR-Cas effector fusion protein is combined with the RNA guide to form a protein-nucleic acid complex; preferably, the protein-nucleic acid complex is non-naturally occurring or Modified; preferably, at least one component of the protein-nucleic acid complex is non-naturally occurring or modified.

In gene editing systems, RNA guides and Cas proteins can form protein-nucleic acid complexes. The RNA guide can be modified or the Cas protein can be modified, or an unmodified protein-nucleic acid complex can be directly used (that is, the RNA guide and Cas protein are both natural), both of which can play a role in gene editing.

In a preferred embodiment, by CRISPR-Cas effector protein or CRISPR-Cas effector fusion protein and The RNA guide targets the target nucleic acid and modifies the target nucleic acid; preferably, the modification includes but is not limited to cleavage or nicking; preferably, the modification results in: (1) a change in the expression of the cell containing at least one gene product; or (2) the cell comprises an alteration in the expression of at least one gene product, wherein the expression of at least one gene product is increased; or (3) the cell comprises an alteration in the expression of at least one gene product, wherein the expression of at least one gene product Reduced; or (4) the cell contains an edited genome; preferably, the modification results in cytotoxicity; preferably, the modification results in inhibition of gene expression, reduction in gene expression, or enhancement of gene expression.

Using the above-mentioned CRISPR-Cas effector protein or CRISPR-Cas effector fusion protein and RNA guide to target the target nucleic acid, the target nucleic acid can be modified, including cutting or nicking. Cleavage is the break of single-stranded DNA or double-stranded DNA. A nick is a break in one of the DNA strands of double-stranded DNA. The above-mentioned modifications to cellular genes can lead to changes in the expression of gene products in cells, including increases or decreases, and can also lead to editing of the cell genome. The above modifications can act on the cell's own genome, or on foreign genes such as plasmids in the cell. The above modifications can cause cytotoxicity, inhibit gene expression, reduce gene expression, or enhance gene expression.

In a preferred embodiment, the gene editing system includes a target nucleic acid or a nucleic acid encoding the target nucleic acid, and the target nucleic acid includes a homology arm fragment and a donor template nucleic acid; preferably, the target nucleic acid includes a sequence capable of hybridizing with the spacer sequence; Preferably, the homology arm fragment includes a 5' homology arm and a 3' homology arm, and the target nucleic acid consists of a 5' homology arm, a donor template nucleic acid and a 3' homology arm sequentially connected.

The above gene editing system may also include a target nucleic acid or a nucleic acid encoding the target nucleic acid. The target nucleic acid includes a sequence that can hybridize with the spacer sequence, that is, the target nucleic acid can be positioned at a specific nucleic acid site (such as a target nucleic acid). In this application, the target nucleic acid is the nucleic acid modified by the gene editing system; the donor template nucleic acid is the template for modifying the target nucleic acid. Further, the target nucleic acid includes a homology arm fragment and a donor template nucleic acid. The homology arm fragment can specifically bind to the nucleic acid surrounding a specific nucleic acid site. The donor template nucleic acid carries specific genetic information and can be synthesized through homology. Source recombination or other mechanisms, after the above-mentioned CRISPR-Cas effector protein or CRISPR-Cas effector fusion protein cleaves the target nucleic acid, specific genetic information is integrated into the position of the target nucleic acid, thereby completing the modification of the target nucleic acid, including Insertion, deletion or replacement of bases to complete gene editing.

"Donor template nucleic acid" means that one or more cellular proteins (such as homologous recombinase) can use it to change the target nucleic acid after the Cas enzyme has changed (modified, including the insertion, deletion or replacement of bases) the target nucleic acid. The structure of a nucleic acid molecule. For example, after using Cas protein to modify the target nucleic acid, homologous recombinase is used to modify and change the target nucleic acid by inserting, deleting, or replacing the donor template nucleic acid as a template. The donor template nucleic acid can be double-stranded nucleic acid or single-stranded nucleic acid; the donor template nucleic acid can be linear or circular (for example, a plasmid can be used); the donor template nucleic acid can be an exogenous nucleic acid molecule. Donor template nucleic acids include DNA or RNA.

In a preferred embodiment, the gene editing system exists in a deliverable form, and the delivery system is used to bring the gene editing system into contact with the target nucleic acid; preferably, the delivery system delivers the gene editing system into cells containing the target nucleic acid; Preferably, deliverable forms include, but are not limited to, nanoparticles, liposomes, exosomes, microvesicles, protein capsids, or particles for gene guns.

For target nucleic acids present in cells, the gene editing system can be delivered into the cells through the above delivery system. The contact between the gene editing system and the target nucleic acid is completed. The delivery system can also be further positioned at specific cell types, internal cell structures, etc., to achieve the purpose of precise delivery of the gene editing system and improve the accuracy of gene editing.

In a seventh typical embodiment of the present application, a gene editing vector is provided, which contains the above-mentioned nucleic acid encoding an RNA guide.

In a preferred embodiment, the gene editing vector also contains the above-mentioned DNA molecule; preferably, the DNA molecule and the nucleic acid encoding the RNA guide are located on the same or different vectors; preferably, the DNA molecule is connected to the first regulatory element; Preferably, the nucleic acid encoding the RNA guide is connected to a second regulatory element; preferably, the first regulatory element and the second regulatory element are each independently selected from one of an inducible promoter, a constitutive promoter or a tissue-specific promoter. One or more; preferably, the first regulatory element and the second regulatory element are independently selected from the group including but not limited to T7, SP6, T3, CMV, EF1a, SV40, PGK1, human β-actin, CAG, U6, H1, T7 , T7lac, araBAD, trp, lac or one or more of Ptac.

The above-mentioned gene editing vector contains a nucleic acid capable of encoding an RNA guide, capable of replicating the nucleic acid encoding the RNA guide, and encoding the RNA guide in the cell. The above-mentioned recombinant vector containing DNA molecules and the gene editing vector can be used to express CRISPR-Cas effector protein or CRISPR-Cas effector fusion protein and RNA guide respectively, that is, the above-mentioned gene editing system can be expressed. The gene editing vector may also contain the above-mentioned DNA molecule, and the DNA molecule and the nucleic acid encoding the RNA guide are located on the same or different vectors. If they are on the same vector, the gene editing vector includes one vector; if they are on different vectors, multiple vectors make up the gene editing vector. Using the above gene editing vector, the above gene editing system can be expressed in cells. On the gene editing vector, a variety of regulatory elements such as promoters can be flexibly set independently to help RNA guides and/or proteins complete transcription, translation, purification and other tasks.

In an eighth typical embodiment of the present application, a method for binding the above-mentioned gene editing system to a target nucleic acid in a cell is provided. The method includes: delivering the gene editing system to the cell, and the cell includes the target nucleic acid; allowing CRISPR- Cas effector proteins or CRISPR-Cas effector fusion proteins combine with the RNA guide to bind the spacer sequence to the target nucleic acid.

In a preferred embodiment, the target nucleic acid is double-stranded DNA or single-stranded DNA; preferably, the combination of the gene editing system and the target nucleic acid in the cell causes a change in the expression state of the target nucleic acid; preferably, the gene editing system and the cell The binding of the target nucleic acid results in the target nucleic acid being cleaved; preferably, the target nucleic acid is cleaved leading to the destruction of the target nucleic acid, or the replacement of a specific site of the target nucleic acid, or the removal of the target nucleic acid site, or the change of the function of the target nucleic acid region, or A sequence inversion between two sites on a target nucleic acid.

Target nucleic acid disruption, e.g., target mutation, e.g., resulting in gene knockout; Target nucleic acid replacement, e.g., resulting in target correction; Removal of target sites, e.g., resulting in target deletion; Changes in function, e.g., target nucleic acid activity or accessibility, resulting For example (transcriptional and/or epigenetic) gene or genomic region activation or gene or genomic region silencing. Sequence inversion between two sites on the target nucleic acid can be achieved, for example, by cutting the target nucleic acid into two sites and then using the Donor sequence or Cre-loxP recombinase system.

The above-mentioned delivery includes, but is not limited to, existing technologies such as plasmid transformation, microinjection, nanoparticles, liposomes, exosomes, microvesicles, protein capsids or gene guns. Target nucleic acid destruction includes target nucleic acid mutation or gene knockout. Target nucleic acid site-specific replacement includes correction of incorrect bases in the target nucleic acid. Removal of the target nucleic acid site can lead to deletion of the target nucleic acid site. Changes in regional function include but not Limited to reactivation of target nucleic acid expression activity, or inactivation of target nucleic acid expression activity, or increase in expression level, or expression The amount is reduced, or the expression product is changed. Changes in the expression state of the target nucleic acid, including but not limited to gene silencing or gene expression activation.

In a ninth typical embodiment of the present application, a cell containing a gene editing system is provided. The cell containing a gene editing system includes the above-mentioned gene editing system or gene editing vector.

In a preferred embodiment, the cell containing the gene editing system contains a modified target target locus, and the target target locus is a locus modified using the gene editing system; preferably, the modification of the target target locus results in: ( 1) The cells containing the gene editing system contain changes in the expression of at least one gene product; or (2) The cells containing the gene editing system contain changes in the expression of at least one gene product, wherein the expression of at least one gene product is increased; or (3) the cell containing the gene editing system contains an alteration in the expression of at least one gene product, wherein the expression of at least one gene product is reduced; or (4) the cell containing the gene editing system contains an edited genome; preferably, Cells containing gene editing systems include eukaryotic cells or prokaryotic cells; preferably, eukaryotic cells include but are not limited to animal cells, plant cells or human cells; preferably, animal cells include but are not limited to mammalian cells.

The above-mentioned cells containing the gene editing system contain the above-mentioned gene editing system or gene editing vector. Cells in various states such as gene editing has not occurred, gene editing is occurring, gene editing has been completed, etc., all belong to the above-mentioned cells containing the gene editing system. The above-mentioned cells where gene editing is occurring and where gene editing has been completed contain modified target loci. It can lead to changes in the expression of gene products in cells, including increases or decreases, and can also lead to editing of the cell genome. The above modifications can act on the cell's own genome, or on foreign genes such as plasmids in the cell. The above modifications can cause cytotoxicity, inhibit gene expression, reduce gene expression, or enhance gene expression.

The term "target locus" encompasses any DNA segment or region of a polynucleotide that is desired to be edited. In some embodiments, the target locus is a genomic locus. The target locus may be native to the cell, or alternatively may comprise heterologous or exogenous DNA segments. Heterologous or exogenous DNA segments may include transgenes, expression cassettes, polynucleotides encoding selectable markers, or heterologous or exogenous DNA regions. In specific embodiments, the targeted loci may comprise genes from prokaryotes, eukaryotes, animals or plants, including non-human mammals, non-human cells, rodents, humans, mice, primates or any other The native, heterologous or exogenous genomic nucleic acid sequence of the target organism, or a combination thereof.

In a tenth typical embodiment of the present application, a method for targeting and editing a target nucleic acid is provided, which method includes contacting the target nucleic acid with the above-mentioned gene editing system.

In an eleventh typical embodiment of the present application, a method for non-specific degradation of single-stranded DNA after identifying a target nucleic acid is provided, which method includes contacting the target nucleic acid with the above-mentioned gene editing system. The above method can be realized by utilizing the activity similar to the side-cleaving effect of Cas12i. Cas12i proteins can have incidental activity, that is, under certain circumstances, the activated Cas12i protein remains active after binding to the target sequence and continues to non-specifically cleave non-target oligonucleotides. This incidental activity is called "side-cleaving activity" or "random cleavage activity", and using this activity, the Cas12i system can be used to detect the presence of specific target oligonucleotides. For example, the Cas12i system is engineered to nonspecifically cleave ssDNA or transcripts. For example, Cas12i is provided or expressed transiently or stably in in vitro systems or cells, and targets or triggers non-specific cleavage of cellular nucleic acids, such as ssDNA, such as viral ssDNA.

In the existing technology, the above-mentioned side-cleaving activity can be applied to a highly sensitive and specific nucleic acid detection platform called SHERLOCK, which can be used in many clinical diagnoses. When using "by-cleaving activity" for detection, a reporter nucleic acid is used. "Reporter nucleic acid" refers to a molecule that can be cleaved or otherwise inactivated by the activated CRISPR system protein. Reporter nucleic acids contain nucleic acid elements that can be cleaved by CRISPR proteins. Cleavage of the nucleic acid element releases the agent or produces a conformational change that allows a detectable signal to be generated. Prior to cleavage, or when the reporter nucleic acid is in an "active" state, the reporter nucleic acid prevents the generation or detection of a positive detectable signal. For example, minimal background signal can be generated in the presence of an active reporter nucleic acid. A positive detectable signal can be any signal detectable using optical, fluorescent, chemiluminescent, electrochemical, or other detection methods known in the art. For example, in certain embodiments, a first signal (i.e., a negative detectable signal) can be detected when a reporter nucleic acid is present, which is then converted to a target molecule upon detection and cleavage or inactivation by an activated CRISPR protein. Second signal (eg, positive detectable signal). The above application can be realized using the above method or the above CRISPR-Cas effector protein.

In a twelfth typical embodiment of the present application, a method of targeting and nicking the non-spacer complementary strand of a double-stranded target DNA after identifying the spacer complementary strand of the double-stranded target DNA is provided. Methods include contacting double-stranded target DNA with the gene editing system described above.

In a thirteenth typical embodiment of the present application, a method for targeting and cutting double-stranded target DNA is provided, which method includes contacting the double-stranded target DNA with the above-mentioned gene editing system.

In a preferred embodiment, before nicking the spacer complementary strand of the double-stranded DNA, the non-spacer complementary strand of the double-stranded target DNA is nicked.

In a fourteenth typical embodiment of the present application, a method for specifically editing double-stranded nucleic acids is provided, which method includes contacting the following under sufficient conditions for a sufficient amount of time, (1) the above-mentioned CRISPR-Cas an effector protein, or a CRISPR-Cas effector fusion protein, another enzyme having sequence-specific nicking activity, and an RNA guide, the RNA guide directs the CRISPR-Cas effector protein or CRISPR-Cas effector fusion protein, relative to The activity of another sequence-specific nicking enzyme nicks the opposite strand; and (2) double-stranded nucleic acids; the above method results in the formation of double-stranded breaks.

In the above method, two of the above-mentioned CRISPR-Cas effector proteins, or a CRISPR-Cas effector fusion protein, or one of the above-mentioned proteins and another Cas protein with nicking activity (two Cas proteins are combined with a pair of RNA Guides targeted to the opposite strand of the target locus) can create double-stranded breaks with overhangs. This approach reduces the potential for off-target modifications because double-strand breaks are expected to occur only at loci where both enzymes nick, thereby increasing genome editing specificity. This approach is also known as the "double-nicking" or "paired-nickase" strategy.

In a fifteenth typical embodiment of the present application, a method for editing double-stranded nucleic acids is provided, which method includes contacting the following under sufficient conditions for a sufficient amount of time: (1) the above-mentioned CRISPR-Cas effector Protein, or CRISPR-Cas effector fusion protein, and a fusion protein of a protein domain with DNA modification activity, and an RNA guide targeting double-stranded nucleic acid; and (2) double-stranded nucleic acid; CRISPR-Cas effect of the fusion protein The molecule is modified to nick the non-target strand of the double-stranded nucleic acid. The above method can be realized by utilizing the activity similar to the side-cleaving effect of Cas12i. The above-mentioned double-stranded nucleic acid includes, but is not limited to, viral DNA (eg, Pasteur virus, hepatitis virus, herpes virus, adenovirus, poxvirus, parvovirus, etc.).

In a preferred embodiment, the two strands of the double-stranded nucleic acid are cleaved at different sites, resulting in staggered cleavage; preferably, the two strands of the double-stranded nucleic acid are cleaved at the same site, resulting in flat double-stranded breaks (DSB).

In a sixteenth typical embodiment of the present application, a method for targeting and cutting single-stranded target DNA is provided, which method includes contacting the target nucleic acid with the above-mentioned gene editing system.

The above gene editing system is in contact with the target nucleic acid, and can use the gene editing system to edit the target nucleic acid. Within sufficient contact time, the gene editing system can target nucleic acids for editing. The above-mentioned contacts can be achieved both inside and outside the cell. Sufficient conditions and a sufficient amount of contact time indicate that the above method can proceed or complete the reaction conditions and reaction time, which can be flexibly adjusted according to the specific implementation of the above method. The above gene editing system is in contact with the target nucleic acid and can use the gene editing system to edit the target nucleic acid to achieve different gene editing effects. Within sufficient contact time, the gene editing system can target nucleic acids for editing. The above-mentioned contact can be achieved inside or outside the cell, and the target nucleic acid includes but is not limited to the genome and isolated single-stranded or double-stranded DNA.

In a seventeenth typical embodiment of the present application, a method for inducing changes in cell state is provided. The method conservatively brings the above gene editing system into contact with the target nucleic acid in the cell.

In a preferred embodiment, the cell state includes but is not limited to apoptosis or dormancy; preferably, the cells include eukaryotic cells or prokaryotic cells; preferably, the cells include but are not limited to mammalian cells or plant disease cells; preferably, the cells include but are not limited to mammalian cells or plant disease cells; , cells include but are not limited to cancer cells; preferably, cells include but are not limited to infectious cells or cells infected by infectious agents; preferably, cells include but are not limited to cells infected by viruses and cells infected by prions; preferably, cells include but are not limited to cells infected by viruses and prions. In particular, cells include, but are not limited to, fungal cells, protozoan or parasite cells.

The above-mentioned method of inducing cell state changes uses a gene editing system to contact target nucleic acids in cells that regulate growth, metabolism and other functions, thereby modifying the target nucleic acids over time, thereby inducing changes in cell state. For example, the characteristic target nucleic acids in cancer cells, parasite cells, etc. can be modified to change the state of the cells, causing them to undergo apoptosis or dormancy, thereby achieving the purpose of eliminating such cells.

In an eighteenth typical embodiment of the present application, there is provided an application of using the above gene editing system in preparing a drug for treating a subject's illness or disease.

In a preferred embodiment, the application includes administering the gene editing system to a subject or ex vivo cells of a subject; preferably, the spacer sequence is at least 15 nucleotides of a target nucleic acid associated with the disorder or disease. Complementary, CRISPR-Cas effector protein or CRISPR-Cas effector fusion protein cleaves the target nucleic acid; Preferably, the disorder or disease includes but is not limited to cancer, infectious disease, metabolic disease or genetic disease; Preferably, cancer includes but Not limited to Wilms tumor, Ewing sarcoma, neuroendocrine tumors, glioblastoma, neuroblastoma, melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, kidney cancer, pancreatic cancer , lung cancer, biliary tract cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, medullary thyroid cancer, ovarian cancer, glioma, lymphoma, leukemia, myeloma, acute lymphoblastic leukemia, acute myeloid cell One or more of chronic lymphocytic leukemia, chronic myelogenous leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma or urinary bladder cancer; preferably, the metabolic disease includes familial hypertension cholesterolemia (FH); preferably, the genetic disease includes transthyretin amyloidosis (ATTR), primary hyperoxaluric acidosis (PH1), hereditary angioedema (HAE); preferably, the disorder or Diseases include, but are not limited to, cystic fibrosis, progressive Duchenne muscular dystrophy, Baker muscular dystrophy, alpha-1-antitrypsin deficiency, Pompe disease, myotonic dystrophy, Huntington's disease, fragile X syndrome , Friedreich's ataxia, amyotrophic lateral sclerosis, frontotemporal dementia, hereditary chronic kidney disease, hyperlipidemia, hypercholesterolemia, Leber's congenital amaurosis, sickle cell disease, or One or more beta thalassemias; preferably Specifically, the infectious agents of infectious diseases include but are not limited to one or more of human immunodeficiency virus, herpes simplex virus-1, hepatitis B (HEPATITIS B) or herpes simplex virus-2.

Utilizing the above gene editing system, it can be applied to the preparation of drugs for treating conditions or diseases in subjects. Such applications include the application of gene editing systems to subjects or their isolated cells. The application to subjects includes local application, systemic application or targeted application, etc., so as to achieve the effect of the drug. The spacer sequence is complementary to at least 15 nucleotides of the target nucleic acid related to the disease or disease, which can ensure stable and specific binding between the spacer sequence and the target nucleic acid and prevent off-target situations. The above conditions or diseases include but are not limited to cancer or infectious diseases. Through this application, target nucleic acids such as cancer cell genomes or subject defective genes can be modified to achieve the effect of drugs.

In a nineteenth typical embodiment of the present application, a eukaryotic cell line is provided, which includes the above-mentioned cells containing the gene editing system, or is the progeny of the cells containing the gene editing system.

In a twentieth typical embodiment of the present application, a multicellular organism is provided, which includes the above-mentioned cell containing the gene editing system.

In a preferred embodiment, multicellular organisms include but are not limited to model animals or model plants.

The above-mentioned multicellular organisms are multicellular organisms modified using the above-mentioned gene editing system, and heritable or non-heritable genetic modifications are obtained under the action of the above-mentioned gene editing system. The above-mentioned genetic modification includes gene insertion, deletion or replacement using a gene editing system, the result of genetic modification, and the genetic modification of the multicellular organism are controllable and predictable.

In the twenty-first typical embodiment of the present application, a method for obtaining plants with target traits is provided. The above-mentioned gene editing system is used to contact plant cells to modify the genes of plant cells or introduce target genes. Modify or The target gene can express the target traits, obtain modified plant cells, and use the modified plant cells for regeneration to obtain plants with the target traits.

In the twenty-second typical embodiment of the present application, a method for identifying target traits in plants is provided. The target genes in plant cells can express the target traits, and the above gene editing system is used to contact the plant cells, thereby identifying the target traits. Gene.

In the twenty-third typical embodiment of the present application, a kit is provided, which includes one or more components selected from the following: the above-mentioned CRISPR-Cas effector protein, CRISPR-Cas effector Fusion proteins, DNA molecules, recombinant vectors, host cells, gene editing systems, gene editing vectors, cells containing gene editing systems, eukaryotic cell lines, multicellular organisms; the components of the kit are distributed in the same or different containers middle.

In the twenty-fourth typical embodiment of the present application, a container is provided, which container contains the above-mentioned kit.

In a preferred embodiment, the container includes a sterile container; preferably, the container includes a syringe.

In the twenty-fifth typical embodiment of the present application, an implantable device is provided, and the implantable device includes the above-mentioned gene editing system.

In a preferred embodiment, the gene editing system is within the matrix; preferably, the gene editing system is within the reservoir.

The CRISPR-Cas effector protein, CRISPR-Cas effector fusion protein of the present invention, the RNP of the present disclosure, the nucleic acid of the present disclosure (for example, the above-mentioned DNA molecules, recombinant vectors, gene editing vectors, CRISPR- Cas effector guide RNA, nucleic acid encoding CRISPR-Cas effector guide RNA, nucleic acid encoding CRISPR-Cas effector protein, donor template, etc.) or the CRISPR-Cas effector system of the present disclosure is delivered to target cells (e.g., in vivo Target cells, where target cells are target cells in circulation, target cells in tissues, target cells in organs, etc.). Suitable for delivering a CRISPR-Cas effector polypeptide of the disclosure, a CRISPR-Cas effector fusion polypeptide of the disclosure, an RNP of the disclosure, a nucleic acid of the disclosure, or a CRISPR-Cas effector system of the disclosure to a target cell (e.g., Implantable devices that target cells in vivo, wherein the target cells are target cells in circulation, target cells in tissue, target cells in organs, etc.) may include a container (e.g., reservoir, matrix, etc.) that contains CRISPR -Cas effector protein, CRISPR-Cas effector CRISPR-Cas effector fusion protein, RNP or CRISPR-Cas effector system (or components thereof, such as nucleic acids of the present disclosure).

Suitable implantable devices may include, for example, a polymeric substrate (such as a matrix) serving as the body of the device, and in some cases additional scaffolding materials (such as metals or additional polymers), as well as materials that enhance visibility and imaging . Implantable delivery devices may be advantageous in providing localized and prolonged release, where the polypeptide and/or nucleic acid to be delivered is released directly to the target site, such as the extracellular matrix (ECM), vasculature surrounding tumors, diseased tissue wait. Suitable implantable delivery devices include devices suitable for delivery to a cavity (such as the peritoneal cavity) and/or any other type of administration where the drug delivery system is not anchored or attached, including devices that are biostable and/or implantable. A degradable and/or bioabsorbable polymeric substrate, which may optionally be the matrix, for example.

Such matrices include materials that are efficiently processed and/or modified upon contact with a biological environment without forming biologically active, toxic and/or harmful by-products. Including but not limited to bioabsorbable matrices, materials that can be used for bioabsorbable matrices include, for example, biopolymers (e.g., proteins, peptides, carbohydrates, polynucleotides, etc.), synthetic polymers, proteins, polysaccharides, silk, polydecane Glyceryl acid esters (PGS), polydioxanone, polylactic acid-co-glycolic acid (PLGA), polylactic acid (PLA), collagen, chitosan, silk protein and combinations thereof. Silk materials that can be used in the bioabsorbable matrix include, for example, silk fibroin, modified silk fibroin, spider silk, insect silk, recombinant silk, and any combination thereof. The above-mentioned reservoir reservoir composition includes a matrix of bioerodible, biocompatible polymers, including but not limited to polymer gel reservoirs or membrane-controlled reservoirs.

In some cases, suitable implantable drug delivery devices include degradable polymers where the primary release mechanism is bulkerosion. In some cases, suitable implantable drug delivery devices contain non-degradable or slowly degrading polymers in which the primary release mechanism is diffusion rather than bulk erosion, such that the outer portion acts as a membrane and its inner portion serves as a drug reservoir, In fact, the drug reservoir is not affected by the surrounding environment for long periods of time (eg, from about one week to about several months). Combinations of different polymers with different release mechanisms may also optionally be used. Over the lifetime of the total release period, the concentration gradient can remain effectively constant, and therefore the diffusion rate is effectively constant (referred to as "zero-mode" diffusion). The term "constant" means that the diffusion rate remains above a lower threshold for therapeutic effectiveness, but that it is still optionally characterized by an initial burst and/or may fluctuate, such as increase and decrease to a certain extent. The diffusion rate can be maintained like this for long periods of time, and the diffusion rate can be considered constant to a certain level to optimize the duration of treatment, such as an effective silent period.

In some cases, implantable delivery systems are designed to protect nucleotide-based therapeutics from degradation, whether chemical in nature or due to attack by enzymes and other factors within the subject's body. The implantation site or target site of the device can be selected for maximum therapeutic efficacy.

In the twenty-sixth exemplary embodiment of the present application, a method of treating a disease or condition in a subject in need is provided, the method comprising administering to the subject any one or more of the following:

The above-mentioned DNA molecules;

The above recombinant vector;

The above gene editing system;

The above gene editing vector;

The above-mentioned cells containing gene editing systems;

The above-mentioned eukaryotic cell lines;

The above-mentioned multicellular organisms;

The above-mentioned kit;

The above-mentioned container; or the above-mentioned implantable device.

In a preferred embodiment, the subject includes a subject suffering from a disorder or disease, including cancer, infectious disease, metabolic disease or genetic disease;

In a preferred embodiment, the cancer includes Wilms tumor, Ewing sarcoma, neuroendocrine tumor, glioblastoma, neuroblastoma, melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, Prostate cancer, liver cancer, kidney cancer, pancreatic cancer, lung cancer, biliary tract cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, medullary thyroid cancer, ovarian cancer, glioma, lymphoma, leukemia, bone marrow One or more of neoplasms, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or urinary bladder cancer;

In a preferred embodiment, the metabolic disease includes familial hypercholesterolemia (FH);

In a preferred embodiment, the genetic diseases include transthyretin amyloidosis (ATTR), primary hyperoxaluric acidosis (PH1), and hereditary angioedema (HAE);

In a preferred embodiment, the infectious agent of the infectious disease includes one or more of human immunodeficiency virus, herpes simplex virus-1, hepatitis B (HEPATITIS B) or herpes simplex virus-2;

In a preferred embodiment, the disorder or disease includes cystic fibrosis, progressive Duchenne muscular dystrophy, Becker muscular dystrophy, alpha-1-antitrypsin deficiency, Pompe disease, myotonic dystrophy Dysplasia, Huntington's disease, Fragile X syndrome syndrome, Friedreich's ataxia, amyotrophic lateral sclerosis, frontotemporal dementia, hereditary chronic kidney disease, hyperlipidemia, hypercholesterolemia, Leber's congenital amaurosis, sickle cell disease or one or more of beta thalassemias.

In the twenty-seventh typical embodiment of the present application, the use of the above gene editing system in treating disorders or diseases is provided.

In a preferred embodiment, the condition or disease includes cancer, infectious disease, metabolic disease or genetic disease;

In a preferred embodiment, the disorder or disease includes cystic fibrosis, progressive Duchenne muscular dystrophy, Becker muscular dystrophy, alpha-1-antitrypsin deficiency, Pompe disease, myotonic Malnutrition, Huntington's disease, fragile X syndrome, Friedreich's ataxia, amyotrophic lateral sclerosis, frontotemporal dementia, hereditary chronic kidney disease, hyperlipidemia, hypercholesterolemia, Leber's disease One or more of congenital amaurosis, sickle cell disease, or beta thalassemia.

The beneficial effects of the present application will be further explained in detail below in conjunction with specific embodiments.

Example 1

Acquisition of CasY1, CasY2, CasY3, CasY4 and CasY5 (i.e. CasY1-CasY5) genes and RNA guides.

Downloading microbial genome and metagenomic data from NCBI and JGI databases, a total of 20TB of high-quality data was obtained.

TBLASTN (https://blast.ncbi.nlm.nih.gov/) was used to perform local alignment of metagenomic data, and five new CRISPR-Cas effector proteins were obtained, namely CasY1 (SEQ ID NO: 1) and CasY2 (SEQ ID NO: 2), CasY1 (SEQ ID NO: 3), CasY4 (SEQ ID NO: 4), CasY5 (SEQ ID NO: 5).

Figure 1 shows a schematic diagram of the protein domain of CasY1-CasY5, in which D, E, and D represent the catalytic residues of the three conserved motifs I, II, and III of the RuvC domain, and h represents the bridge helix structure. D-E-D represents Asp-Glu-Asp amino acids, which are conserved amino acid residues of the RuvC domain.

The coding DNA sequence of CasY1 is shown in SEQ ID NO: 6, the coding DNA sequence of CasY2 is shown in SEQ ID NO: 7, the coding DNA sequence of CasY3 is shown in SEQ ID NO: 8, and the coding DNA sequence of CasY4 is shown in SEQ ID NO. NO:9 is shown, and the coding DNA sequence of CasY5 is shown as SEQ ID NO:10.

The direct repeat sequences corresponding to CasY1-CasY5 are shown in SEQ ID NO: 24, 27, 30, 33, and 36 respectively. The sequences of the stem-loop structure are SEQ ID NO: 25, 28, 31, 34, and 37 respectively. RNA guide (Pre-crRNA sequences) are shown in SEQ ID NO: 26, 29, 32, 35, and 38 respectively.

The secondary structure of the direct repeat sequences of CasY1, CasY2, CasY3, CasY4 and CasY5 was analyzed, and the results are shown in Figure 2.

Example 2

Determination of PAM sequences of CasY1-CasY5 genes.

1. Clone the nucleic acid sequence of CasY1 protein (SEQ ID NO: 6) and CasY1 crRNA-TTR sequence (SEQ ID NO: 11) into the expression vector pACYCDuet-1 (SEQ ID NO: 16) to construct the recombinant plasmid pACYCDuet1- CasY1-crRNA, the recombinant plasmid sequence is shown in SEQ ID NO: 17.

Similarly, the nucleic acid sequence of the CasY2 protein (SEQ ID NO: 7) and the CasY2 crRNA-TTR sequence (SEQ ID NO: 12) were cloned into the expression vector pACYCDuet-1 to construct the recombinant plasmid pACYCDuet1-CasY2-crRNA, as described The recombinant plasmid sequence is shown in SEQ ID NO: 18.

The nucleic acid sequence of the CasY3 protein (SEQ ID NO: 8) and the CasY3 crRNA-TTR sequence (SEQ ID NO: 13) were cloned into the expression vector pACYCDuet-1 to construct the recombinant plasmid pACYCDuet1-CasY3-crRNA. The recombinant plasmid sequence As shown in SEQ ID NO: 19.

The nucleic acid sequence of the CasY4 protein (SEQ ID NO: 9) and the CasY4 crRNA-TTR sequence (SEQ ID NO: 14) were cloned into the expression vector pACYCDuet-1 to construct the recombinant plasmid pACYCDuet1-CasY4-crRNA. The recombinant plasmid sequence As shown in SEQ ID NO:20.

The nucleic acid sequence of the CasY5 protein (SEQ ID NO: 10) and the CasY5 crRNA-TTR sequence (SEQ ID NO: 15) were cloned into the expression vector pACYCDuet-1 to construct the recombinant plasmid pACYCDuet1-CasY5-crRNA. The recombinant plasmid sequence As shown in SEQ ID NO: 21.

2. Clone the synthesized PAM library sequence into the pUC19 vector. The recombinant plasmid pUC19-PAM is shown in SEQ ID NO: 22.

The random sequence in SEQ ID NO: 22 of this application includes 6 random bases (n), that is, the types of random base sequences finally constructed reach 4 ⁶ , with a total of 4096 permutations and combinations.

3. Co-transfect pACYCDuet1-CasY1-crRNA and pUC19-PAM library plasmid into DH5α competent cells. The control group is co-transfection of pACYCDuet-1 and pUC19-PAM library plasmid.

Similarly, pACYCDuet1-CasY2-crRNA and pUC19-PAM library plasmids were also co-transfected into DH5α competent cells; pACYCDuet1-CasY3-crRNA and pUC19-PAM library plasmids were co-transfected into DH5α competent cells; pACYCDuet1-CasY4 -crRNA and pUC19-PAM library plasmid were co-transfected into DH5α competent cells; pACYCDuet1-CasY5-crRNA and pUC19-PAM library plasmid were co-transfected into DH5α competent cells.

DH5α cells were transformed with two plasmids and treated at 37°C for 1 hour. The plasmids were extracted and the PAM region sequence was subjected to PCR amplification and high-throughput sequencing.

Figure 3 is a schematic diagram of in vivo screening of effectors and library plasmid design. The pACYC-Effector-crRNA plasmid is the plasmid (effector plasmid) shown in SEQ ID NO: 17-21, and a pUC19-PAM library targeting the PAM library was designed. plasmid.

The experimental process for negative selection screening is as follows:

1) Construct effector plasmid;

2) Transform the effector plasmid and library plasmid into E. coli, then grow and conduct antibiotic screening;

3) Use targeted sequencing to identify depleted library plasmids, and use small RNA sequencing to identify mature crRNA.

Figure 4 is a schematic diagram of the negative selection screening workflow.

4. Count the occurrence times of 4096 combinations of PAM sequences in the experimental group and the control group respectively, and standardize them with the number of PAM sequences. For a PAM sequence, when log2 (standardized value of the control group/standardized value of the experimental group) > 3.5, it is considered This PAM sequence is significantly consumed, and the PAM domain is predicted from the significantly consumed PAM sequence.

Through experimental results, it was observed that CasY1-CasY5 effectively edits target sequences with 5’-TTA, 5’-TTT, and 5’-TTC PAMs. The PAM sequence of the CasY1-CasY5 protein is a 5’-TTN structure. Statistics show that the editing activity of CRISPR/CasY1, CRISPR/CasY2, CRISPR/CasY3, CRISPR/CasY4, and CRISPR/CasY5 systems for 5’-TTA, 5’-TTT, and 5’-TTC is much higher than that of 5’-TTG. The PAM domain analysis results of CasY1, CasY2, CasY3, CasY4, and CasY5 are shown in Figure 5. "Bits" in Figure 5 refers to "bits", and "Position" refers to "position".

Example 3

Determination of bacterial cleavage activity in vivo

1. Construct the ccdb virus plasmid. The ccdb virus plasmid is shown in SEQ ID: 23. The ccdb toxic plasmid (SEQ ID NO: 23) was transferred into Top10 competent cells. Through gradient experiments, it was found that when 64mM L-arabinose (L-ara) was added to the plate, the bacteria were lethal, indicating that this dose was a lethal dose.

2. Co-transfer pACYCDuet1-CasY1-crRNA recombinant plasmid (SEQ ID NO: 17) and ccdb toxic plasmid into Top10 competent cells, and then add 64mM L-arabinose (L-ara), some bacteria survive, indicating that in The CasY1 protein in the surviving bacteria cut the ccdb virulence plasmid.

Similarly, pACYCDuet1-CasY2-crRNA recombinant plasmid (SEQ ID NO: 18), pACYCDuet1-CasY3-crRNA recombinant plasmid (SEQ ID NO: 19), pACYCDuet1-CasY4-crRNA recombinant plasmid (SEQ ID NO: 20), pACYCDuet1 -CasY5-crRNA recombinant plasmid (SEQ ID NO: 21) and ccdb toxic plasmid were co-transfected into Top10 competent cells, and when 64mM L-arabinose (L-ara) was added, some bacteria survived, indicating that in the surviving part In bacteria, CasY2-CasY5 proteins cut the ccdb virulence plasmid respectively.

The schematic diagram of CasY1-CasY5 protein cleavage in bacteria is shown in Figure 6, and the cleavage activity identification results are shown in Figure 7.

Example 4

In vitro cleavage activity of CasY1-CasY5 proteins

1. Expression and purification of CasY1-CasY5 proteins

Rosetta (DE3) pLyseS (EMD Millipore) cells expressing CasY1-CasY5 respectively were inoculated into 10 ml LB medium and cultured at 37°C overnight. When the OD ₆₀₀ of the bacteria reaches 0.2, lower the culture temperature to 21°C and continue culturing until the OD ₆₀₀ reaches 0.6. Then add IPTG at a final concentration of 500 μM to induce Cas protein expression. Collect the cells after 14-18 hours of induction culture, and resuspend the cells in 200ml lysis buffer (50mM HEPES [pH7], 2M NaCl, 5mM MgCl, 20mM imidazole), which contains protease inhibitors (Roche complete, EDTA-free, COEDTAF -RO) and lysozyme (Sigma, 10837059001) for homogenization. Cells were lysed using sonication (Branson Sonifier 450). Centrifuge at 10,000 × g for 1 hour to clear lysate. The lysate was filtered through a 0.22 μm filter (Millipore, Stericup) before being transferred to a nickel column (HisTrap FF, 5 ml) and eluted with an imidazole gradient. Proteins containing the expected sizes were pooled together, TEV protease (Sigma, T4455-10KU) was added, and the samples were dialyzed overnight in TEV buffer (500mM NaCl, 50mM HEPES [pH 7], 5mM MgCl, 2mM DTT). After dialysis, the sample was concentrated to 500 μl and stored frozen at -80°C.

2. In vitro cleavage assay

The purified protein (25nM) was reacted in cleavage buffer (NEBuffer 3, 5mM DTT) at 37°C for 20 minutes. Use 500ng of synthesized TTR-2 crRNA (pre-crRNA, SEQ ID NO: 40, 43, 46, 49, 52) (synthesized by Nanjing Genscript Biotechnology Co., Ltd.) and 200ng of target DNA (dsDNA, such as SEQ ID NO: 57) for cleavage reaction, the protein concentrations were 0nM, 50nM, 100nM, 200nM, 500nM and 1μM respectively. reaction product The materials were recovered using a purification and recovery kit (QIAGEN). Cleavage efficiency was tested on TBE-urea 6% polyacrylamide gel (Invitrogen).

CasY1-CasY5 proteins have obvious cleavage activity under 37°C. Figure 8 shows the results of in vitro cleavage activity, in which the in vitro cleavage activities of CasY1, CasY2, CasY3, CasY4, and CasY5 on target dsDNA are shown in Figure 8, Figure a, Figure b, Figure c, Figure d, and Figure e, respectively.

Example 5

293T cell cleavage activity

1. Inoculate HEK293T cells (purchased from ATCC) into DMEM medium (Gibco, 11965092) supplemented with 10% FBS (v/v), containing 1% Penicillin Streptomycin (v/v) (Gibco, 15140122), Culture was performed in a 37 °C cell culture incubator containing 5% _CO2 . LONZA transfection reagent (Lonza, Cat #V4XP-3032) was used for transfection according to the instructions, and the cells were counted to 2×10 ⁶ .

CasY1 protein: crRNA (chemically synthesized by Genscript Biotechnology Co., Ltd.) was mixed at a mass of 3 μg: 1.5 μg. Similarly, CasY2-CasY5 protein and Lbcpf1 protein (QRU95066.1, obtained after expression and purification using existing technology) were mixed with a variety of crRNA (chemically synthesized by Genscript Biotechnology Co., Ltd.) in the above proportions according to the above method, As shown in Table 1.

Table 1

After 48 hours of electroporation, cells were collected to detect cutting efficiency.

Cells were subjected to genome extraction (TIANGEN, DP304-03). Using the genome as a template, PCR amplification was performed on sequences near the target site, and the amplified PCR products were used for high-throughput deep sequencing (Jinweizhi Biotechnology Co., Ltd.). The system used for target site sequence amplification is as follows: 2×Taq Master Mix (Vazyme, P112-03) 25 μL; Primer-F (10 pmol/μL) 1 μL; Primer-R (10 pmol/μL) 1 μL; template 1 μL; ddH ₂ O. Make up to 50 μL.

2. The cleavage activities of CasY1, CasY2, CasY3, CasY4, CasY5 proteins and LbCpf1 in 293T cells are shown in Figure 9, indel represents the insertion and deletion rate. Shown is the indel activity induced by CasY1-CasY5 and LbCpf1 CRISPR effectors targeting the TTR locus and PCSK9 in the 293T cell line 48 hours after transient transfection of effector proteins and RNA guides. Different RNA guide designs were tested and showed varying degrees of efficacy. Error bars represent S.E.M. of 3 replicates.

This shows that CasY1-CasY5 proteins can achieve efficient cleavage at different sites in 293T cells, and the cleavage efficiency is better than LbCpf1.

From the above description, it can be seen that the above-mentioned embodiments of the present invention achieve the following technical effects: the above-mentioned CasY1, CasY2, CasY3, CasY4, and CasY5 proteins can be used in systems, methods, and compositions for manipulating nucleic acids in a targeted manner. . Non-naturally occurring engineered CRISPR-Cas systems for targeted modification of nucleic acids (e.g., DNA) consisting of the above-described proteins and RNA guides, each system including one or more protein components that together target the nucleic acid and One or more nucleic acid components can exert the function and activity of the CRISPR-Cas system both inside and outside the cell.

The above descriptions are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection scope of the present invention.

Claims

A CRISPR-Cas effector protein, characterized in that the CRISPR-Cas effector protein includes at least 70% identity with the amino acid sequence described in any one of SEQ ID NO: 2, 1, 3-5 protein.
The CRISPR-Cas effector protein according to claim 1, characterized in that the CRISPR-Cas effector protein includes an amino acid sequence having 80 % or more, preferably 90% or more, more preferably 95% or more, even more preferably 99% or more identical proteins;

Preferably, the CRISPR-Cas effector protein includes a RuvC domain.
The CRISPR-Cas effector protein according to claim 2, wherein the CRISPR-Cas effector protein includes:

a) The protein shown in any one of SEQ ID NO: 1 to 5; or

b) Based on the amino acid sequence shown in SEQ ID NO: 1, a protein with one or more point mutations as follows:

N21X, N23X, R25X, K26X, Q482X, S484X, R486X, S489X, R493X, H511X, C513X, H515X, N516X, R518X, R540X, K558X, Y560X, K562X, K565X, T600X, T672X, D6 76X, Q680X, Y683X, L686X, D693X, Y731X, G767X, R772X, K832X, K833X, Q836X, M896X; or

c) Based on the amino acid sequence shown in SEQ ID NO: 2, a protein with one or more point mutations as follows:

or

d) Based on the amino acid sequence shown in SEQ ID NO: 3, a protein with one or more point mutations as follows:

OR

e) Based on the amino acid sequence shown in SEQ ID NO: 4, a protein with one or more point mutations as follows:

OR

f) Based on the amino acid sequence shown in SEQ ID NO: 5, a protein with one or more point mutations as follows:

M9X, V16X, D18X, K21X, K518X, W531X, F550X, K553X, R609X, Y612X, R616X, Y730X, L733X, Y781X, N879X, K880X, E883X, K884X, K887X, K936X, F943X;

where X is any amino acid.
A CRISPR-Cas effector fusion protein, characterized in that the CRISPR-Cas effector fusion protein includes the CRISPR-Cas effector protein according to any one of claims 1 to 3, or the CRISPR-Cas effector Derivatives of the daughter protein or functional fragments of the CRISPR-Cas effector protein, as well as heterologous functional domains.
The CRISPR-Cas effector fusion protein according to claim 4, wherein the heterologous functional domain is located at the N-terminal, C-terminal or inside the CRISPR-Cas effector fusion protein;

Preferably, the heterologous functional domain includes a positioning signal, a reporter protein, a CRISPR-Cas effector protein targeting portion, a DNA binding domain, an epitope tag, a transcription activation domain, a transcription inhibition domain, a nuclease, and a deamination domain. , one or more of methylase, demethylase, transcription release factor, HDAC, cleavage active polypeptide, and ligase;

Preferably, the localization signal includes a nuclear localization signal and/or a nuclear export signal;

Preferably, the nuclear export signal includes human protein tyrosine kinase 2;

Preferably, the reporter protein includes glutathione-S-transferase, horseradish peroxidase, chloramphenicol acetyltransferase, β-galactosidase, β-glucuronidase or autofluorescence one or more proteins;

Preferably, the autofluorescent protein includes one or more of green fluorescent protein, HcRed, DsRed, cyan fluorescent protein, yellow fluorescent protein or blue fluorescent protein;

Preferably, the DNA binding domain includes one or more of methylation binding protein, Lex A DBD or Gal4 DBD;

Preferably, the epitope tag includes one or more of histidine tag, V5 tag, FLAG tag, influenza virus hemagglutinin tag, Myc tag, VSV-G tag or thioredoxin tag;

Preferably, the transcription activation domain includes VP64 and/or VPR;

Preferably, the transcription repression domain includes KRAB and/or SID;

Preferably, the nuclease includes FokI;

Preferably, the deamination domain includes one or more of ADAR1, ADAR2, APOBEC, AID or TAD;

Preferably, the cleavage active polypeptide includes a polypeptide with single-stranded RNA cleavage activity, a polypeptide with double-stranded RNA cleavage activity, a polypeptide with single-stranded DNA cleavage activity or a polypeptide with double-stranded DNA cleavage activity;

Preferably, the ligase includes DNA ligase and/or RNA ligase.
A DNA molecule, characterized in that the DNA molecule encodes the CRISPR-Cas effector protein described in any one of claims 1 to 3, or the CRISPR-Cas effector fusion protein described in claim 4 or 5.
The DNA molecule according to claim 6, characterized in that the DNA molecule is a DNA molecule that is codon-optimized according to the codon preference of the host cell;

Preferably, the host cell includes a prokaryotic cell or a eukaryotic cell;

Preferably, the DNA molecule includes more than 70% of the nucleotide sequence described in any one of SEQ ID NO: 6 to 10, preferably more than 90%, more preferably more than 95%, further preferably 99%, and further Nucleotides that are 100% identical are preferred.
A recombinant vector, characterized in that the recombinant vector contains the DNA molecule described in claim 6 or 7.
The recombinant vector according to claim 8, wherein the DNA molecule is connected to a promoter;

Preferably, the promoter includes one or more of an inducible promoter, a constitutive promoter or a tissue-specific promoter;

Preferably, the promoter includes one or more of T7, SP6, T3, CMV, EF1a, SV40, PGK1, human β-actin, CAG, U6, H1, T7, T7lac, araBAD, trp, lac or Ptac ;

Preferably, the recombinant vector includes a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated virus vector, a herpes simplex vector or a phagemid vector;

Preferably, the recombinant vector includes a plasmid vector.
A host cell, characterized in that the host cell is transformed with the recombinant vector according to claim 8 or 9.
A gene editing system, characterized in that the gene editing system includes:

a) an RNA guide or a nucleic acid encoding the RNA guide, which includes a direct repeat sequence and a spacer sequence for hybridizing to a target nucleic acid;

b) The CRISPR-Cas effector protein of any one of claims 1 to 3, or the CRISPR-Cas effector fusion protein of claim 4 or 5, or the DNA molecule of claim 6 or 7, or the The recombinant vector described in claim 8 or 9, or the host cell described in claim 10;

The DNA molecule, the recombinant vector or the host cell is capable of expressing the CRISPR-Cas effector protein or the CRISPR-Cas effector fusion protein,

In the gene editing system, the CRISPR-Cas effector protein or the CRISPR-Cas effector fusion protein, after being combined with the RNA guide, targets the spacer sequence and the target nucleic acid. The hybridization sequence formed by hybridization.
The gene editing system according to claim 11, characterized in that the gene editing system does not contain tracrRNA.
The gene editing system according to claim 11, wherein the RNA guide includes one or more.
The gene editing system according to claim 11, wherein the target nucleic acid includes DNA;

Preferably, the DNA includes DNA derived from eukaryotic organisms or DNA derived from prokaryotes;

Preferably, the eukaryotes include animals or plants;

Preferably, the DNA includes non-human mammalian DNA, human DNA, insect DNA, avian DNA, reptile DNA, amphibian DNA, rodent DNA, fish DNA, worm DNA, nematode DNA or yeast DNA;

Preferably, the non-human mammalian DNA includes non-human primate DNA.
The gene editing system according to claim 11, wherein the 3' end of the direct repeat sequence includes a stem-loop structure, and the stem-loop structure includes a first stem nucleotide chain and a cyclic nucleotide connected in sequence. chain and a second stem nucleotide chain, the first stem nucleotide chain and the second stem nucleotide chain hybridize to each other to form the stem of the stem-loop structure, and the cyclic nucleotide chain forms the Rings of stem-loop structure;

Preferably, the length of the first stem nucleotide chain is 5 or 6 nucleotides;

Preferably, the length of the second stem nucleotide chain is 5 nucleotides;

Preferably, the cyclic nucleotide chain is 6, 7 or 8 nucleotides in length.
The gene editing system according to claim 15, wherein the stem-loop structure includes the nucleotide sequence described in SEQ ID NO: 25, 28, 31, 34 or 37.
The gene editing system according to claim 11, wherein the direct repeat sequence includes a core with at least 80% identity to the nucleotide sequence described in SEQ ID NO: 24, 27, 30, 33 or 36. nucleotide sequence;

Preferably, the direct repeat sequence includes at least 85% or more, more preferably more than 90%, and further preferably more than 95% identity with the nucleotide sequence described in SEQ ID NO: 24, 27, 30, 33 or 36. Nucleotide sequence;

Preferably, the direct repeat sequence includes the nucleotide sequence described in SEQ ID NO: 24, 27, 30, 33 or 36.
The gene editing system according to claim 11, wherein more than 80% of the spacer sequence is complementary to the target nucleic acid;

Preferably, more than 90% of the spacer sequence, more preferably more than 95%, further preferably more than 99%, even more preferably 100% is complementary to the target nucleic acid;

Preferably, the length of the spacer sequence is 18-41 nt;

Preferably, the length of the spacer sequence is 18-37nt;

Preferably, the length of the spacer sequence is 18-26 or 34-36nt;

Preferably, the length of the spacer sequence is 20nt.
The gene editing system according to claim 11, wherein the direct repeat sequence includes a first direct repeat sequence and a second direct repeat sequence;

Preferably, the RNA guide includes the first direct repeat sequence, the spacer sequence connected in sequence. subsequence and said second direct repeat sequence;

Preferably, said first direct repeat sequence and said second direct repeat sequence are identical.
The gene editing system according to claim 11, wherein the target nucleic acid includes a pre-spacer adjacent motif, and the CRISPR-Cas effector protein or the CRISPR-Cas effector fusion protein can recognize the a pre-spacer adjacent motif, the pre-spacer adjacent motif comprising the nucleic acid sequence 5'-TTN-3', wherein N is any nucleotide;

Preferably, the N is A, C or T.
The gene editing system of claim 11, wherein the CRISPR-Cas effector protein or the CRISPR-Cas effector fusion protein combines with the RNA guide to form a protein-nucleic acid complex;

Preferably, the protein-nucleic acid complex is non-naturally occurring or modified;

Preferably, at least one component of the protein-nucleic acid complex is non-naturally occurring or modified.
The gene editing system according to claim 11, characterized in that the targeting of the target nucleic acid by the CRISPR-Cas effector protein or the CRISPR-Cas effector fusion protein and the RNA guide Function to modify the target nucleic acid;

Preferably, said modification includes cutting or incision;

Preferably, said modification results in:

(1) The cell contains an alteration in the expression of at least one gene product; or

(2) the cell comprises an alteration in the expression of at least one gene product, wherein the expression of the at least one gene product is increased; or

(3) the cell comprises an alteration in the expression of at least one gene product, wherein the expression of the at least one gene product is reduced; or

(4) The cell contains an edited genome;

Preferably, said modification results in cytotoxicity;

Preferably, the above modification results in inhibition of gene expression, reduction in gene expression or enhancement of gene expression.
The gene editing system according to claim 11, characterized in that the gene editing system includes a target nucleic acid or a nucleic acid encoding the target nucleic acid, and the target nucleic acid includes a homology arm fragment and a donor template nucleic acid;

Preferably, the target nucleic acid comprises a sequence capable of hybridizing to the spacer sequence;

Preferably, the homology arm fragment includes a 5' homology arm and a 3' homology arm, and the target nucleic acid is sequenced by the 5' homology arm, the donor template nucleic acid and the 3' homology arm. Connection composition.
The gene editing system according to claim 11, characterized in that the gene editing system exists in a deliverable form, and the delivery system is used to bring the gene editing system into contact with the target nucleic acid;

Preferably, the delivery system delivers the gene editing system into cells containing the target nucleic acid;

Preferably, the deliverable form includes nanoparticles, liposomes, exosomes, microvesicles, protein capsids or particles for gene guns.
A gene editing vector, characterized in that the gene editing vector contains the nucleic acid encoding the RNA guide in the gene editing system according to any one of claims 11 to 24.
The gene editing vector according to claim 25, characterized in that the gene editing vector further comprises the DNA molecule according to claim 6 or 7;

Preferably, the DNA molecule and the nucleic acid encoding the RNA guide are located on the same or different vectors;

Preferably, the DNA molecule is connected to a first regulatory element;

Preferably, the nucleic acid encoding said RNA guide is linked to a second regulatory element;

Preferably, the first regulatory element and the second regulatory element are independently selected from one or more of inducible promoters, constitutive promoters or tissue-specific promoters;

Preferably, the first regulatory element and the second regulatory element are independently selected from T7, SP6, T3, CMV, EF1a, SV40, PGK1, human β-actin, CAG, U6, H1, T7, T7lac, araBAD, One or more of trp, lac or Ptac.
A method for binding the gene editing system to a target nucleic acid in a cell according to any one of claims 11 to 24, characterized in that the method includes:

delivering the gene editing system to the cell, the cell including the target nucleic acid;

causing the CRISPR-Cas effector protein or the CRISPR-Cas effector fusion protein to bind to the RNA guide,

The spacer sequence is allowed to bind to the target nucleic acid.
The method of claim 27, wherein the target nucleic acid is double-stranded DNA or single-stranded DNA;

Preferably, the combination of the gene editing system with the target nucleic acid in the cell results in a change in the expression state of the target nucleic acid;

Preferably, the binding of the gene editing system to the target nucleic acid in the cell causes the target nucleic acid to be cleaved;

Preferably, the target nucleic acid is cleaved resulting in the destruction of the target nucleic acid, or the replacement of a specific site on the target nucleic acid, or the removal of the target nucleic acid site, or the change in the function of the target nucleic acid region, or the change between two sites on the target nucleic acid. Sequence inversion.
A cell containing a gene editing system, characterized in that the cell containing a gene editing system includes the gene editing system of any one of claims 11 to 24, or the gene editing vector of claims 25 or 26 .
The cell containing a gene editing system according to claim 29, wherein the cell containing a gene editing system contains a modified target target locus, and the target target locus is modified using the gene editing system. loci;

Preferably, said modification of said target locus of interest results in:

(1) The cells containing the gene editing system contain changes in the expression of at least one gene product; or

(2) the cell containing the gene editing system comprises an alteration in the expression of at least one gene product, wherein the expression of the at least one gene product is increased; or

(3) The cells containing the gene editing system comprise changes in the expression of at least one gene product, wherein the expression of the at least one gene product is reduced; or

(4) The cells containing the gene editing system contain the edited genome;

Preferably, the cells containing the gene editing system include eukaryotic cells or prokaryotic cells;

Preferably, the eukaryotic cells include animal cells, plant cells or human cells;

Preferably, the animal cells comprise mammalian cells.
A method of targeting and editing a target nucleic acid, characterized in that the method includes contacting the target nucleic acid with the gene editing system of any one of claims 11 to 24.
A method for non-specific degradation of single-stranded DNA after identifying a target nucleic acid, characterized in that the method includes contacting the target nucleic acid with the gene editing system of any one of claims 11 to 24.
A method for targeting and nicking the non-spacer complementary strand of a double-stranded target DNA after identifying the spacer complementary strand of the double-stranded target DNA, characterized in that the method includes making the double-stranded target DNA The DNA is contacted with the gene editing system of any one of claims 11 to 24.
A method of targeting and cutting double-stranded target DNA, characterized in that the method includes contacting the double-stranded target DNA with the gene editing system of any one of claims 11 to 24.
The method according to claim 34, characterized in that, before nicking the spacer complementary strand of the double-stranded DNA, the non-spacer complementary strand of the double-stranded target DNA is nicked.
A method for specifically editing double-stranded nucleic acids, characterized by:

The method includes contacting under sufficient conditions for a sufficient amount of time,

(1) The CRISPR-Cas effector protein of any one of claims 11 to 24, or the CRISPR-Cas effector fusion protein of claim 4 or 5, or another enzyme with sequence-specific nicking activity, as well as the an RNA guide that directs the CRISPR-Cas effector protein or the CRISPR-Cas effector fusion protein to nick an opposing strand relative to the activity of the other sequence-specific nickase; and

(2) the double-stranded nucleic acid;

The method results in the formation of double-strand breaks.
A method of editing double-stranded nucleic acids, characterized in that the method includes contacting the following under sufficient conditions for a sufficient amount of time:

(1) Fusion of the CRISPR-Cas effector protein according to any one of claims 11 to 24, or the CRISPR-Cas effector fusion protein according to claim 4 or 5, and a protein domain having DNA modification activity a protein, and the RNA guide targeting the double-stranded nucleic acid; and

(2) the double-stranded nucleic acid;

The CRISPR-Cas effector of the fusion protein is modified to nick the non-target strand of the double-stranded nucleic acid.
The method of claim 37, wherein the two strands of the double-stranded nucleic acid are cleaved at different sites, resulting in staggered cleavage.
The method of claim 37, wherein the two strands of the double-stranded nucleic acid are cleaved at the same site, resulting in a flat double-stranded break.
A method for targeting and cutting single-stranded target DNA, characterized in that the method includes contacting the target nucleic acid with the gene editing system of any one of claims 11 to 24.
A method for inducing changes in cell state, characterized in that the method includes contacting the gene editing system of any one of claims 11 to 24 with the target nucleic acid in the cell.
The method of claim 41, wherein the cell state includes apoptosis or dormancy;

Preferably, the cells include eukaryotic cells or prokaryotic cells;

Preferably, the cells include mammalian cells or plant disease cells;

Preferably, the cells include cancer cells;

Preferably, the cells include infectious cells or cells infected by an infectious agent;

Preferably, the cells include virus-infected cells and prion-infected cells;

Preferably, the cells comprise fungal, protozoan or parasitic cells.
Use of the gene editing system according to any one of claims 11 to 24 in the preparation of medicines for treating conditions or diseases in a subject.
The application according to claim 43, characterized in that the application comprises administering the gene editing system to the subject or the ex vivo cells of the subject;

Preferably, the spacer sequence is complementary to at least 15 nucleotides of the target nucleic acid associated with the disorder or disease, which is cleaved by the CRISPR-Cas effector protein or the CRISPR-Cas effector fusion protein. The target nucleic acid;

Preferably, the condition or disease includes cancer, infectious disease, metabolic disease or genetic disease;

Preferably, the cancer includes Wilms tumor, Ewing sarcoma, neuroendocrine tumor, glioblastoma, neuroblastoma, melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, Kidney cancer, pancreatic cancer, lung cancer, biliary tract cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, medullary thyroid cancer, ovarian cancer, glioma, lymphoma, leukemia, myeloma, acute lymphocyte One or more of leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or urinary bladder cancer;

Preferably, the infectious agent of the infectious disease includes one or more of human immunodeficiency virus, herpes simplex virus-1, hepatitis B or herpes simplex virus-2;

Preferably, the metabolic disease includes familial hypercholesterolemia;

Preferably, the genetic disease includes transthyretin amyloidosis, primary hyperoxaluric acidosis, and hereditary angioedema;

Preferably, the disorder or disease includes cystic fibrosis, progressive Duchenne muscular dystrophy, Baker muscular dystrophy, alpha-1-antitrypsin deficiency, Pompe disease, myotonic dystrophy, Huntington's disease, Fragile One or more of syncytosis or beta thalassemia.
A eukaryotic cell line, characterized in that the eukaryotic cell line contains the cell containing the gene editing system of claim 29 or 30, or is a progeny of the cell containing the gene editing system.
A multicellular organism, characterized in that the multicellular organism contains the cell containing the gene editing system of claim 29 or 30.
The multicellular organism according to claim 46, characterized in that the multicellular organism includes a model animal or a model plant.
A method for obtaining plants with target traits, characterized by using the gene editing system of any one of claims 11 to 24 to contact plant cells, modifying the genes of the plant cells or introducing target genes, so The modification or target gene can express the target trait, and a modified plant cell is obtained,

The modified plant cells are used for regeneration to obtain plants with the desired traits.
A method for identifying target traits in plants, characterized in that the target genes in plant cells are capable of expressing the target traits, and the gene editing system of any one of claims 11 to 24 is used to contact the plant cells, Thus, the target gene is identified.
A kit, characterized in that the kit includes one or more components selected from the following: the CRISPR-Cas effector protein according to any one of claims 1 to 3, claim 4 or 5 Described CRISPR-Cas effector fusion protein, the DNA molecule described in claim 6 or 7, the recombinant vector described in claim 8 or 9, the host cell described in claim 10, the method described in claims 11 to 24 Gene editing system, the gene editing vector according to claim 25 or 26, the cell containing the gene editing system according to claim 29 or 30, the eukaryotic cell line according to claim 45, the gene editing vector according to claim 46 or 47 multicellular organisms;

The components of the kit are in the same or different containers.
A container, characterized in that the container contains the kit according to claim 50.
The container of claim 51, wherein said container comprises a sterile container;

Preferably, the container includes a syringe.
An implantable device, characterized in that the implantable device includes the gene editing system according to any one of claims 11 to 24.
The implantable device of claim 53, wherein the gene editing system is within a matrix;

Preferably, the gene editing system is within a reservoir.
A method of treating a disease or condition in a subject in need thereof, characterized in that the method includes administering to the subject any one or more of the following:

The CRISPR-Cas effector protein of any one of claims 1 to 3, or a derivative of the CRISPR-Cas effector protein or a functional fragment of the CRISPR-Cas effector protein, and a heterologous functional domain , and an RNA guide or a nucleic acid encoding said RNA guide;

The CRISPR-Cas effector fusion protein of claim 4 or 5, and an RNA guide or a nucleic acid encoding the RNA guide;

The DNA molecule of claim 6 or 7;

The recombinant vector according to claim 8 or 9;

The gene editing system according to any one of claims 11 to 24;

The gene editing vector according to claim 25 or 26;

The cell containing the gene editing system according to claim 29 or 30;

The eukaryotic cell line of claim 45;

The multicellular organism of claim 46 or 47;

The kit according to claim 50;

A container as claimed in claim 51 or 52; or

The implantable device of claim 53 or 54.
The method of claim 55, wherein the subject includes a subject suffering from a disorder or disease, including cancer, infectious disease, metabolic disease, or genetic disease;

Preferably, the cancer includes Wilms tumor, Ewing sarcoma, neuroendocrine tumor, glioblastoma, neuroblastoma, melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, Kidney cancer, pancreatic cancer, lung cancer, biliary tract cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, medullary thyroid cancer, ovarian cancer, glioma, lymphoma, leukemia, myeloma, acute lymphocyte One or more of leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or urinary bladder cancer;

Preferably, the infectious agent of the infectious disease includes one or more of human immunodeficiency virus, herpes simplex virus-1 or herpes simplex virus-2;

Preferably, the metabolic disease includes familial hypercholesterolemia;

Preferably, the genetic disease includes transthyretin amyloidosis, primary hyperoxaluric acidosis, and hereditary angioedema;

Preferably, the disorder or disease includes cystic fibrosis, progressive Duchenne muscular dystrophy, Baker muscular dystrophy, alpha-1-antitrypsin deficiency, Pompe disease, myotonic dystrophy, Huntington's disease, Fragile One or more of syncytosis or beta thalassemia.
Use of the gene editing system of any one of claims 11 to 24 in the treatment of a condition or disease.
Use according to claim 57, characterized in that the condition or disease includes cancer, infectious diseases, metabolic diseases or genetic diseases;

The cancers include Wilms tumor, Ewing sarcoma, neuroendocrine tumors, glioblastoma, neuroblastoma, melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, kidney cancer, Pancreatic cancer, lung cancer, biliary tract cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, medullary thyroid cancer, ovarian cancer, glioma, lymphoma, leukemia, myeloma, acute lymphoblastic leukemia, acute One or more of myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or urinary bladder cancer;

Preferably, the infectious agent of the infectious disease includes one or more of human immunodeficiency virus, herpes simplex virus-1, hepatitis B or herpes simplex virus-2;

Preferably, the metabolic disease includes familial hypercholesterolemia;

Preferably, the genetic disease includes transthyretin amyloidosis, primary hyperoxaluric acidosis, and hereditary angioedema;

Preferably, the disorder or disease includes cystic fibrosis, progressive Duchenne muscular dystrophy, Baker muscular dystrophy, alpha-1-antitrypsin deficiency, Pompe disease, myotonic dystrophy, Huntington's disease, Fragile One or more of syncytosis or beta thalassemia.