CN114480383A

CN114480383A - Homodromous repetitive sequence with base mutation and application thereof

Info

Publication number: CN114480383A
Application number: CN202110635989.3A
Authority: CN
Inventors: 段志强
Original assignee: Shandong Shunfeng Biotechnology Co Ltd
Current assignee: Shandong Shunfeng Biotechnology Co Ltd
Priority date: 2021-06-08
Filing date: 2021-06-08
Publication date: 2022-05-13
Anticipated expiration: 2041-06-08
Also published as: CN116751780A; CN114480383B

Abstract

The present invention relates to the field of gene editing, in particular to the technical field of clustered regularly interspaced short palindromic repeats (CRISPR/Cas). Specifically, the present invention provides a base-mutated direct repeat sequence, and a gRNA comprising the base-mutated direct repeat sequence can improve the activity of a Cas enzyme.

Description

Homodromous repetitive sequence with base mutation and application thereof

Technical Field

The present invention relates to the field of gene editing, in particular to the technical field of clustered regularly interspaced short palindromic repeats (CRISPR/Cas). Specifically, the invention relates to a base mutation-containing direct repeat sequence and application thereof, and particularly relates to a gRNA (ribonucleic acid) containing the base mutation-containing direct repeat sequence and suitable for a Cas12 protein family and application thereof.

Background

The CRISPR/Cas system is an acquired immune system in bacteria, which is used against foreign DNA, plasmids and phages of the invading bacteria. The CRISPR/Cas system is used for gene editing, has the characteristics of accuracy, low price and easy use, and is widely applied to gene engineering.

CRISPR/Cas systems are divided into 3 classes, the most common being class 2 CRISPR/Cas systems, whose programmable single effector nucleases become convenient tools for gene editing and nucleic acid detection. The single-effect nuclease-Cas protein in the CRISPR/Cas system of the class 2 can cut nucleic acid under the guidance of guide RNA, and the cutting site is mainly positioned near PAM. The guide RNA is double-stranded RNA formed by base pairing of crRNA and tracrRNA, or a gRNA formed by combining crRNA and tracrRNA by artificial modification, and is required to form a stem-loop structure for the guide function. The stem-loop structure is the backbone region where the guide RNA binds to the Cas enzyme. The class 2 CRISPR/Cas system is divided into a type II, a type V and a type VI and is respectively based on a Cas9 protein, a Cas12 protein and a Cas13 protein, wherein the Cas12 protein of the type V CRISPR/Cas system has specific cis cleavage activity and non-specific trans cleavage activity, and the activity provides a new research idea for nucleic acid detection or clinical diagnosis.

In order to improve the activity of the Cas enzyme, the inventor of the application optimizes the stem-loop structure of the gRNA, particularly the direct repeat sequence of the gRNA, and obtains the gRNA which can improve the ratio of trans cleavage activity to cis cleavage activity of the Cas enzyme.

Disclosure of Invention

Through a large number of experiments and repeated investigations, the inventors of the present application found a gRNA that can improve Cas enzyme activity.

In one aspect, the present invention provides a direct repeat sequence having a base mutation relative to a parent direct repeat sequence, wherein the parent direct repeat sequence and the direct repeat sequence having a base mutation can form a stem-loop structure, the stem-loop structure can bind to a Cas protein, the parent direct repeat sequence and the direct repeat sequence having a base mutation comprise a first segment, a second segment, a third segment and a fourth segment which are sequentially connected from 5 'to 3', and the second segment and the fourth segment can form a stem region of the stem-loop structure through base pairing;

the stem region of the direct repeat sequence with the base mutation has at least 1 unpaired base pair relative to the stem region of the parent direct repeat sequence; preferably, the stem region of the direct repeat having the base mutation has 1 unpaired base pair relative to the stem region of the parent direct repeat.

In the present invention, the direct repeat sequence is an important part constituting a gRNA, and it is known in the art that a gRNA generally includes a direct repeat sequence (DR) and a guide sequence targeting a target nucleic acid; the direct repeat sequence, also referred to as the backbone region of the gRNA, can interact or bind with the Cas protein, thereby directing the Cas protein to the targeting region of the target nucleic acid.

In one embodiment, the second segment and the fourth segment of the parental direct repeat sequence and the direct repeat sequence with base mutation respectively comprise 4-10 bases; preferably, the second segment and the fourth segment each comprise 5 bases.

In one embodiment, the stem-loop structure formed by the direct repeat sequence can bind to a V-type Cas protein; preferably, the V-type Cas protein is selected from Cas proteins of the Cas12 family, e.g., Cas12a (Cpf1), Cas12b, Cas12g, Cas12i, Cas12j, and the like.

In one embodiment, any 1 base of the second and fourth segments of the direct repeat having a base mutation is mutated relative to the parent direct repeat, resulting in a stem region of the direct repeat having a base mutation that has 1 unpaired base pair relative to the stem region of the parent direct repeat.

In one embodiment, the second and fourth segments of the parental direct repeat sequence are, from 5 'to 3', gugg and CACAC, respectively; further, the sequences of the first segment and the third segment from the 5 'end to the 3' end of the parental homodromous sequence are AGAGAAU and CAUAGU respectively.

In a preferred embodiment, the direct repeat sequence having a base mutation has one or more mutations at the nucleotides corresponding to the following positions of SEQ ID No.1, relative to the parent direct repeat sequence: g8, U9, G10, U11, G12, C19, a20, C21, a22, C23; preferably, the direct repeat sequence having a base mutation has one or more base mutations at the following positions in the sequence corresponding to SEQ ID No.1, relative to the parental direct repeat sequence: C19G, a20U, C21G, a22U, C23G.

In a preferred embodiment, the direct repeat sequence having a base mutation comprises a sequence shown in any one of SEQ ID Nos. 2 to 6; preferably, the direct repeat sequence is a sequence shown in any one of SEQ ID No. 2-6.

In another aspect, the present invention provides a gRNA including the above direct repeat having a base mutation and a guide sequence (alternatively referred to as a guide sequence) that hybridizes to a target nucleic acid;

preferably, the gRNA includes the direct repeat sequence having a base mutation described above and a targeting sequence hybridizing to a target nucleic acid, which are sequentially connected from 5 'to 3'.

In another aspect, the present invention provides a nucleic acid encoding the above gRNA, or encoding a precursor of the above gRNA, or encoding the above direct repeat having a base mutation.

In another aspect, the present invention provides a composition or CRISPR system comprising the above gRNA and Cas protein.

In another aspect, the present invention provides an activated CRISPR complex comprising a gRNA as described above, a Cas protein, and a target nucleic acid bound to the gRNA as described above.

In another aspect, the present invention provides a vector comprising the gRNA or the nucleic acid described above.

In another aspect, the present invention provides a vector system comprising one or more vectors, the one or more vectors comprising:

(i) a first regulatory element operably linked to the gRNA,

(ii) a second regulatory element operably linked to the Cas protein;

wherein components (i) and (ii) are on the same or different carriers.

In another aspect, the invention provides an application of the gRNA described above in improving the activity of a Cas protein; preferably, the improving the activity of the Cas protein is increasing the ratio of trans cleavage activity to cis cleavage activity of the Cas protein.

It is known in the art that Cas proteins, particularly V-type Cas proteins (e.g., Cas12a, Cas12b, Cas12i, Cas12j) may exhibit cis cleavage activity and may also exhibit trans cleavage activity when subjected to gene editing.

In a preferred embodiment, the Cas protein is a V-type Cas protein; preferably, the V-type Cas protein is selected from Cas proteins of the Cas12 family, e.g., Cas12a (Cpf1), Cas12b, Cas12g, Cas12i, Cas12j, and the like.

In a specific embodiment, the Cas protein is Cas12 i; preferably, the Cas12i is selected from any one of the following groups:

(1) SEQ ID NO: 14, or a pharmaceutically acceptable salt thereof;

(2) converting SEQ ID NO: 14 or an active fragment thereof, by substitution, deletion or addition of one or more (e.g. 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid residues, and is substantially identical to SEQ ID NO: 14 proteins having substantially the same function;

(3) and SEQ ID NO: 14 has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 14 proteins having substantially the same function.

The above-mentioned substantially same function means the same function as that of SEQ ID NO: 14 have the same or similar gene editing activities, including cis and trans cleavage activities.

In the present invention, the ratio of the trans cleavage activity to the cis cleavage activity of the Cas protein is increased, in which the ratio of the trans cleavage activity to the cis cleavage activity exhibited by the Cas protein when the optimized gRNA performs gene editing with the Cas protein (trans cleavage activity/cis cleavage activity) is higher than the ratio of the trans cleavage activity to the cis cleavage activity exhibited by the Cas protein when the wild-type gRNA performs gene editing with the Cas protein (trans cleavage activity/cis cleavage activity), as compared with the gRNA including the parental homologous recombination sequence (wild-type gRNA).

In a specific embodiment, a gRNA of the invention having a base-mutated direct repeat, when used in combination with a Cas protein, has: a significant decrease in cis activity of the Cas protein of at least 60% (e.g., at least 70%, at least 80%, at least 90%); the trans activity of the Cas protein (particularly the trans activity exhibited when binding to a single-stranded target nucleic acid) can be retained by at least 60% (e.g., at least 70%, at least 80%, at least 90%).

The single-stranded nucleic acid is preferably single-stranded dna (ssdna), and the double-stranded nucleic acid is preferably double-stranded dna (dsdna).

In another aspect, the invention provides a method of improving Cas protein activity or increasing the ratio of trans cleavage activity to cis cleavage activity of a Cas protein, the method comprising gene editing or gene cleavage using a Cas protein and the above-described gRNA.

In another aspect, the invention provides the use of the gRNA, the nucleic acid, the composition or the CRISPR system, the activated CRISPR complex, the vector or the vector system for gene editing, gene targeting or gene cleavage;

the gene editing comprises modifying a gene, knocking out a gene, changing the expression of a gene product, repairing a mutation, and/or inserting a polynucleotide, a gene mutation;

the editing can be performed in prokaryotic cells and/or eukaryotic cells.

In another aspect, the present invention provides a method of editing, targeting, or cleaving a target nucleic acid, the method including the step of contacting the target nucleic acid with the gRNA, the nucleic acid, the composition or CRISPR system described above, the vector described above, or the vector system described above.

In another aspect, the invention provides the use of the gRNA, the nucleic acid, the composition or the CRISPR system, the activated CRISPR complex, the vector or the vector system for nucleic acid detection or diagnosis.

In another aspect, the invention provides the use of the gRNA, the nucleic acid, the composition or the CRISPR system, the activated CRISPR complex, the vector or the vector system for non-specific cleavage of single-stranded nucleic acids.

In another aspect, the invention provides a method of cleaving a single-stranded nucleic acid, the method comprising, contacting a nucleic acid population with a Cas protein and the above-described grnas, wherein the nucleic acid population comprises a target nucleic acid and a non-target single-stranded nucleic acid, the Cas protein cleaving the non-target single-stranded nucleic acid;

the contacting may be in vitro, ex vivo, or inside a cell in vivo;

preferably, the cleaved single-stranded nucleic acid is non-specific cleavage.

In another aspect, the present invention provides a kit for gene editing, the kit including the above gRNA and Cas protein.

In another aspect, the present invention provides a kit for detecting a target nucleic acid in a sample, the kit comprising: (i) the gRNA, or a nucleic acid encoding the gRNA, or a precursor RNA of the gRNA, or a nucleic acid encoding the precursor RNA; (ii) a Cas protein, or a nucleic acid encoding the Cas protein; and (iii) a single-stranded nucleic acid detector that is single-stranded and does not hybridize to the gRNA.

In another aspect, the present invention provides the use of the gRNA, the nucleic acid, the composition or the CRISPR system, the activated CRISPR complex, the vector or the vector system for the preparation of a formulation for use in any one or any of the following (i) - (v):

(i) gene or genome editing;

(ii) target nucleic acid detection and/or diagnosis;

(iii) editing a target sequence in a target locus to modify an organism or non-human organism;

(iv) treatment of diseases;

(v) targeting a target gene;

preferably, the gene or genome editing is gene or genome editing in a cell;

preferably, the target nucleic acid detection and/or diagnosis is in vitro;

preferably, the treatment of the disease is the treatment of a condition caused by a defect in the target sequence in the target locus.

In some embodiments, the disorder or disease is cancer or an infectious disease. For example, the cancer may be selected from Wilms 'tumor, Ewing's 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 lymphocytic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hodgkin's lymphoma, non-hodgkin's lymphoma, and bladder cancer.

In another aspect, the invention provides a method of detecting a target nucleic acid in a sample, the method comprising contacting the sample with a Cas protein, a gRNA as described above comprising a region that binds to the Cas protein and a guide sequence that hybridizes to the target nucleic acid, and a single-stranded nucleic acid detector; detecting a detectable signal generated by the Cas protein-cleaved single-stranded nucleic acid detector, thereby detecting a target nucleic acid; the single-stranded nucleic acid detector does not hybridize to the gRNA;

in the present invention, the detectable signal is realized by: vision-based detection, fluorescence signal-based detection, sensor-based detection, color detection, gold nanoparticle-based detection, fluorescence polarization or fluorescence signal, colloidal phase transition/dispersion, electrochemical detection, and semiconductor-based detection.

In some embodiments, the methods of the invention further comprise the step of measuring a detectable signal produced by the CRISPR/CAS effector protein (CAS protein). The Cas protein, upon recognition or hybridization to the target nucleic acid, can activate the cleavage activity of single-stranded nucleic acids (trans cleavage activity), thereby cleaving the single-stranded nucleic acid detector and thereby generating a detectable signal.

In the present invention, the detectable signal may be any signal generated when the single-stranded nucleic acid detector is cleaved. For example, detection based on gold nanoparticles, fluorescence polarization, colloidal phase transition/dispersion, electrochemical detection, semiconductor-based sensing. The detectable signal may be read by any suitable means, including but not limited to: measurement of a detectable fluorescent signal, gel electrophoresis detection (by detecting a change in a band on the gel), detection of the presence or absence of a color based on vision or a sensor, or a difference in the presence of a color (e.g., based on gold nanoparticles) and a difference in an electrical signal.

In a preferred embodiment, the detectable signal is achieved by: the 5 'end and the 3' end of the single-stranded nucleic acid detector are respectively provided with different reporter groups, and when the single-stranded nucleic acid detector is cut, a detectable reporter signal can be shown; for example, a single-stranded nucleic acid detector having a fluorophore and a quencher disposed at opposite ends thereof, when cleaved, can exhibit a detectable fluorescent signal.

In one embodiment, the fluorescent group is selected from one or any of FAM, FITC, VIC, JOE, TET, CY3, CY5, ROX, Texas Red or LC Red 460; the quenching group is selected from one or more of BHQ1, BHQ2, BHQ3, Dabcy1 or Tamra.

In other embodiments, the detectable signal may also be achieved by: the 5 'end and the 3' end of the single-stranded nucleic acid detector are respectively provided with different marker molecules, and a reaction signal is detected in a colloidal gold detection mode.

In one embodiment, the target nucleic acid comprises DNA, RNA, preferably single-stranded nucleic acid or double-stranded nucleic acid or nucleic acid modification.

In one embodiment, the target nucleic acid is derived from a sample of a virus, bacterium, microorganism, soil, water source, human, animal, plant, or the like. Preferably, the target nucleic acid is a product enriched or amplified by PCR, NASBA, RPA, SDA, LAMP, HAD, NEAR, MDA, RCA, LCR, RAM and the like.

In one embodiment, the method further comprises the step of obtaining the target nucleic acid from the sample.

In one embodiment, the target nucleic acid is a viral nucleic acid, a bacterial nucleic acid, a specific nucleic acid associated with a disease, such as a specific mutation site or SNP site or a nucleic acid that is different from a control; preferably, the virus is a plant virus or an animal virus, e.g., papilloma virus, hepatic DNA virus, herpes virus, adenovirus, poxvirus, parvovirus, coronavirus; preferably, the virus is a coronavirus, preferably SARS, SARS-CoV2(COVID-19), HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, Mers-CoV.

In some embodiments, the target nucleic acid is derived from a cell, e.g., from a cell lysate.

In some embodiments, the measurement of the detectable signal may be quantitative, and in other embodiments, the measurement of the detectable signal may be qualitative.

Preferably, the single stranded nucleic acid detector produces a first detectable signal prior to cleavage by the Cas protein and produces a second detectable signal different from the first detectable signal after cleavage.

In other embodiments, the single-stranded nucleic acid detector comprises one or more modifications, such as base modifications, backbone modifications, sugar modifications, and the like, to provide new or enhanced features (e.g., improved stability) to the nucleic acid. Examples of suitable modifications include modified nucleic acid backbones and non-natural internucleoside linkages, and nucleic acids having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. Suitable modified oligonucleotide backbones containing phosphorus atoms therein include phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates. In some embodiments, the single stranded nucleic acid detector comprises one or more phosphorothioate and/or heteroatomic nucleotide linkages. In other embodiments, the single stranded nucleic acid detector can be a nucleic acid mimetic; in certain embodiments, the nucleic acid mimetics are Peptide Nucleic Acids (PNAs), another class of nucleic acid mimetics is based on linked morpholino units having a heterocyclic base attached to a morpholino ring (morpholino nucleic acids), and other nucleic acid mimetics further include cyclohexenyl nucleic acids (CENAs), further including ribose or deoxyribose chains.

The cells of the invention are selected from the group consisting of: prokaryotic or eukaryotic cells, for example, archaeal cells, bacterial cells, eukaryotic unicellular organisms, somatic cells, germ cells, stem cells, plant cells, algal cells, animal cells, invertebrate cells, vertebrate cells, fish cells, frog cells, bird cells, mammalian cells, pig cells, cow cells, goat cells, sheep cells, rodent cells, rat cells, mouse cells, non-human primate cells, and human cells.

In some cases, the gene or genome may be modified by introduction of the gene editing methods of the invention into a cell, such that the cell and its progeny are altered. Preferably, the cell or progeny thereof obtained by the method described above, wherein said cell contains a modification not present in its wild type.

In one embodiment, the cell is a prokaryotic cell.

In one embodiment, the cell is a eukaryotic cell.

In one embodiment, the cell is a mammalian cell.

In one embodiment, the cell is a human cell. In certain embodiments, the cell is a non-human mammalian cell, e.g., a non-human primate, bovine, ovine, porcine, canine, monkey, rabbit, rodent (e.g., rat or mouse) cell.

In one embodiment, the cell is a non-mammalian eukaryotic cell, such as a cell of a poultry bird (e.g., chicken), fish, or crustacean (e.g., clam, shrimp).

In one embodiment, the cell is a plant cell; preferably, the plant is a monocotyledon or dicotyledon; including but not limited to arabidopsis, tobacco, rice, corn, sorghum, barley, wheat, millet, soybean, tomato, potato, quinoa, lettuce, canola, cabbage, strawberry.

In one embodiment, the cell is a stem cell or stem cell line.

General definition:

in the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, the procedures of molecular genetics, nucleic acid chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA, etc., used herein, are all conventional procedures widely used in the corresponding field. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

Cas protein

In the present invention, the expression "Cas protein" refers to a CRISPR protein, the terms "CRISPR/Cas protein", "Cas effector protein", "Cas protein", "single-acting nuclease" are used interchangeably.

The Cas protein, once bound to the signature sequence (target sequence) to be detected (i.e., forming a ternary complex of Cas protein-gRNA-target sequence), can induce its trans cleavage activity, i.e., random cleavage of non-targeted single-stranded nucleotides (i.e., single-stranded nucleic acid detector described herein, preferably single-stranded DNA (ssdna), single-stranded DNA-RNA hybrids, single-stranded RNA). When the Cas protein is combined with the characteristic sequence, the protein can cut or not cut the characteristic sequence to induce the trans cutting activity; preferably, it induces its trans cleavage activity by cleaving the signature sequence; more preferably, it induces its trans activity by cleaving the single-stranded signature sequence. The Cas protein recognizes the characteristic sequence by recognizing PAM (protospacer adjacenttoment motif) adjacent to the characteristic sequence.

The Cas protein of the present invention is a protein having at least trans cleavage activity, preferably, the Cas protein is a protein having cis cleavage activity and trans cleavage activity. The cis cleavage activity refers to the activity that the Cas protein can recognize a PAM site and specifically cleave a target sequence under the action of the gRNA.

The Cas protein is selected from any one or any combination of V-type Cas family proteins; preferably, the Cas family protein is selected from one or any combination of Cas12i, Cas12j, Cas12a, Cas12b, Cas12d, Cas12e, Cas12f, Cas12g and Cas12 h; more preferably, the Cas protein is one or a combination of any two of Cas12i, Cas12j, Cas12a and Cas12 b. More preferably, the Cas protein is Cas12i or Cas12 j.

CRISPR/Cas system

As used herein, the terms "clustered regularly interspaced short palindromic repeats (CRISPR/Cas)", "CRISPR system", "CRISPR/Cas" are used interchangeably and have the meaning commonly understood by those skilled in the art, which typically comprise a transcript or other element that is associated with the expression of a CRISPR-associated ("Cas") gene, or a transcript or other element that is capable of directing the activity of said Cas gene.

Guide RNA (guide RNA, gRNA)

As used herein, the terms "guide RNA", "mature crRNA", "guide sequence" are used interchangeably and have the meaning commonly understood by those skilled in the art. The gRNA may be a double-stranded RNA formed by base pairing of crRNA and tracrRNA, or a gRNA formed by combining crRNA and tracrRNA into one gRNA through artificial modification.

In general, the guide RNA may comprise, consist essentially of, or consist of a Direct Repeat (DR) and a guide sequence (also referred to as a spacer (spacer) in the context of an endogenous CRISPR system).

The direct repeat sequence is also called "framework region", "protein binding segment", "protein binding sequence"; the guide sequence may also be referred to as a "targeting sequence for targeting a nucleic acid" or a "targeting segment of a targeting nucleic acid.

The targeting sequence of the targeting nucleic acid or the targeting segment of the targeting nucleic acid comprises a nucleotide sequence that is complementary to a sequence in the target nucleic acid. In other words, the targeting sequence of the targeting nucleic acid or the targeting segment of the targeting nucleic acid interacts with the target nucleic acid in a sequence-specific manner through hybridization (i.e., base pairing). Thus, the targeting sequence of the targeting nucleic acid or the targeting segment of the targeting nucleic acid may be altered or modified to hybridize to any desired sequence within the target nucleic acid. The nucleic acid is selected from DNA or RNA.

The percent complementarity between the targeting sequence of the targeting nucleic acid or the targeting segment of the targeting nucleic acid and the target sequence of the target nucleic acid can be at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%).

The "framework region," "protein-binding segment," "protein-binding sequence," or "direct repeat" of a gRNA can interact with a CRISPR protein (or, Cas protein). In the present invention, the direct repeat sequence may form a stem-loop structure, the stem-loop structure is capable of binding with the Cas protein, and the parent direct repeat sequence and the direct repeat sequence with base mutation comprise a first segment, a second segment, a third segment, and a fourth segment connected in sequence from 5 'to 3', and the second segment and the fourth segment may form a stem region of the stem-loop structure through base pairing. The gRNA, through the action of the targeting sequence of the targeting nucleic acid, directs its interacting Cas protein to a specific nucleotide sequence within the target nucleic acid.

Preferably, the guide RNA comprises in the 5 'to 3' direction a direct repeat (direct repeat) and a guide sequence.

In the present invention, the targeting sequence of the targeting nucleic acid or the targeting segment of the targeting nucleic acid is preferably a targeting sequence of the targeting DNA or a targeting segment of the targeting DNA.

In certain instances, the guide sequence is any polynucleotide sequence that is sufficiently complementary to the target sequence to hybridize to the target sequence and direct specific binding of the CRISPR/Cas complex to the target sequence. In one embodiment, the degree of complementarity between a guide sequence and its corresponding target sequence is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%, when optimally aligned. Determining the optimal alignment is within the ability of one of ordinary skill in the art. For example, there are published and commercially available alignment algorithms and programs such as, but not limited to, ClustalW, the Smith-Waterman algorithm in matlab (Smith-Waterman), Bowtie, Geneius, Biopython, and SeqMan.

In the context of forming a CRISPR/Cas complex, a "target sequence" refers to a polynucleotide targeted by a guide sequence that is designed to be targeted, e.g., a sequence that is complementary to the guide sequence, wherein hybridization between the target sequence and the guide sequence will promote formation of the CRISPR/Cas complex. Complete complementarity is not necessary as long as there is sufficient complementarity to cause hybridization and promote formation of a CRISPR/Cas complex. The target sequence may comprise any polynucleotide, such as DNA or RNA. In some cases, the target sequence is located intracellularly or extracellularly. In some cases, the target sequence is located in the nucleus or cytoplasm of the cell. In some cases, the target sequence may be located within an organelle of the eukaryotic cell, such as a mitochondrion or chloroplast. Sequences or templates that can be used for recombination into a target locus containing the target sequence are referred to as "editing templates" or "editing polynucleotides" or "editing sequences". In one embodiment, the editing template is an exogenous nucleic acid. In one embodiment, the recombination is homologous recombination.

Single-stranded nucleic acid detector

The single-stranded nucleic acid detector of the present invention refers to a sequence containing 2 to 200 nucleotides, preferably, 2 to 150 nucleotides, preferably, 3 to 100 nucleotides, preferably, 3 to 30 nucleotides, preferably, 4 to 20 nucleotides, and more preferably, 5 to 15 nucleotides. Preferably a single-stranded DNA molecule, a single-stranded RNA molecule or a single-stranded DNA-RNA hybrid.

The single-stranded nucleic acid detector comprises different reporter groups or marker molecules at both ends, and does not present a reporter signal when in an initial state (i.e., an uncleaved state), and presents a detectable signal when the single-stranded nucleic acid detector is cleaved, i.e., presents a detectable difference after cleavage from before cleavage. In the present invention, if a detectable difference can be detected, it is reflected that the target nucleic acid contains a characteristic sequence to be detected; alternatively, if the detectable difference is not detectable, it indicates that the target nucleic acid does not contain the signature sequence to be detected.

Parent or wild type

As used herein, the term "parent" or "wild-type" has the meaning commonly understood by those skilled in the art to mean a typical form of an organism, strain, gene, or characteristic that, when it exists in nature, is distinguished from a mutant or variant form, which may be isolated from a source in nature and which has not been intentionally modified by man.

Carrier

The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it is linked. Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules comprising one or more free ends, free ends (e.g., circular); nucleic acid molecules comprising DNA, RNA, or both; and other various polynucleotides known in the art. The vector may be introduced into a host cell by transformation, transduction, or transfection, and the genetic material elements carried thereby are expressed in the host cell. A vector can be introduced into a host cell to thereby produce a transcript, protein, or peptide, including from a protein, fusion protein, isolated nucleic acid molecule, etc. (e.g., a CRISPR transcript, such as a nucleic acid transcript, protein, or enzyme) as described herein. A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may contain a replication initiation site.

One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be inserted, for example, by standard molecular cloning techniques.

Another type of vector is a viral vector, in which the virus-derived DNA or RNA sequences are present in a vector for packaging of viruses (e.g., retroviruses, replication-defective retroviruses, adenoviruses, replication-defective adenoviruses, and adeno-associated viruses). Viral vectors also comprise polynucleotides carried by viruses for transfection into a host cell. Certain vectors (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors) are capable of autonomous replication in a host cell into which they are introduced.

Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "expression vectors".

Host cell

As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, and includes, but is not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, eukaryotic cells such as microbial cells, fungal cells, animal cells, and plant cells.

One skilled in the art will appreciate that the design of an expression vector may depend on factors such as the choice of host cell to be transformed, the level of expression desired, and the like.

Regulatory element

As used herein, the term "regulatory element" is intended to include promoters, enhancers, Internal Ribosome Entry Sites (IRES), and other expression control elements (e.g., transcription termination signals such as polyadenylation signals and poly-U sequences), which are described in detail with reference to gordel (Goeddel), "gene expression technology: METHODS IN ENZYMOLOGY (GENE EXPRESSION TECHNOLOGY: METHOD IN ENZYMOLOGY)185, Academic Press, San Diego, Calif. (1990). In some cases, regulatory elements include those sequences that direct constitutive expression of a nucleotide sequence in many types of host cells as well as those sequences that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters may primarily direct expression in a desired tissue of interest, such as muscle, neuron, bone, skin, blood, a particular organ (e.g., liver, pancreas), or a particular cell type (e.g., lymphocyte). In certain instances, the regulatory element may also direct expression in a time-dependent manner (e.g., in a cell cycle-dependent or developmental stage-dependent manner), which may or may not be tissue or cell type specific. In certain instances, the term "regulatory element" encompasses enhancer elements, such as WPRE; a CMV enhancer; the R-U5' fragment in the LTR of HTLV-I ((mol. cell. biol., Vol.8 (1), pp.466-472, 1988); the SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit β -globin (Proc. Natl. Acad. Sci. USA., Vol.78 (3), pp.1527-31, 1981).

Promoters

As used herein, the term "promoter" has a meaning well known to those skilled in the art and refers to a non-coding nucleotide sequence located upstream of a gene that promotes expression of a downstream gene. Constitutive (constitutive) promoters are nucleotide sequences that: when operably linked to a polynucleotide that encodes or defines a gene product, it results in the production of the gene product in the cell under most or all physiological conditions of the cell. An inducible promoter is a nucleotide sequence that, when operably linked to a polynucleotide that encodes or defines a gene product, causes the gene product to be produced intracellularly substantially only when an inducer corresponding to the promoter is present in the cell. A tissue-specific promoter is a nucleotide sequence that: when operably linked to a polynucleotide that encodes or defines a gene product, it results in the production of the gene product in the cell substantially only if the cell is of the tissue type to which the promoter corresponds.

Is operably connected to

As used herein, the term "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the one or more regulatory elements in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).

Complementarity

As used herein, the term "complementarity" refers to the ability of a nucleic acid to form one or more hydrogen bonds with another nucleic acid sequence by means of a conventional watson-crick or other unconventional type. Percent complementarity refers to the percentage of residues (e.g., 5, 6, 7, 8, 9, 10 out of 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementary) in a nucleic acid molecule that can form hydrogen bonds (e.g., watson-crick base pairing) with a second nucleic acid sequence. "completely complementary" means that all consecutive residues of one nucleic acid sequence hydrogen bond with the same number of consecutive residues in a second nucleic acid sequence. As used herein, "substantially complementary" refers to a degree of complementarity of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more nucleotides, or to two nucleic acids that hybridize under stringent conditions.

Stringent conditions

As used herein, "stringent conditions" for hybridization refer to conditions under which a nucleic acid having complementarity to a target sequence predominantly hybridizes to the target sequence and does not substantially hybridize to non-target sequences. Stringent conditions are generally sequence dependent and vary depending on a number of factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence. Non-limiting examples of stringent conditions are described In Thyson (Tijssen) (1993) Laboratory technology-Nucleic Acid Probe Hybridization In biochemistry and Molecular Biology, section I, chapter II, "brief summary of Hybridization principles and Nucleic Acid Probe analysis strategy" ("Overview of principles of Hybridization and Hybridization of Nucleic Acid Probe assay"), Emericella (Elsevier), New York.

Hybridization of

As used herein, the term "hybridization" refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding of bases between the nucleotide residues. Hydrogen bonding can occur by means of watson-crick base pairing, Hoogstein binding, or in any other sequence specific manner. The complex may comprise two strands forming a duplex, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these. The hybridization reaction may constitute a step in a broader process, such as the initiation of PCR, or the cleavage of a polynucleotide by an enzyme. Sequences that are capable of hybridizing to a given sequence are referred to as "complements" of the given sequence.

Expression of

As used herein, the term "expression" refers to the process by which a polynucleotide is transcribed from a DNA template (e.g., into mRNA or other RNA transcript) and/or the process by which transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. The transcripts and encoded polypeptides may be collectively referred to as "gene products". If the polynucleotide is derived from genomic DNA, expression may include splicing of mRNA in eukaryotic cells.

Joint

As used herein, the term "linker" refers to a linear polypeptide formed from a plurality of amino acid residues joined by peptide bonds. The linker of the present invention may be an artificially synthesized amino acid sequence, or a naturally occurring polypeptide sequence, such as a polypeptide having a hinge region function. Such linker polypeptides are well known in the art (see, e.g., Holliger, P. et al (1993) Proc. Natl. Acad. Sci. USA 90: 6444-.

Animal(s) production

For example, a mammal, such as a bovine, equine, ovine, porcine, canine, feline, lagomorph, rodent (e.g., mouse or rat), non-human primate (e.g., macaque or cynomolgus monkey), or human. In certain embodiments, the subject (e.g., human) has a disorder (e.g., a disorder resulting from a deficiency in a disease-associated gene).

Plant and method for producing the same

The term "plant" is to be understood as including any differentiated multicellular organism capable of photosynthesis, in including crop plants at any stage of maturity or development, in particular monocotyledonous or dicotyledonous plants, vegetable crops, including artichokes, corm cabbages, sesames, leeks, asparagus, lettuce (e.g. head lettuce, leaf lettuce), bok choy, yellow croaker, melons (e.g. melons, watermelons, crow's melon, honeydew melon, cantaloupe), rape crops (e.g. brussels sprouts, cabbage, cauliflower, broccoli, collards, headless cabbages, chinese cabbages, cephalanoplos, carrots, cabbage (napa), okra, onions, celery, chickpea, parsnip, endive, potato, cucurbits (e.g. zucchini, cucurbits, etc, Squash, pumpkin), radish, dried onion, turnip cabbage, purple eggplant (also called eggplant), salsify, endive, shallot, endive, garlic, spinach, green onion, squash, leafy vegetables (greens), beets (sugar and feed beets), sweet potato, lettuce, horseradish, tomato, turnip, and spices; fruit and/or vine crops such as apple, apricot, cherry, nectarine, peach, pear, plum, prune, cherry, quince, almond, chestnut, hazelnut, pecan, pistachio, walnut, citrus, blueberry, boysenberry (boysenberry), raspberry, gooseberry, loganberry, raspberry, strawberry, blackberry, grape, avocado, banana, kiwi, persimmon, pomegranate, pineapple, tropical fruit, pome, melon, mango, papaya, and lychee; field crops, such as clover, alfalfa, evening primrose, meadowfoam, corn/maize (fodder corn, sweet corn, popcorn), hops, jojoba, peanuts, rice, safflower, small grain crops (barley, oats, rye, wheat, etc.), sorghum, tobacco, kapok, legumes (beans, lentils, peas, soybeans), oleaginous plants (oilseed rape, mustard, poppy, olives, sunflowers, coconut, castor oil plants, cocoa beans, groundnuts), arabidopsis, fibrous plants (cotton, flax, hemp, jute), lauraceae (cinnamon, camphor), or a plant such as coffee, sugar cane, tea, and natural rubber plants; and/or bedding plants, such as flowering plants, cactus, fleshy plants and/or ornamental plants, and trees, such as forests (broad leaf and evergreen trees, such as conifers), fruit trees, ornamental trees, and nut-bearing trees, as well as shrubs and other plantlets.

Advantageous effects of the invention

Compared with the prior art, the gRNA containing the base mutation direct repeat sequence can improve the activity of the Cas enzyme.

Embodiments of the present invention will be described in detail below with reference to the drawings and examples, but those skilled in the art will understand that the following drawings and examples are only for illustrating the present invention and do not limit the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of the preferred embodiments.

Sequence information

The partial sequence information related to the present invention is provided as follows:

drawings

Fig. 1 stem-loop structure formed by wild-type DR of gRNA of Cas12 i.

Figure 2 agarose gel electrophoresis, using different grnas, detects cis cleavage activity of Cas12 i.

Figure 3 fluorescent assay, using different grnas, detects trans cleavage activity of Cas12 i.

Detailed description of the preferred embodiments

The invention will now be described with reference to the following examples, which are intended to illustrate the invention, but not to limit it.

Unless otherwise indicated, the experiments and procedures described in the examples were performed essentially according to conventional methods well known in the art and described in various references. For example, conventional techniques in immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA used in the present invention can be found in Sambrook (Sambrook), friesch (Fritsch), and manitis (manitis), molecular cloning: a laboratory Manual (Molecula clone: Alborory Manual), 2 nd edition (1989); a Current Manual of MOLECULAR BIOLOGY experiments (Current PROTOCOLS IN MOLECULAR BIOLOGY BIOLOGY) (edited by F.M. Otsubel et al, (1987)); METHODS IN ENZYMOLOGY (METHODS IN Enzymology) series (academic Press): PCR 2: practical methods (PCR 2: APRACTICAL APPROACH) (m.j. macpherson), b.d. sames (b.d. hames) and g.r. taylor (g.r. taylor) editions (1995)), Harlow (Harlow) and la nei (Lane) editions (1988): antibodies: a laboratory Manual (ANTIBODIES, ALABORATORY MANUAL), and animal cell CULTURE (ANIMAL CELL CURTURE) (edited by R.I. Freusch (R.I. Freshney) (1987)).

In addition, those whose specific conditions are not specified in the examples are conducted under the conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available. The examples are given by way of illustration and are not intended to limit the scope of the invention as claimed. All publications and other references mentioned herein are incorporated by reference in their entirety.

Example 1 optimization of gRNA for Cas12 protein

In this example, in order to improve the activity of Cas12i (the amino acid sequence is shown in SEQ ID No. 14), base mutation is performed on the direct repeat sequence of its gRNA, the direct repeat sequence of the different grnas after mutation is shown in SEQ ID nos. 2-11, and the sites of the mutation of the different direct repeat sequences are shown in the following table:

TABLE 1 direct repeat sequences of different types of gRNAs

The wild-type direct repeat (DR-WT) can form a stem-loop structure as shown in FIG. 1, whose sequence from 5 'to 3' end is AGAGAAUGUGUGCAUAGUCACAC; the device sequentially comprises a first section AGAGAAU, a second section GUGUG, a third section CAUAGU and a fourth section CACACAC; wherein the second segment and the fourth segment may form a stem region of a stem-loop structure. DR-14, DR-15, DR-16, DR-17, and DR-18 have 1 base mutation at different bases of the fourth segment, respectively, compared with DR-WT, resulting in 1 mismatched base pair in the stem region portion of the stem-loop structure formed by them.

Example 2 agarose gel electrophoresis method to detect cis cleavage activity of Cas12i under the action of different gRNAs

1. Design of target sequences

Amplifying a sequence shown as SEQ ID No.12 by PCR to obtain a PCR double-chain product; and selecting a targeting sequence of gRNA on the PCR product as follows: AUGCAGAGUUCACUUUUG, respectively; the gRNA containing DR area shown in SEQ ID No.1-11 is adopted, the homodromous repetitive sequence is connected with the target sequence to obtain different gRNAs, and the homodromous repetitive sequence is arranged at the 5' end of the target sequence.

Cis cleavage Activity detection

Samples were loaded as follows except that different grnas comprising DR regions as shown in SEQ ID nos. 1 to 11 were added to the samples, respectively. After the completion of the sample addition, the reaction was carried out at 37 ℃ for 30min, and then the protein was inactivated at 80 ℃ for 20 min.

After completion of the reaction, gels were run on a 2% PAGE gel and the results are shown in FIG. 2:

the top band is the PCR product which is not cut by the Cas protein, the total length is 613bp, the middle band and the bottom band are the products of the PCR product which is cut by the Cas protein, and the middle band and the bottom band are respectively about 400bp and about 210bp, which are in line with the expectation of the designed target sequence. The rightmost lane is the PCR product control, only the top band, and very dark, no other miscellaneous bands, can prove the middle band and the lowest band is PCR product cutting and formation.

Comparing different lanes corresponding to different gRNAs, after the same time of cleavage reaction, PCR products of DR-WT (i 3-WT in the figure) and DR-7, DR-8, DR-9, DR-10 and DR-11 all have three bands, but only one obvious bright band is existed in DR-14, DR-15, DR-16, DR-17 and DR-18, namely, full-length PCR products, and the cleavage product band is very weak. That is, the cis-cleavage activity of Cas12i did not change much for grnas comprising mutations DR-7, DR-8, DR-9, DR-10, DR-11 when used with Cas12i for gene editing compared to wild-type grnas; gRNAs comprising mutated DR-14, DR-15, DR-16, DR-17, DR-18 have significantly reduced cis cleavage activity of Cas12i when used with Cas12i for gene editing.

Example 3 fluorescence assay to detect trans cleavage activity of Cas12i by different gRNAs

This embodiment employs the following protocol to detect changes in trans cleavage activity of Cas12i under the action of different grnas. The present embodiment is based on the principle that the gRNA is used to guide Cas12i protein to recognize and bind to a target nucleic acid; subsequently, the Cas12i protein activates trans cleavage activity, a single-stranded nucleic acid detector in the cleavage system; the two ends of the single-stranded nucleic acid detector are respectively provided with a fluorescent group and a quenching group, if the single-stranded nucleic acid detector is cut, fluorescence is excited, and the trans cutting activity of the Cas12i is reflected by a fluorescence value.

1. Design of target sequences

The targeting sequence of gRNA is: AUGCAGAGUUCACUUUUG are provided. The gRNA containing DR area shown in SEQ ID No.1-11 is adopted, the homodromous repetitive sequence is connected with the target sequence to obtain different gRNAs, and the homodromous repetitive sequence is arranged at the 5' end of the target sequence.

Trans cleavage Activity detection

Samples were loaded as follows except that different gRNAs containing DR regions as shown in SEQ ID NOS: 1-11 were added to the samples, respectively. After the completion of the sample addition, the reaction was carried out at 37 ℃ for 30min, and fluorescence was taken every one minute using a fluorescent PCR instrument.

In this embodiment, dsDNA (shown in SEQ ID No. 12) and ssDNA (shown in SEQ ID No. 13) are used to detect trans cleavage activity against a double-stranded target nucleic acid (dsDNA) and a single-stranded target nucleic acid (ssDNA) of different grnas, respectively.

The Reporter is a single-stranded nucleic acid detector.

The results are shown in FIG. 3: each gRNA corresponds to three fluorescent strips, NTC is the fluorescence value of the blank, the fluorescence value of ssDNA represents the trans activity of Cas protein with ssDNA as target nucleic acid, and the fluorescence value of dsDNA represents the trans activity of Cas protein with dsDNA as target nucleic acid.

Comparing fluorescence values corresponding to different gRNAs, fluorescence values of dsDNA corresponding to DR-WT (WT in the drawing) and DR-7, DR-8, DR-9, DR-10 and DR-11 are not obviously different from fluorescence values of ssDNA; however, the fluorescence values of dsDNA of DR-14, DR-15, DR-16, DR-17, DR-18 decreased significantly compared to DR-WT (WT in the figure), but the fluorescence values of ssDNA remained or decreased slightly.

In connection with examples 2-3, it was shown that grnas comprising mutations DR-7, DR-8, DR-9, DR-10, DR-11 did not significantly change the cis and trans cleavage activity of Cas12i when used with Cas12i for gene editing, compared to wild-type grnas.

gRNAs comprising mutated DR-14, DR-15, DR-16, DR-17, DR-18, when used with Cas12i for gene editing, significantly reduced cis cleavage activity of Cas12i (FIG. 2); however, the trans activity can be largely retained, at least the trans activity is reduced to a much lower extent than the cis activity, especially at least 60% when ssDNA is used as the target nucleic acid. To some extent, when the gRNA containing the mutated DR-14, DR-15, DR-16, DR-17, DR-18 is used together with Cas12i for gene editing, the ratio of trans cleavage activity to cis cleavage activity of Cas12i (trans cleavage activity/cis cleavage activity) is significantly increased, which can expand the application space of the Cas protein, especially for the application scenario when the cis activity of the Cas protein needs to be inhibited while the trans activity thereof is retained.

While specific embodiments of the invention have been described in detail, those skilled in the art will understand that: various modifications and changes in detail can be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. A full appreciation of the invention is gained by taking the entire specification as a whole in the light of the appended claims and any equivalents thereof.

SEQUENCE LISTING

<110> Shunheng Biotech Co., Ltd

<120> direct repeat sequence having base mutation and use thereof

<130> 20210527

<160> 14

<170> PatentIn version 3.5

<210> 1

<211> 23

<212> RNA

<213> Artificial Sequence

<220>

<223> DR-WT

<400> 1

agagaaugug ugcauaguca cac 23

<210> 2

<211> 23

<212> RNA

<213> Artificial Sequence

<220>

<223> DR-14

<400> 2

agagaaugug ugcauaguca cag 23

<210> 3

<211> 23

<212> RNA

<213> Artificial Sequence

<220>

<223> DR-15

<400> 3

agagaaugug ugcauaguca cuc 23

<210> 4

<211> 23

<212> RNA

<213> Artificial Sequence

<220>

<223> DR-16

<400> 4

agagaaugug ugcauaguca gac 23

<210> 5

<211> 23

<212> RNA

<213> Artificial Sequence

<220>

<223> DR-17

<400> 5

agagaaugug ugcauagucu cac 23

<210> 6

<211> 23

<212> RNA

<213> Artificial Sequence

<220>

<223> DR-18

<400> 6

agagaaugug ugcauaguga cac 23

<210> 7

<211> 23

<212> RNA

<213> Artificial Sequence

<220>

<223> DR-7

<400> 7

agagaaucug ugcauaguca cag 23

<210> 8

<211> 23

<212> RNA

<213> Artificial Sequence

<220>

<223> DR-8

<400> 8

agagaaugcg ugcauaguca cgc 23

<210> 9

<211> 23

<212> RNA

<213> Artificial Sequence

<220>

<223> DR-9

<400> 9

agagaaucug cgcauagucg cag 23

<210> 10

<211> 23

<212> RNA

<213> Artificial Sequence

<220>

<223> DR-10

<400> 10

agagaaugcg cgcauagucg cgc 23

<210> 11

<211> 23

<212> RNA

<213> Artificial Sequence

<220>

<223> DR-11

<400> 11

agagaauccg cgcauagucg cgg 23

<210> 12

<211> 613

<212> DNA

<213> Artificial Sequence

<220>

<223> double-stranded product

<400> 12

gctgtgttct taactcgcac agcacagctg agaccactct tgatagtttc ttcagcaggg 60

cgggattagt tggagagata gacctccccc ttgagggcac aactaaccca aatggttatg 120

ccaactggga catagatata acaggttacg cgcaaatgcg taggaaggtg gagctattca 180

cttacatgcg ctttgatgca gagttcactt ttgttgcgtg cacacccacc ggggaagttg 240

ttccacaatt gctccaatat atgtttgtgc cacctggagc ccctaagcca gattcaaggg 300

aatcccttgc atggcaaact gccactaacc cctcagtttt tgtcaagctg tcagaccctc 360

cagcgcaggt ttcagtgcca ttcatgtcac ctgcgagtgc ttatcaatgg ttttatgacg 420

gatatcccac attcggagaa cacaaacagg agaaggatct tgaatacggg gcatgtccta 480

ataacatgat gggtacgttc tcagtgcgga ctgtggggac ctccaagtcc aagtaccctt 540

tagtggttag gatctacatg agaatgaagc acgtcagggc gtggatacct cgcccgatgc 600

gtaaccagaa cta 613

<210> 13

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> Single-stranded product

<400> 13

gtgcacgcaa caaaagtgaa ctctgcatca aagcgcatgt 40

<210> 14

<211> 1045

<212> PRT

<213> Artificial Sequence

<220>

<223> Cas12i amino acid sequence

<400> 14

Met Lys Lys Val Glu Val Ser Arg Pro Tyr Gln Ser Leu Leu Leu Pro

1 5 10 15

Asn His Arg Lys Phe Lys Tyr Leu Asp Glu Thr Trp Asn Ala Tyr Lys

20 25 30

Ser Val Lys Ser Leu Leu His Arg Phe Leu Val Cys Ala Tyr Gly Ala

35 40 45

Val Pro Phe Asn Lys Phe Val Glu Val Val Glu Lys Val Asp Asn Asp

50 55 60

Gln Leu Val Leu Ala Phe Ala Val Arg Leu Phe Arg Leu Val Pro Val

65 70 75 80

Glu Ser Thr Ser Phe Ala Lys Val Asp Lys Ala Asn Leu Ala Lys Ser

85 90 95

Leu Ala Asn His Leu Pro Val Gly Thr Ala Ile Pro Ala Asn Val Gln

100 105 110

Ser Tyr Phe Asp Ser Asn Phe Asp Pro Lys Lys Tyr Met Trp Ile Asp

115 120 125

Cys Ala Trp Glu Ala Asp Arg Leu Ala Arg Glu Met Gly Leu Ser Ala

130 135 140

Ser Gln Phe Ser Glu Tyr Ala Thr Thr Met Leu Trp Glu Asp Trp Leu

145 150 155 160

Pro Leu Asn Lys Asp Asp Val Asn Gly Trp Gly Ser Val Ser Gly Leu

165 170 175

Phe Gly Glu Gly Lys Lys Glu Asp Arg Gln Gln Lys Val Lys Met Leu

180 185 190

Asn Asn Leu Leu Asn Gly Ile Lys Lys Asn Pro Pro Lys Asp Tyr Thr

195 200 205

Gln Tyr Leu Lys Ile Leu Leu Asn Ala Phe Asp Ala Lys Ser His Lys

210 215 220

Glu Ala Val Lys Asn Tyr Lys Gly Asp Ser Thr Gly Arg Thr Ala Ser

225 230 235 240

Tyr Leu Ser Glu Lys Ser Gly Glu Ile Thr Glu Leu Met Leu Glu Gln

245 250 255

Leu Met Ser Asn Ile Gln Arg Asp Ile Gly Asp Lys Gln Lys Glu Ile

260 265 270

Ser Leu Pro Lys Lys Asp Val Val Lys Lys Tyr Leu Glu Ser Glu Ser

275 280 285

Gly Val Pro Tyr Asp Gln Asn Leu Trp Ser Gln Ala Tyr Arg Asn Ala

290 295 300

Ala Ser Ser Ile Lys Lys Thr Asp Thr Arg Asn Phe Asn Ser Thr Leu

305 310 315 320

Glu Lys Phe Lys Asn Glu Val Glu Leu Arg Gly Leu Leu Ser Glu Gly

325 330 335

Asp Asp Val Glu Ile Leu Arg Ser Lys Phe Phe Ser Ser Glu Phe His

340 345 350

Lys Thr Pro Asp Lys Phe Val Ile Lys Pro Glu His Ile Gly Phe Asn

355 360 365

Asn Lys Tyr Asn Val Val Ala Glu Leu Tyr Lys Leu Lys Ala Glu Ala

370 375 380

Thr Asp Phe Glu Ser Ala Phe Ala Thr Val Lys Asp Glu Phe Glu Glu

385 390 395 400

Lys Gly Ile Lys His Pro Ile Lys Asn Ile Leu Glu Tyr Ile Trp Asn

405 410 415

Asn Glu Val Pro Val Glu Lys Trp Gly Arg Val Ala Arg Phe Asn Gln

420 425 430

Ser Glu Glu Lys Leu Leu Arg Ile Lys Ala Asn Pro Thr Val Glu Cys

435 440 445

Asn Gln Gly Met Thr Phe Gly Asn Ser Ala Met Val Gly Glu Val Leu

450 455 460

Arg Ser Asn Tyr Val Ser Lys Lys Gly Ala Leu Val Ser Gly Glu His

465 470 475 480

Gly Gly Arg Leu Ile Gly Gln Asn Asn Met Ile Trp Leu Glu Met Arg

485 490 495

Leu Leu Asn Lys Gly Lys Trp Glu Thr His His Val Pro Thr His Asn

500 505 510

Met Lys Phe Phe Glu Glu Val His Ala Tyr Asn Pro Ser Leu Ala Asp

515 520 525

Ser Val Asn Val Arg Asn Arg Leu Tyr Arg Ser Glu Asp Tyr Thr Gln

530 535 540

Leu Pro Ser Ser Ile Thr Asp Gly Leu Lys Gly Asn Pro Lys Ala Lys

545 550 555 560

Leu Leu Lys Arg Gln His Cys Ala Leu Asn Asn Met Thr Ala Asn Val

565 570 575

Leu Asn Pro Lys Leu Ser Phe Thr Ile Asn Lys Lys Asn Asp Asp Tyr

580 585 590

Thr Val Ile Ile Val His Ser Val Glu Val Ser Lys Pro Arg Arg Glu

595 600 605

Val Leu Val Gly Asp Tyr Leu Val Gly Met Asp Gln Asn Gln Thr Ala

610 615 620

Ser Asn Thr Tyr Ala Val Met Gln Val Val Lys Pro Lys Ser Thr Asp

625 630 635 640

Ala Ile Pro Phe Arg Asn Met Trp Val Arg Phe Val Glu Ser Gly Ser

645 650 655

Ile Glu Ser Arg Thr Leu Asn Ser Arg Gly Glu Tyr Val Asp Gln Leu

660 665 670

Asn His Asp Gly Val Asp Leu Phe Glu Ile Gly Asp Thr Glu Trp Val

675 680 685

Asp Ser Ala Arg Lys Phe Phe Asn Lys Leu Gly Val Lys His Lys Asp

690 695 700

Gly Thr Leu Val Asp Leu Ser Thr Ala Pro Arg Lys Ala Tyr Ala Phe

705 710 715 720

Asn Asn Phe Tyr Phe Lys Thr Met Leu Asn His Leu Arg Ser Asn Glu

725 730 735

Val Asp Leu Thr Leu Leu Arg Asn Glu Ile Leu Arg Val Ala Asn Gly

740 745 750

Arg Phe Ser Pro Met Arg Leu Gly Ser Leu Ser Trp Thr Thr Leu Lys

755 760 765

Ala Leu Gly Ser Phe Lys Ser Leu Val Leu Ser Tyr Phe Asp Arg Leu

770 775 780

Gly Ala Lys Glu Met Val Asp Lys Glu Ala Lys Asp Lys Ser Leu Phe

785 790 795 800

Asp Leu Leu Val Ala Ile Asn Asn Lys Arg Ser Asn Lys Arg Glu Glu

805 810 815

Arg Thr Ser Arg Ile Ala Ser Ser Leu Met Thr Val Ala Gln Lys Tyr

820 825 830

Lys Val Asp Asn Ala Val Val His Val Val Val Glu Gly Asn Leu Ser

835 840 845

Ser Thr Asp Arg Ser Ala Ser Lys Ala His Asn Arg Asn Thr Met Asp

850 855 860

Trp Cys Ser Arg Ala Val Val Lys Lys Leu Glu Asp Met Cys Asn Leu

865 870 875 880

Tyr Gly Phe Asn Ile Lys Gly Val Pro Ala Phe Tyr Thr Ser His Gln

885 890 895

Asp Pro Leu Val His Arg Ala Asp Tyr Asp Asp Pro Lys Pro Ala Leu

900 905 910

Arg Cys Arg Tyr Ser Ser Tyr Ser Arg Ala Asp Phe Ser Lys Trp Gly

915 920 925

Gln Asn Ala Leu Ala Ala Val Val Arg Trp Ala Ser Asn Lys Lys Ser

930 935 940

Asn Thr Cys Tyr Lys Val Gly Ala Val Glu Phe Leu Lys Gln His Gly

945 950 955 960

Leu Phe Ala Asp Lys Lys Leu Thr Val Glu Gln Phe Leu Ser Lys Val

965 970 975

Lys Asp Glu Glu Ile Leu Ile Pro Arg Arg Gly Gly Arg Val Phe Leu

980 985 990

Thr Thr His Arg Leu Leu Ala Glu Ser Thr Phe Val Tyr Leu Asn Gly

995 1000 1005

Val Lys Tyr His Ser Cys Asn Ala Asp Glu Val Ala Ala Val Asn

1010 1015 1020

Ile Cys Leu Asn Asp Trp Val Ile Pro Cys Lys Lys Lys Met Lys

1025 1030 1035

Glu Glu Ser Ser Ala Ser Gly

1040 1045

Claims

1. A direct repeat sequence having a base mutation relative to a parent direct repeat sequence, wherein the parent direct repeat sequence and the direct repeat sequence having a base mutation can form a stem-loop structure, the stem-loop structure can be combined with Cas protein, the parent direct repeat sequence and the direct repeat sequence having a base mutation comprise a first segment, a second segment, a third segment and a fourth segment which are sequentially connected from 5 'end to 3' end, and the second segment and the fourth segment can form a stem region of the stem-loop structure through base pairing;

characterized in that the stem region of the direct repeat having the base mutation has at least 1 unpaired base pair relative to the stem region of the parent direct repeat.

2. The direct repeated sequence with base mutation of claim 1, wherein the second segment and the fourth segment of the parental direct repeated sequence and the direct repeated sequence with base mutation respectively comprise 4-10 bases;

preferably, the second segment and the fourth segment each comprise 5 bases.

3. The direct repeat with base mutation of any one of claims 1-2, wherein the stem-loop structure is capable of binding to a V-type Cas protein;

preferably, the V-type Cas protein is selected from Cas proteins of the Cas12 family.

4. The direct repeat having a base mutation according to claim 3, wherein any 1 base of the second segment and the fourth segment of the direct repeat having a base mutation is mutated with respect to the parental direct repeat.

5. A gRNA comprising (i) a direct repeat sequence having a base mutation according to any one of claims 1 to 4; and (ii) a targeting sequence that hybridizes to the target nucleic acid;

preferably, the nucleotide sequence comprises (i) the direct repeat sequence having a base mutation according to any one of claims 1 to 4, which is sequentially linked from the 5 'end to the 3' end; and (ii) a targeting sequence that hybridizes to the target nucleic acid.

6. A nucleic acid encoding a gRNA according to claim 5, or encoding a precursor of a gRNA according to claim 5, or encoding a direct repeat having a base mutation according to any one of claims 1 to 4.

7. A composition or CRISPR system comprising a gRNA of claim 5 and a Cas protein.

8. An activated CRISPR complex comprising a gRNA of claim 5, a Cas protein, and a target nucleic acid bound on the gRNA.

9. A vector comprising a gRNA according to claim 5 or a nucleic acid according to claim 6.

10. A vector system, wherein the vector system comprises one or more vectors, wherein the one or more vectors comprise:

(i) a first regulatory element operably linked to the gRNA of claim 5,

(ii) a second regulatory element operably linked to the Cas protein;

wherein components (i) and (ii) are on the same or different carriers.

11. Use of a gRNA of claim 5 to improve activity of a Cas protein;

preferably, the improving the activity of the Cas protein is increasing the ratio of trans cleavage activity to cis cleavage activity of the Cas protein.

12. A method of improving Cas protein activity or increasing the ratio of trans cleavage activity to cis cleavage activity of a Cas protein, comprising gene editing or gene cleavage using the Cas protein and the gRNA of claim 5.

13. Use of the gRNA of claim 5, the nucleic acid of claim 6, the composition or CRISPR system of claim 7, the activated CRISPR complex of claim 8, the vector of claim 9, or the vector system of claim 10 in gene editing, gene targeting, or gene cleavage.

14. A method of editing, targeting, or cleaving a target nucleic acid, comprising the step of contacting the target nucleic acid with the gRNA of claim 5, the nucleic acid of claim 6, the composition or CRISPR system of claim 7, the vector of claim 9, or the vector system of claim 10.

15. Use of the gRNA of claim 5, the nucleic acid of claim 6, the composition or CRISPR system of claim 7, the activated CRISPR complex of claim 8, the vector of claim 9, or the vector system of claim 10 in nucleic acid detection or diagnosis.

16. Use of the gRNA of claim 5, the nucleic acid of claim 6, the composition or CRISPR system of claim 7, the activated CRISPR complex of claim 8, the vector of claim 9, or the vector system of claim 10 for non-specifically cleaving single stranded nucleic acids.

17. A method of cleaving single-stranded nucleic acid, the method comprising contacting a nucleic acid population with a Cas protein and the gRNA of claim 5, wherein the nucleic acid population comprises a target nucleic acid and a non-target single-stranded nucleic acid, and the Cas protein cleaves the non-target single-stranded nucleic acid.

18. A kit for gene editing, comprising a gRNA of claim 5 and a Cas protein.

19. A kit for detecting a target nucleic acid in a sample, the kit comprising: (i) the gRNA of claim 5, or a nucleic acid encoding the gRNA, or a precursor RNA of the gRNA, or a nucleic acid encoding the precursor RNA; (ii) a Cas protein, or a nucleic acid encoding the Cas protein; and (iii) a single-stranded nucleic acid detector that is single-stranded and does not hybridize to the gRNA.

20. Use of the gRNA of claim 5, the nucleic acid of claim 6, the composition or CRISPR system of claim 7, the activated CRISPR complex of claim 8, the vector of claim 9, or the vector system of claim 10 in the preparation of a formulation for use in any one or any of the following (i) - (v):

(i) gene or genome editing;

(ii) target nucleic acid detection and/or diagnosis;

(iv) treatment of disease;

(v) target genes are targeted.

21. A method of detecting a target nucleic acid in a sample, comprising contacting the sample with a Cas protein, a gRNA of claim 5 comprising a region that binds to the Cas protein and a guide sequence that hybridizes to the target nucleic acid, and a single-stranded nucleic acid detector; detecting a detectable signal generated by the Cas protein-cleaved single-stranded nucleic acid detector, thereby detecting a target nucleic acid; the single-stranded nucleic acid detector does not hybridize to the gRNA.