CN113667718B - Method for detecting target nucleic acid by double-stranded nucleic acid detector - Google Patents

Method for detecting target nucleic acid by double-stranded nucleic acid detector Download PDF

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CN113667718B
CN113667718B CN202110983691.1A CN202110983691A CN113667718B CN 113667718 B CN113667718 B CN 113667718B CN 202110983691 A CN202110983691 A CN 202110983691A CN 113667718 B CN113667718 B CN 113667718B
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CN113667718A (en
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段志强
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Shandong Shunfeng Biotechnology Co Ltd
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Abstract

The present invention provides a method for detecting a target nucleic acid using a double-stranded nucleic acid detector, in particular a method for detecting a target nucleic acid in a sample, the method comprising contacting the sample with a CRISPR-effector protein, a gRNA (guide RNA) comprising a region that binds to the CRISPR-effector protein and a targeting sequence that hybridizes to the target nucleic acid, and a nucleic acid detector; detecting a detectable signal resulting from cleavage of the nucleic acid detector by a CRISPR effect protein, thereby detecting a target nucleic acid, the nucleic acid detector not hybridizing to the gRNA; the nucleic acid detector comprises a nucleic acid that can form a double-stranded complementary pairing structure; the CRISPR effector protein is selected from Mad7 or LbCas12a.

Description

Method for detecting target nucleic acid by double-stranded nucleic acid detector
Technical Field
The invention relates to the field of nucleic acid detection, in particular to a method for detecting target nucleic acid by using a double-stranded nucleic acid detector, and in particular relates to a method for detecting nucleic acid by using a Cas protein, wherein the detector in the detection method is the double-stranded nucleic acid detector.
Background
The method for specifically detecting the nucleic acid molecule (Nucleic acid detection) has important application values, such as pathogen detection, genetic disease detection and the like. In pathogen detection, since each pathogen microorganism has a unique characteristic nucleic acid molecule sequence, nucleic acid molecule detection for specific species, also called nucleic acid diagnosis (NADs, nucleic acid diagnostics), can be developed, and has important significance in the fields of food safety, environmental microorganism pollution detection, human pathogen infection and the like.
The detection of specific nucleic acid molecules established at present usually requires two steps, the first step being the amplification of the nucleic acid of interest and the second step being the detection of the nucleic acid of interest. Existing detection techniques include restriction endonuclease methods, southern, northern, spot hybridization, fluorescent PCR detection techniques, LAMP loop-mediated isothermal amplification techniques, recombinase Polymerase Amplification (RPA), and the like.
After 2012, CRISPR gene editing technology is raised, zhang Feng team developed a new nucleic acid diagnosis technology (shrlock technology) of targeting RNA with Cas13 as a core based on RPA technology, doudna team developed a diagnosis technology (detect technology) with Cas12 enzyme as a core, chinese academy of sciences Shanghai plant physiology and ecology institute king doctor and the like developed a new nucleic acid detection technology (HOLMES technology) based on Cas 12. Nucleic acid detection techniques developed based on CRISPR technology are playing an increasingly important role. The applicant also developed corresponding nucleic acid detection systems based on Cas12i and Cas12j, for example, chinese patent application (CN 111996236a, publication date: 11/27/2020) discloses nucleic acid detection methods based on Cas12i and Cas12j, but the detectors utilized in the above methods are all single-stranded nucleic acids; the present application improves the detector in the above detection method, and proposes a method for detecting nucleic acid using double-stranded nucleic acid as the detector.
Disclosure of Invention
The present invention provides a method, composition, system and kit for nucleic acid detection based on CRISPR technology, and in particular provides a method, composition, system and kit for target nucleic acid detection using an optimized nucleic acid detector.
In one aspect, the invention provides a method of detecting a target nucleic acid in a sample, the method comprising contacting the sample with a CRISPR-effect protein, a gRNA (guide RNA) comprising a region that binds to the CRISPR-effect protein and a targeting sequence that hybridizes to the target nucleic acid, and a nucleic acid detector; detecting a detectable signal resulting from cleavage of the nucleic acid detector by a CRISPR effect protein, thereby detecting a target nucleic acid, the nucleic acid detector not hybridizing to the gRNA;
the nucleic acid detector comprises a nucleic acid that can form a double-stranded complementary pairing structure; the CRISPR effector protein is selected from Mad7 or LbCas12a.
Said Mad7 is described in the patent application (CN 111511906a, publication date: 20200807), in other embodiments said Mad7 may also comprise mutants or orthologs of Mad7, for example, orthologs Mad7v1, mad7v2, mad7v3 and Mad7v4 of Mad7 described in US10704033B1, and mutant Mad70 series proteins of Mad7 described in US10604746B1 (shown in SEQ ID No.8, 9, 10 or 15 of US10604746B 1).
In the present invention, the nucleic acid detector comprises a nucleic acid that can form a double-stranded complementary pairing structure; in one embodiment, the nucleic acid detector is a single stranded nucleic acid having an inverted repeat sequence that can form a double stranded complementary pairing structure by base complementary pairing; in other embodiments, the complementary pairing structure is formed by a double-stranded nucleic acid complementary pairing; in one embodiment, the nucleic acid of the nucleic acid detector is a double-stranded nucleic acid. The nucleic acid of the single-stranded nucleic acid or the double-stranded nucleic acid is DNA or RNA.
In another aspect, the invention also provides a system or composition for detecting a target nucleic acid in a sample, the system or composition comprising a CRISPR-effect protein, a gRNA (guide RNA) and a nucleic acid detector as described above.
In another aspect, the invention also provides a kit for detecting a target nucleic acid in a sample, the kit comprising a CRISPR-effect protein as described above, a gRNA (guide RNA) and a nucleic acid detector.
In another aspect, the invention also provides the use of the above system or composition or kit for detecting a target nucleic acid in a sample.
In another aspect, the invention also provides the use of the above system or composition in the preparation of a reagent or kit for detecting a target nucleic acid in a sample.
In another aspect, the invention also provides a method of cleaving a non-target nucleic acid, the method comprising contacting a population of nucleic acids with a CRISPR effector protein and a gRNA, the population of nucleic acids comprising a target nucleic acid and a plurality of non-target nucleic acids, the gRNA comprising a region that binds to the CRISPR effector protein and a targeting sequence that hybridizes to the target nucleic acid; the CRISPR effect protein cleaves the non-target nucleic acid comprising a nucleic acid that can form a double-stranded complementary pairing structure, the non-target nucleic acid not hybridizing to the gRNA; the CRISPR effector protein is selected from Mad7 or LbCas12a.
The contacting may be inside a cell in vitro, ex vivo or in vivo.
Preferably, the cleaving the non-target nucleic acid is non-specific cleavage of the non-target nucleic acid.
The non-target nucleic acids include nucleic acids that can form a double-stranded complementary pairing structure; in one embodiment, the non-target nucleic acid is a single stranded nucleic acid having an inverted repeat sequence that can form a double stranded complementary pairing structure by base complementary pairing; in other embodiments, the complementary pairing structure is formed by a double-stranded nucleic acid complementary pairing; in one embodiment, the non-target nucleic acid is a double-stranded nucleic acid.
In another aspect, the invention also provides the use of the above CRISPR effector protein and gRNA in non-specific cleavage of the non-target nucleic acid or in the preparation of a reagent or kit for non-specific cleavage of the non-target nucleic acid.
The method of cleaving non-target nucleic acids using Mad7 or LbCas12a described above can be used to remove unwanted non-target nucleic acids or contaminating nucleic acids in actual use, e.g., aerosol contamination during nucleic acid amplification.
In the present invention, the target nucleic acid includes ribonucleotides or deoxyribonucleotides, including single-stranded nucleic acids, double-stranded nucleic acids, e.g., single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA.
In the present invention, the detectable signal is realized by: vision-based detection, gel electrophoresis-based detection, sensor-based detection, color detection, gold nanoparticle-based detection, fluorescence polarization, fluorescence signal detection, 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 effector protein. The CRISPR effector protein, upon recognition or hybridization to the target nucleic acid, can trigger a trans cleavage activity, thereby cleaving the nucleic acid detector to generate a detectable signal.
In the present invention, the detectable signal may be any signal that is generated when the nucleic acid detector is cleaved. For example, gold nanoparticle based detection, fluorescence polarization, colloidal phase change/dispersion, electrochemical detection, semiconductor based sensing. The detectable signal may be read out by any suitable means including, but not limited to: measurement of detectable fluorescent signals, gel electrophoresis detection (by detecting a change in the band on the gel), detection based on the presence or absence of a visual or sensor color, or differences in color (e.g., based on gold nanoparticles), and differences in electrical signals.
In a preferred embodiment, the detectable signal is achieved by: different reporting groups are respectively arranged at the 5 'end and the 3' end of the nucleic acid detector, and when the nucleic acid detector is cut, a detectable reporting signal can be displayed; for example, a nucleic acid detector may exhibit a detectable fluorescent signal when cleaved by providing a fluorescent group and a quenching group at each end of the nucleic acid detector.
In one embodiment, the fluorophore is selected from one or any of FAM, FITC, VIC, JOE, TET, CY, 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 implemented by: different marker molecules are respectively arranged at the 5 'end and the 3' end of the nucleic acid detector, and a reaction signal is detected in a colloidal gold detection mode.
In other embodiments, the detectable signal may also be detected by means of gel electrophoresis: judging whether the nucleic acid detector is cleaved or not by gel electrophoresis.
In one embodiment, the target nucleic acid comprises DNA, RNA, preferably single-stranded nucleic acid or double-stranded nucleic acid.
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 of enrichment or amplification by methods such as PCR, NASBA, RPA, SDA, LAMP, HAD, NEAR, MDA, RCA, LCR, RAM.
In one embodiment, the method further comprises the step of obtaining the target nucleic acid from the sample.
In one embodiment, the sample comprises a sample derived from a virus, bacteria, microorganism, soil, water source, human, animal, plant, or the like.
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 differs from a control; preferably, the virus is a plant virus or an animal virus, for example, papilloma virus, hepadnavirus, 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.
As can be seen from the description of the present invention, cas12i, cas12j or Cas12b is not capable of cleaving the nucleic acid detector (the nucleic acid detector includes a nucleic acid capable of forming a double-strand complementary pairing structure), and is used for nucleic acid detection. That is, the above-mentioned nucleic acid detector is specific for Mad7 or LbCas12 a. Thus, in another aspect, the invention also provides a multiplex nucleic acid detection method, composition, system or kit based on different CRISPR effector proteins and different nucleic acid detectors.
For example, CN112795625a, publication date: 20210514 it is disclosed that a single stranded nucleic acid detector consisting of two bases is specific for Cas12i, a single stranded nucleic acid detector consisting of or consisting of a locked nucleic acid is specific for Cas12b, and Cas12j is capable of specifically cleaving a single stranded nucleic acid detector consisting of the nucleic acid analog-2' oxymethyl RNA.
Accordingly, the present invention also provides a method, system, composition and kit for multiplex nucleic acid detection based on CRISPR technology.
In one aspect, the invention provides a method of multiplex detection of target nucleic acids in a sample, the method comprising contacting the sample with a CRISPR-effect protein, a gRNA (guide RNA) comprising a region that binds to the CRISPR-effect protein and a targeting sequence that hybridizes to the target nucleic acid, and a nucleic acid detector; detecting a detectable signal resulting from cleavage of the nucleic acid detector by a CRISPR effect protein, thereby detecting a target nucleic acid, the nucleic acid detector not hybridizing to the gRNA; the nucleic acid detector comprises a nucleic acid that can form a double-stranded complementary pairing structure; the CRISPR effect protein is selected from Mad7 or LbCAs12a;
the method further comprises contacting the sample with a nucleic acid detection composition comprising a Cas protein, a gRNA, and a single stranded nucleic acid detector; the gRNA includes a region that binds to the Cas protein and a guide sequence that hybridizes to a target sequence on a target nucleic acid; detecting a detectable signal generated by the Cas protein cleaving the single-stranded nucleic acid detector, thereby detecting the target nucleic acid;
the nucleic acid detection composition is selected from any one, any two or three of a first nucleic acid detection composition, a second nucleic acid detection composition and a third nucleic acid detection composition;
The first nucleic acid detection composition comprises Cas12i, a first gRNA that can bind Cas12i and hybridize to a first target sequence on a target nucleic acid, and a first single-stranded nucleic acid detector;
the second nucleic acid detection composition comprises Cas12b (preferably AaCas12 b), a second gRNA that can bind Cas12b and hybridize to a second target sequence on a target nucleic acid, and a second single-stranded nucleic acid detector;
the third nucleic acid detection composition comprises Cas12j, a third gRNA that can bind Cas12j and hybridize to a third target sequence on a target nucleic acid, and a third single-stranded nucleic acid detector;
the first single-stranded nucleic acid detector consists of two consecutive nucleotides; preferably, the nucleotide is one or more of ribonucleotide, deoxyribonucleotide and nucleic acid analogue; the base of the ribonucleotide is selected from one or any of A, U, C, G, T, I; the base of the deoxyribonucleotide is selected from one or any of A, T, C, G, U, I.
Preferably, the nucleic acid of the first single-stranded nucleic acid detector is two consecutive deoxynucleotides, and the base sequence of the deoxynucleotide is TT or CT.
The nucleic acid structure of the second single-stranded nucleic acid detector is a nucleic acid analogue, which is a Locked Nucleic Acid (LNA), and a single-stranded nucleic acid detector comprising a locked nucleic acid is also described in chinese application CN 2020105609327. The base of the locked nucleic acid is selected from one or any of A, T, C, G, U, I.
The nucleic acid structure of the third single-stranded nucleic acid detector is a nucleic acid analogue, the nucleic acid analogue is 2 'oxymethyl RNA, and the base of the 2' oxymethyl RNA is selected from one or more than one of A, T, U, C, G, I.
In another aspect, the invention also provides a reagent or system for detecting a target nucleic acid in a sample, the reagent or system comprising a CRISPR-effect protein, a gRNA (guide RNA) comprising a region that binds to the CRISPR-effect protein and a targeting sequence that hybridizes to the target nucleic acid, and a nucleic acid detector that does not hybridize to the gRNA; the nucleic acid detector comprises a nucleic acid that can form a double-stranded complementary pairing structure; the CRISPR effect protein is selected from Mad7 or LbCAs12a; the reagent or system further comprises any one, any two or three selected from the group consisting of the first nucleic acid detecting composition, the second nucleic acid detecting composition and the third nucleic acid detecting composition described above.
In another aspect, the invention also provides the use of the above reagent or system in the preparation of a kit for detecting a target nucleic acid in a sample.
In another aspect, the invention also provides a kit for detecting a target nucleic acid in a sample, the kit comprising a CRISPR-effect protein, a gRNA (guide RNA) comprising a region that binds to the CRISPR-effect protein and a targeting sequence that hybridizes to the target nucleic acid, and a nucleic acid detector that does not hybridize to the gRNA; the nucleic acid detector comprises a nucleic acid that can form a double-stranded complementary pairing structure; the CRISPR effect protein is selected from Mad7 or LbCAs12a; the system further comprises any one, any two or three selected from the group consisting of the first nucleic acid detection composition, the second nucleic acid detection composition and the third nucleic acid detection composition described above.
In another aspect, the invention also provides the use of the above-described reagents, systems or kits for detecting a target nucleic acid in a sample.
In the present invention, the gRNA includes a sequence (guide sequence) that targets the target nucleic acid and a sequence (orthostatic repeat sequence or portion thereof) that recognizes Cas protein (CRISPR effector protein).
In the invention, the guide sequence comprises 10-40bp; preferably, 12-25bp; preferably, 15-23bp; preferably 16-18bp.
In the present invention, the gRNA has a degree of match of at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90% with the sequence to be hybridized.
In one embodiment, the Cas protein to gRNA molar ratio is used in an amount of (0.8-1.2): 1.
in one embodiment, the Cas protein is used in a final concentration of 20-200nM, preferably 30-100nM, more preferably 40-80nM, more preferably 50nM.
In one embodiment, the final concentration of the gRNA is used in an amount of 20-200nM, preferably 30-100nM, more preferably 40-80nM, more preferably 50nM.
In one embodiment, the target nucleic acid is used in a final concentration of 5-100nM, preferably 10-50nM.
In one embodiment, the nucleic acid detector is used in a final concentration of 100-1000nM, preferably 150-800nM, preferably 200-500nM, preferably 200-300nM.
In one embodiment, the nucleic acid detector is double-stranded DNA.
In a specific embodiment, the amino acid sequence of Mad7 is shown in SEQ ID No.1 and the amino acid sequence of LbCas12a is shown in SEQ ID No.2, or a protein having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, while still maintaining Mad7 or LbCas12a activity.
The term "hybridization" or "complementary" or "substantially complementary" means that a nucleic acid (e.g., RNA, DNA) comprises a nucleotide sequence that enables it to bind non-covalently, i.e., form base pairs and/or G/U base pairs with another nucleic acid in a sequence-specific, antiparallel manner (i.e., the nucleic acid specifically binds to the complementary nucleic acid), "anneal" or "hybridize". Hybridization requires that the two nucleic acids contain complementary sequences, although there may be mismatches between bases. Suitable conditions for hybridization between two nucleic acids depend on the length of the nucleic acids and the degree of complementarity, variables well known in the art. Typically, the hybridizable nucleic acid is 8 nucleotides or more in length (e.g., 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 22 nucleotides or more, 25 nucleotides or more, or 30 nucleotides or more).
It will be appreciated that the sequence of a polynucleotide need not be 100% complementary to the sequence of its target nucleic acid to specifically hybridize. Polynucleotides may comprise 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence complementarity to a target region in a target nucleic acid sequence to which it hybridizes.
General definition:
unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.
The term "amino acid" refers to a carboxylic acid containing an amino group. Various proteins in living bodies are composed of 20 basic amino acids.
The terms "polynucleotide", "nucleotide sequence", "nucleic acid molecule" and "nucleic acid" are used interchangeably and include DNA, RNA or hybrids thereof, which may be double-stranded or single-stranded.
The term "oligonucleotide" refers to a sequence of 3-100 nucleotides, preferably 3-30 nucleotides, preferably 4-20 nucleotides, more preferably 5-15 nucleotides.
The term "homology" or "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. Between the two sequences. Typically, the comparison is made when two sequences are aligned to produce maximum identity. Such an alignment may be determined by computerized operation algorithms (GAP, BESTFIT, FASTA in Wisco nsin Genetics software package, and TFASTA, genetics Comp uter Group) using, for example, the identity of amino acid sequences may be determined by conventional methods, with reference to, for example, the teachings of Smith and Waterman,1981,Adv.Appl.Math.2:482Pearson&Lip man,1988,Proc.Natl.Acad.Sci.USA 85:2444,Thompsonetal, 1994,Nucleic Acids Res 22:467380, etc. The default parameters may also be used for determination using the BLAST algorithm available from the national center for Biotechnology information (NCBI www.nc bi.nlm.nih.gov /).
As used herein, the "CRISPR" refers to clustered, regularly interspaced short palindromic repeats (Clustered regularly interspaced short palindromic repeats) from the immune system of a microorganism.
As used herein, "biotin" is also known as vitamin H, a small molecule vitamin having a molecular weight of 244 Da. "avidin" is also known as avidin, which is an alkaline glycoprotein having 4 binding sites with very high affinity for biotin, and is commonly known as streptavidin. The extremely strong affinity of biotin for avidin can be used to amplify or enhance the detection signal in a detection system. For example, biotin is easily combined with protein (such as antibody) by covalent bond, while avidin molecule combined with enzyme reacts with biotin molecule combined with specific antibody, thus playing the role of multi-stage amplification, and achieving the purpose of detecting unknown antigen (or antibody) molecule due to the catalytic action of enzyme when encountering corresponding substrate.
Target nucleic acid
As used herein, the term "target nucleic acid" refers to a polynucleotide molecule extracted from a biological sample (sample to be tested). The biological sample is any solid or fluid sample obtained, excreted or secreted from any organism, including but not limited to single cell organisms such as bacteria, yeasts, protozoa, amoebas and the like, multicellular organisms (e.g. plants or animals, including samples from healthy or surface healthy human subjects or human patients to be diagnosed or investigated for the effects of a disorder or disease, e.g. infection by a pathogenic microorganism such as a pathogenic bacterium or virus). For example, the biological sample may be a biological fluid obtained from, for example, blood, plasma, serum, urine, stool, sputum, mucus, lymph, synovial fluid, bile, ascites, pleural effusion, seroma, saliva, cerebrospinal fluid, aqueous or vitreous humor, or any bodily secretion, exudate (e.g., fluid obtained from an abscess or any other site of infection or inflammation) or a fluid obtained from a joint (e.g., a normal joint or a joint affected by a disease, such as rheumatoid arthritis, osteoarthritis, gout, or septic arthritis), or a swab of a skin or mucosal surface. The sample may also be a sample obtained from any organ or tissue (including a biopsy or autopsy specimen, such as a tumor biopsy) or may comprise cells (primary cells or cultured cells) or a medium conditioned by any cell, tissue or organ. Exemplary samples include, but are not limited to, cells, cell lysates, blood smears, cell centrifuge preparations, cytological smears, bodily fluids (e.g., blood, plasma, serum, saliva, sputum, urine, bronchoalveolar lavage, semen, etc.), tissue biopsies (e.g., tumor biopsies), fine needle aspirates, and/or tissue sections (e.g., cryostat tissue sections and/or paraffin embedded tissue sections).
In other embodiments, the biological sample may be a plant cell, a callus, a tissue or organ (e.g., root, stem, leaf, flower, seed, fruit), or the like.
In the present invention, the target nucleic acid further includes a DNA molecule formed by reverse transcription of RNA, and further, the target nucleic acid may be amplified by using a technique known in the art, such as isothermal amplification technique and non-isothermal amplification technique, and the isothermal amplification may be nucleic acid sequencing-based amplification (NASBA), recombinase Polymerase Amplification (RPA), loop-mediated isothermal amplification (LAMP), strand Displacement Amplification (SDA), helicase-dependent amplification (HDA), or Nicking Enzyme Amplification Reaction (NEAR). In certain exemplary embodiments, non-isothermal amplification methods may be used, including, but not limited to, PCR, multiple Displacement Amplification (MDA), rolling Circle Amplification (RCA), ligase Chain Reaction (LCR), or derivative amplification methods (RAM).
Further, the detection method of the present invention further comprises a step of amplifying the target nucleic acid; the detection system further comprises a reagent for amplifying the target nucleic acid. The amplified reagents include one or more of the following group: DNA polymerase, strand displacing enzyme, helicase, recombinase, single-stranded binding protein, and the like.
Cas proteins
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 and trans-cleavage activity. The Cis activity refers to the activity that the Cas protein can recognize the PAM locus and specifically cut the target sequence under the action of gRNA.
In embodiments, cas proteins referred to herein, such as Cas12, also encompass functional variants of Cas or homologs or orthologs thereof. "functional variant" of a protein as used herein refers to a variant of such a protein that retains, at least in part, the activity of the protein. Functional variants may include mutants (which may be insertion, deletion or substitution mutants), including polymorphs and the like. Functional variants also include fusion products of such proteins with another nucleic acid, protein, polypeptide or peptide that is not normally associated. Functional variants may be naturally occurring or may be artificial. Advantageous embodiments may relate to engineered or non-naturally occurring V-type DNA targeting effector proteins.
In one embodiment, one or more nucleic acid molecules encoding a Cas protein, such as Cas12, or an ortholog or homolog thereof, may be codon optimized for expression in eukaryotic cells. Eukaryotes may be as described herein. One or more nucleic acid molecules may be engineered or non-naturally occurring.
In one embodiment, the Cas12 protein or an ortholog or homolog thereof may comprise one or more mutations (and thus the nucleic acid molecule encoding the same may have one or more mutations).
In one embodiment, the Cas protein may be from: cilium, listeria, corynebacterium, sart, legionella, treponema, actinomyces, eubacterium, streptococcus, lactobacillus, mycoplasma, bacteroides, flaviivola, flavobacterium, azospirillum, sphaerochaeta, gluconacetobacter, neisseria, rochanterium, parvibacum, staphylococcus, nifctifraactor, mycoplasma, campylobacter and chaetobacter.
The Cas protein can be obtained by recombinant expression vector technology, namely, a nucleic acid molecule for encoding the protein is constructed on a proper vector and then is transformed into a host cell, so that the encoding nucleic acid molecule is expressed in the cell, and the corresponding protein is obtained. The protein can be secreted by the cell or disrupted by conventional extraction techniques to obtain the protein. The coding nucleic acid molecule may or may not be integrated into the genome of the host cell for expression. The vector may further comprise regulatory elements that facilitate sequence integration, or self-replication. The vector may be, for example, a plasmid, virus, cosmid, phage, etc., which are well known to those skilled in the art, and preferably the expression vector in the present invention is a plasmid. The vector further comprises one or more regulatory elements selected from the group consisting of promoters, enhancers, ribosome binding sites for translation initiation, terminators, polyadenylation sequences, and selectable marker genes.
The host cell may be a prokaryotic cell, such as E.coli, streptomyces, agrobacterium: or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant cells. It will be clear to one of ordinary skill in the art how to select appropriate vectors and host cells.
gRNA
As used herein, the "gRNA" is also known as guide RNA or guide RNA, and has the meaning commonly understood by those of skill in the art. In general, the guide RNA can comprise, consist essentially of, or consist of, a direct (direct) repeat sequence and a guide sequence (spacer), also referred to in the context of endogenous CRISPR systems. The gRNA may include crRNA and tracrRNA, or may contain only crRNA, depending on the Cas protein on which it depends, in different CRISPR systems. The crRNA and tracrRNA may be fused by artificial engineering to form single guide RNA (sgRNA). In certain instances, a targeting sequence is any polynucleotide sequence that has sufficient complementarity to a target sequence (a feature sequence described herein) to hybridize to the target sequence and direct specific binding of a CRISPR/Cas complex to the target sequence, typically having a sequence length of 12-25 nt. The co-repeat sequence can be folded to form a specific structure (e.g., a stem-loop structure) for Cas protein recognition to form a complex. The targeting sequence need not be 100% complementary to the feature sequence (target sequence). The targeting sequence is not complementary to the single stranded nucleic acid detector.
In certain embodiments, the degree of complementarity (degree of matching) between a targeting 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. It is within the ability of one of ordinary skill in the art to determine the optimal alignment. For example, there are published and commercially available alignment algorithms and programs such as, but not limited to, the Smith-Waterman algorithm (Smith-Waterman), bowtie, geneious, biopython, and SeqMan in ClustalW, matlab.
The gRNA of the invention can be natural or artificially modified or designed and synthesized.
Nucleic acid detector
The nucleic acid detector of the invention comprises different reporter groups or marker molecules at both ends which, when in an initial state (i.e. not cleaved), exhibit no reporter signal and, when cleaved, exhibit a detectable signal, i.e. a detectable distinction between after cleavage and before cleavage.
In one embodiment, the reporter or marker molecule comprises a fluorophore and a quencher, wherein the fluorophore is selected from one or more of FAM, FITC, VIC, JOE, TET, CY, 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 one embodiment, the nucleic acid detector has a first molecule (e.g., FAM or FITC) attached to the 5 'end and a second molecule (e.g., biotin) attached to the 3' end. The reaction system containing the nucleic acid detector is matched with the flow strip to detect the characteristic sequence (preferably, a colloidal gold detection mode). The flow strip is designed with two capture lines, with an antibody binding to a first molecule (i.e., a first molecular antibody) at the sample contact end (colloidal gold), an antibody binding to the first molecular antibody at the first line (control line), and an antibody binding to a second molecule (i.e., a second molecular antibody, such as avidin) at the second line (test line). When the reaction flows along the strip, the first molecular antibody binds to the first molecule carrying the cleaved or uncleaved oligonucleotide to the capture line, the cleaved reporter will bind to the antibody of the first molecular antibody at the first capture line, and the uncleaved reporter will bind to the second molecular antibody at the second capture line. Binding of the reporter group at each line will result in a strong readout/signal (e.g., color). As more reporter is cut, more signal will accumulate at the first capture line and less signal will appear at the second line. In certain aspects, the invention relates to the use of a flow strip as described herein for detecting nucleic acids. In certain aspects, the invention relates to a method of detecting nucleic acids using a flow strip as defined herein, e.g. a (lateral) flow test or a (lateral) flow immunochromatographic assay. In certain aspects, the molecules in the single stranded nucleic acid detector may be interchanged or the positions of the molecules may be changed, so long as the reporting principle is the same or similar to that of the present invention, and the modified manner is also included in the present invention.
Drawings
FIG. 1 is a schematic diagram of a double-stranded structure formed by a Reporter with inverted repeat sequences used in the examples.
Fig. 2. Fluorescence results of nucleic acid detection using different Cas proteins with reporters having double-stranded structures.
FIG. 3 is a graph comparing the results of nucleic acid detection using Mad7 for a Reporter having a double-stranded structure with a conventional single-stranded Reporter. Wherein line 1 is the experimental set result of the Reporter with the double-stranded structure, line 2 is the experimental set result of the regular single-stranded Reporter, line 3 is the control set result of the Reporter with the double-stranded structure, line 4 is the control set result of the regular single-stranded Reporter, and no target nucleic acid is added in the control set.
FIG. 4 shows a trans-cleavage electrophoresis of a non-specific double-stranded PCR product using Mad7, wherein lane 1: maker; lanes 2-3: mad7+ev71+grna+ostgw6; lanes 4-5: mad7+ev71+ostgw 6 (without addition of gRNA); lanes 6-7: mad7+grna+ostgw6 (no EV71 added); lane 8: osTGW6 (Mad 7, EV71, gRNA was not added).
Fig. 5. Trans cleavage activity of Mad7 and Cas12i was verified using different reporters with double-stranded structures.
Description of the embodiments
The present invention is further described in terms of the following examples, which are given by way of illustration only, and not by way of limitation, of the present invention, and any person skilled in the art may make any modifications to the equivalent examples using the teachings disclosed above. Any simple modification or equivalent variation of the following embodiments according to the technical substance of the present invention falls within the scope of the present invention.
The technical scheme of the invention is based on the following principle that nucleic acid of a sample to be detected is obtained, for example, target nucleic acid can be obtained by an amplification method, and the target nucleic acid is identified and combined by using the gRNA which can be paired with the target nucleic acid to guide Cas protein; subsequently, the Cas protein excites the cleavage activity of the double-stranded nucleic acid detector, thereby cleaving the double-stranded nucleic acid detector in the system; the double-stranded nucleic acid detector is provided with a fluorescent group and a quenching group, and if the double-stranded nucleic acid detector is cleaved, fluorescence is excited; in other embodiments, both ends of the double-stranded nucleic acid detector may also be provided with a label that can be detected by colloidal gold; in other embodiments, the double-stranded nucleic acid detector is not provided with any reporter group, and it can be checked by gel electrophoresis whether the double-stranded nucleic acid detector is cleaved.
In this embodiment, the Cas proteins employed are Mad7, lbCas12a, asCas12a, aaCas12b, cas12i, and Cas12j.
Said Mad7 is described in the patent application (CN 111511906a, publication date: 20200807), in other embodiments said Mad7 may also comprise mutants or orthologs of Mad7, for example, orthologs Mad7v1, mad7v2, mad7v3 and Mad7v4 of Mad7 described in US10704033B1, and mutant Mad70 series proteins of Mad7 described in US10604746B1 (shown in SEQ ID No.8, 9, 10 or 15 of US10604746B 1).
In this embodiment, the amino acid sequences of Mad7, lbCas12a, asCas12a, aaCas12b, cas12i, and Cas12j are shown in SEQ ID nos. 1-6, respectively.
Example 1 nucleic acid detection Using different Cas proteins Using a Reporter containing double-stranded Structure
In this embodiment, the sequence of the Reporter is designed to be 5'-CY5-TGTCTTATTCCAATAAGACA-3' BHQ1, and since the 5 'and 3' ends of the Reporter have inverted repeat sequences, the two ends are labeled CY5 and BHQ respectively, and after annealing, the self-complementary pairing can form a hairpin structure with double strands or a double-strand structure formed by pairing with each other, as shown in fig. 1.
To verify whether different Cas proteins can be used for nucleic acid detection using the reporters described above, gRNA was involved based on different Cas proteins as follows:
TABLE 1 gRNA and target nucleic acids for different Cas proteins
The reaction system used is as follows: different Cas proteins, corresponding gRNA, target nucleic acid and Reporter (5 '-CY5-TGTCTTATTCCAATAAGACA-3' BHQ1) are respectively added into a reaction system; wherein, the final concentration of Cas protein is 50nM, the final concentration of gRNA is 50nM, the final concentration of target nucleic acid is 500nM, and the final concentration of reporter is 500nM. Reacting at 37 ℃, and taking primary fluorescence on Q6 for 20 s; the results are shown in FIG. 2; of the Cas proteins tested above, only Mad7 and LbCas12a can exhibit fluorescent signals using the reporters described above, and thus are used for nucleic acid detection, and other Cas proteins cannot use the reporters described above for nucleic acid detection.
The above results are unexpected because Cas12i, cas12j, cas12a, cas12b, mad7 can all utilize single-stranded nucleic acid detectors as reporters (e.g., 5'-FAM-TTGTT-3' bhq) for nucleic acid detection; however, cas12i, cas12j, asCas12a, aaCas12b cannot detect nucleic acids using the Reporter; this may be because the reporters described above form a structure containing double-stranded nucleic acids, resulting in different Cas enzymes exhibiting different cleavage activities; this suggests that Mad7 and LbCas12a can cleave double-stranded nucleic acid detectors and are useful for nucleic acid detection.
In addition, we compared the activity of the nucleic acid detector that can cleave double strands against Mad7 and LbCas12a with that of the conventional single-stranded nucleic acid detector (5 '-FAM-TTGTT-3' bhq1); as a result, as shown in FIG. 3, the detection activity of the nucleic acid detector using the above-mentioned double-strand-containing structure was superior to that of the conventional single-strand nucleic acid detector.
EXAMPLE 2 cleavage of nonspecific double-stranded nucleic acids Using Mad7 and LbCAs12a
In this embodiment, the property of Mad7 and LbCas12a to cleave non-specific double-stranded nucleic acids was further verified. Respectively adding Mad7, target nucleic acid ssDNA, gRNA and nonspecific dsDNA into a detection system; mad7 was 50nM final, gRNA was 50nM final, ssDNA was 500nM final, and dsDNA was 300ng final. And (5) after enzyme digestion for 30min, gel electrophoresis is used for verification.
In this embodiment, the target nucleic acid selected is EV71, the non-specific dsDNA is the PCR product of OsTGW6, and the gRNA used can target EV71 but will not target OsTGW6.
As a result, as shown in FIG. 4, when Mad7, target nucleic acid ssDNA, gRNA and non-specific dsDNA were added to the reaction system, it was evident that the non-specific dsDNA was significantly degraded; while the other controls (no gRNA, no EV71 or electrophoresis with only non-specific dsDNA) the non-specific dsDNA was not degraded; this further illustrates that in the case of nucleic acid detection using the trans-cleavage activity of Mad7, nucleic acid detection can be performed using double-stranded nucleic acid as a detector. In addition, the Mad7 can be used for removing aerosol pollution during nucleic acid amplification by utilizing the Trans-cleavage effect of double-stranded nucleic acid.
Example 3 optimization of Reporter containing double-stranded Structure
In order to further optimize the cleavage effect of Mad7 and Cas12i on the reporters in example 1, in this embodiment, the reporters with inverted repeat sequences (5 '-CY5-TGTCTTATTCCAATAAGACA-3' bhq 1) in example 1 were further optimized, and the optimized sequences were as follows:
2C (Reporter in example 1): CY5-TGTCTTATTccAATAAGACA-BHQ1;
3C：CY5-TGTCTTATcccATAAGACA-BHQ1；
4C：CY5-TGTCTTATccccATAAGACA-BHQ1；
5C：CY5-TGTCTTATcccccATAAGACA-BHQ1；
3C, 4C, 5C increased the number of bases in the unpaired region in the middle of the sequence compared to 2C.
The cutting effect of Mad7 on different reporters was verified in the same way as in example 1.
As shown in FIG. 5, mad7 has good cleavage effect on 2C/3C/4C/5C reporters, and can be used for nucleic acid detection; meanwhile, cas12i was used for detection, and Cas12i had no cleavage activity for the reporters (as shown in fig. 5).
Sequence listing
<110> Shunfeng biotechnology Co., ltd
<120> method for detecting target nucleic acid by double-stranded nucleic acid detector
<130> P2021-2279
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<170> PatentIn version 3.5
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Asn Arg Asp Glu Ser Phe Tyr Gly Asp Phe Val Leu Ala Tyr Asp Ile
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Thr Ile Leu Arg Tyr Gly Ser Lys Tyr Tyr Leu Ala Ile Met Asp Lys
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Asp Ser Ile Ser Arg Tyr Pro Lys Trp Ser Asn Ala Tyr Asp Phe Asn
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Arg Gly Glu Arg Asn Leu Leu Tyr Ile Val Val Val Asp Gly Lys Gly
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Asn Ile Val Glu Gln Tyr Ser Leu Asn Glu Ile Ile Asn Asn Phe Asn
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Glu Leu Val Glu Lys Tyr Asp Ala Val Ile Ala Leu Glu Asp Leu Asn
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Lys Phe Glu Lys Met Leu Ile Asp Lys Leu Asn Tyr Met Val Asp Lys
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Lys Ser Asn Pro Cys Ala Thr Gly Gly Ala Leu Lys Gly Tyr Gln Ile
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Thr Asn Lys Phe Glu Ser Phe Lys Ser Met Ser Thr Gln Asn Gly Phe
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Ile Phe Tyr Ile Pro Ala Trp Leu Thr Ser Lys Ile Asp Pro Ser Thr
995 1000 1005
Gly Phe Val Asn Leu Leu Lys Thr Lys Tyr Thr Ser Ile Ala Asp
1010 1015 1020
Ser Lys Lys Phe Ile Ser Ser Phe Asp Arg Ile Met Tyr Val Pro
1025 1030 1035
Glu Glu Asp Leu Phe Glu Phe Ala Leu Asp Tyr Lys Asn Phe Ser
1040 1045 1050
Arg Thr Asp Ala Asp Tyr Ile Lys Lys Trp Lys Leu Tyr Ser Tyr
1055 1060 1065
Gly Asn Arg Ile Arg Ile Phe Arg Asn Pro Lys Lys Asn Asn Val
1070 1075 1080
Phe Asp Trp Glu Glu Val Cys Leu Thr Ser Ala Tyr Lys Glu Leu
1085 1090 1095
Phe Asn Lys Tyr Gly Ile Asn Tyr Gln Gln Gly Asp Ile Arg Ala
1100 1105 1110
Leu Leu Cys Glu Gln Ser Asp Lys Ala Phe Tyr Ser Ser Phe Met
1115 1120 1125
Ala Leu Met Ser Leu Met Leu Gln Met Arg Asn Ser Ile Thr Gly
1130 1135 1140
Arg Thr Asp Val Asp Phe Leu Ile Ser Pro Val Lys Asn Ser Asp
1145 1150 1155
Gly Ile Phe Tyr Asp Ser Arg Asn Tyr Glu Ala Gln Glu Asn Ala
1160 1165 1170
Ile Leu Pro Lys Asn Ala Asp Ala Asn Gly Ala Tyr Asn Ile Ala
1175 1180 1185
Arg Lys Val Leu Trp Ala Ile Gly Gln Phe Lys Lys Ala Glu Asp
1190 1195 1200
Glu Lys Leu Asp Lys Val Lys Ile Ala Ile Ser Asn Lys Glu Trp
1205 1210 1215
Leu Glu Tyr Ala Gln Thr Ser Val Lys His
1220 1225
<210> 3
<211> 1307
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> AsCas12a
<400> 3
Met Thr Gln Phe Glu Gly Phe Thr Asn Leu Tyr Gln Val Ser Lys Thr
1 5 10 15
Leu Arg Phe Glu Leu Ile Pro Gln Gly Lys Thr Leu Lys His Ile Gln
20 25 30
Glu Gln Gly Phe Ile Glu Glu Asp Lys Ala Arg Asn Asp His Tyr Lys
35 40 45
Glu Leu Lys Pro Ile Ile Asp Arg Ile Tyr Lys Thr Tyr Ala Asp Gln
50 55 60
Cys Leu Gln Leu Val Gln Leu Asp Trp Glu Asn Leu Ser Ala Ala Ile
65 70 75 80
Asp Ser Tyr Arg Lys Glu Lys Thr Glu Glu Thr Arg Asn Ala Leu Ile
85 90 95
Glu Glu Gln Ala Thr Tyr Arg Asn Ala Ile His Asp Tyr Phe Ile Gly
100 105 110
Arg Thr Asp Asn Leu Thr Asp Ala Ile Asn Lys Arg His Ala Glu Ile
115 120 125
Tyr Lys Gly Leu Phe Lys Ala Glu Leu Phe Asn Gly Lys Val Leu Lys
130 135 140
Gln Leu Gly Thr Val Thr Thr Thr Glu His Glu Asn Ala Leu Leu Arg
145 150 155 160
Ser Phe Asp Lys Phe Thr Thr Tyr Phe Ser Gly Phe Tyr Glu Asn Arg
165 170 175
Lys Asn Val Phe Ser Ala Glu Asp Ile Ser Thr Ala Ile Pro His Arg
180 185 190
Ile Val Gln Asp Asn Phe Pro Lys Phe Lys Glu Asn Cys His Ile Phe
195 200 205
Thr Arg Leu Ile Thr Ala Val Pro Ser Leu Arg Glu His Phe Glu Asn
210 215 220
Val Lys Lys Ala Ile Gly Ile Phe Val Ser Thr Ser Ile Glu Glu Val
225 230 235 240
Phe Ser Phe Pro Phe Tyr Asn Gln Leu Leu Thr Gln Thr Gln Ile Asp
245 250 255
Leu Tyr Asn Gln Leu Leu Gly Gly Ile Ser Arg Glu Ala Gly Thr Glu
260 265 270
Lys Ile Lys Gly Leu Asn Glu Val Leu Asn Leu Ala Ile Gln Lys Asn
275 280 285
Asp Glu Thr Ala His Ile Ile Ala Ser Leu Pro His Arg Phe Ile Pro
290 295 300
Leu Phe Lys Gln Ile Leu Ser Asp Arg Asn Thr Leu Ser Phe Ile Leu
305 310 315 320
Glu Glu Phe Lys Ser Asp Glu Glu Val Ile Gln Ser Phe Cys Lys Tyr
325 330 335
Lys Thr Leu Leu Arg Asn Glu Asn Val Leu Glu Thr Ala Glu Ala Leu
340 345 350
Phe Asn Glu Leu Asn Ser Ile Asp Leu Thr His Ile Phe Ile Ser His
355 360 365
Lys Lys Leu Glu Thr Ile Ser Ser Ala Leu Cys Asp His Trp Asp Thr
370 375 380
Leu Arg Asn Ala Leu Tyr Glu Arg Arg Ile Ser Glu Leu Thr Gly Lys
385 390 395 400
Ile Thr Lys Ser Ala Lys Glu Lys Val Gln Arg Ser Leu Lys His Glu
405 410 415
Asp Ile Asn Leu Gln Glu Ile Ile Ser Ala Ala Gly Lys Glu Leu Ser
420 425 430
Glu Ala Phe Lys Gln Lys Thr Ser Glu Ile Leu Ser His Ala His Ala
435 440 445
Ala Leu Asp Gln Pro Leu Pro Thr Thr Leu Lys Lys Gln Glu Glu Lys
450 455 460
Glu Ile Leu Lys Ser Gln Leu Asp Ser Leu Leu Gly Leu Tyr His Leu
465 470 475 480
Leu Asp Trp Phe Ala Val Asp Glu Ser Asn Glu Val Asp Pro Glu Phe
485 490 495
Ser Ala Arg Leu Thr Gly Ile Lys Leu Glu Met Glu Pro Ser Leu Ser
500 505 510
Phe Tyr Asn Lys Ala Arg Asn Tyr Ala Thr Lys Lys Pro Tyr Ser Val
515 520 525
Glu Lys Phe Lys Leu Asn Phe Gln Met Pro Thr Leu Ala Ser Gly Trp
530 535 540
Asp Val Asn Lys Glu Lys Asn Asn Gly Ala Ile Leu Phe Val Lys Asn
545 550 555 560
Gly Leu Tyr Tyr Leu Gly Ile Met Pro Lys Gln Lys Gly Arg Tyr Lys
565 570 575
Ala Leu Ser Phe Glu Pro Thr Glu Lys Thr Ser Glu Gly Phe Asp Lys
580 585 590
Met Tyr Tyr Asp Tyr Phe Pro Asp Ala Ala Lys Met Ile Pro Lys Cys
595 600 605
Ser Thr Gln Leu Lys Ala Val Thr Ala His Phe Gln Thr His Thr Thr
610 615 620
Pro Ile Leu Leu Ser Asn Asn Phe Ile Glu Pro Leu Glu Ile Thr Lys
625 630 635 640
Glu Ile Tyr Asp Leu Asn Asn Pro Glu Lys Glu Pro Lys Lys Phe Gln
645 650 655
Thr Ala Tyr Ala Lys Lys Thr Gly Asp Gln Lys Gly Tyr Arg Glu Ala
660 665 670
Leu Cys Lys Trp Ile Asp Phe Thr Arg Asp Phe Leu Ser Lys Tyr Thr
675 680 685
Lys Thr Thr Ser Ile Asp Leu Ser Ser Leu Arg Pro Ser Ser Gln Tyr
690 695 700
Lys Asp Leu Gly Glu Tyr Tyr Ala Glu Leu Asn Pro Leu Leu Tyr His
705 710 715 720
Ile Ser Phe Gln Arg Ile Ala Glu Lys Glu Ile Met Asp Ala Val Glu
725 730 735
Thr Gly Lys Leu Tyr Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ala Lys
740 745 750
Gly His His Gly Lys Pro Asn Leu His Thr Leu Tyr Trp Thr Gly Leu
755 760 765
Phe Ser Pro Glu Asn Leu Ala Lys Thr Ser Ile Lys Leu Asn Gly Gln
770 775 780
Ala Glu Leu Phe Tyr Arg Pro Lys Ser Arg Met Lys Arg Met Ala His
785 790 795 800
Arg Leu Gly Glu Lys Met Leu Asn Lys Lys Leu Lys Asp Gln Lys Thr
805 810 815
Pro Ile Pro Asp Thr Leu Tyr Gln Glu Leu Tyr Asp Tyr Val Asn His
820 825 830
Arg Leu Ser His Asp Leu Ser Asp Glu Ala Arg Ala Leu Leu Pro Asn
835 840 845
Val Ile Thr Lys Glu Val Ser His Glu Ile Ile Lys Asp Arg Arg Phe
850 855 860
Thr Ser Asp Lys Phe Phe Phe His Val Pro Ile Thr Leu Asn Tyr Gln
865 870 875 880
Ala Ala Asn Ser Pro Ser Lys Phe Asn Gln Arg Val Asn Ala Tyr Leu
885 890 895
Lys Glu His Pro Glu Thr Pro Ile Ile Gly Ile Asp Arg Gly Glu Arg
900 905 910
Asn Leu Ile Tyr Ile Thr Val Ile Asp Ser Thr Gly Lys Ile Leu Glu
915 920 925
Gln Arg Ser Leu Asn Thr Ile Gln Gln Phe Asp Tyr Gln Lys Lys Leu
930 935 940
Asp Asn Arg Glu Lys Glu Arg Val Ala Ala Arg Gln Ala Trp Ser Val
945 950 955 960
Val Gly Thr Ile Lys Asp Leu Lys Gln Gly Tyr Leu Ser Gln Val Ile
965 970 975
His Glu Ile Val Asp Leu Met Ile His Tyr Gln Ala Val Val Val Leu
980 985 990
Glu Asn Leu Asn Phe Gly Phe Lys Ser Lys Arg Thr Gly Ile Ala Glu
995 1000 1005
Lys Ala Val Tyr Gln Gln Phe Glu Lys Met Leu Ile Asp Lys Leu
1010 1015 1020
Asn Cys Leu Val Leu Lys Asp Tyr Pro Ala Glu Lys Val Gly Gly
1025 1030 1035
Val Leu Asn Pro Tyr Gln Leu Thr Asp Gln Phe Thr Ser Phe Ala
1040 1045 1050
Lys Met Gly Thr Gln Ser Gly Phe Leu Phe Tyr Val Pro Ala Pro
1055 1060 1065
Tyr Thr Ser Lys Ile Asp Pro Leu Thr Gly Phe Val Asp Pro Phe
1070 1075 1080
Val Trp Lys Thr Ile Lys Asn His Glu Ser Arg Lys His Phe Leu
1085 1090 1095
Glu Gly Phe Asp Phe Leu His Tyr Asp Val Lys Thr Gly Asp Phe
1100 1105 1110
Ile Leu His Phe Lys Met Asn Arg Asn Leu Ser Phe Gln Arg Gly
1115 1120 1125
Leu Pro Gly Phe Met Pro Ala Trp Asp Ile Val Phe Glu Lys Asn
1130 1135 1140
Glu Thr Gln Phe Asp Ala Lys Gly Thr Pro Phe Ile Ala Gly Lys
1145 1150 1155
Arg Ile Val Pro Val Ile Glu Asn His Arg Phe Thr Gly Arg Tyr
1160 1165 1170
Arg Asp Leu Tyr Pro Ala Asn Glu Leu Ile Ala Leu Leu Glu Glu
1175 1180 1185
Lys Gly Ile Val Phe Arg Asp Gly Ser Asn Ile Leu Pro Lys Leu
1190 1195 1200
Leu Glu Asn Asp Asp Ser His Ala Ile Asp Thr Met Val Ala Leu
1205 1210 1215
Ile Arg Ser Val Leu Gln Met Arg Asn Ser Asn Ala Ala Thr Gly
1220 1225 1230
Glu Asp Tyr Ile Asn Ser Pro Val Arg Asp Leu Asn Gly Val Cys
1235 1240 1245
Phe Asp Ser Arg Phe Gln Asn Pro Glu Trp Pro Met Asp Ala Asp
1250 1255 1260
Ala Asn Gly Ala Tyr His Ile Ala Leu Lys Gly Gln Leu Leu Leu
1265 1270 1275
Asn His Leu Lys Glu Ser Lys Asp Leu Lys Leu Gln Asn Gly Ile
1280 1285 1290
Ser Asn Gln Asp Trp Leu Ala Tyr Ile Gln Glu Leu Arg Asn
1295 1300 1305
<210> 4
<211> 1129
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> AaCas12b
<400> 4
Met Ala Val Lys Ser Ile Lys Val Lys Leu Arg Leu Asp Asp Met Pro
1 5 10 15
Glu Ile Arg Ala Gly Leu Trp Lys Leu His Lys Glu Val Asn Ala Gly
20 25 30
Val Arg Tyr Tyr Thr Glu Trp Leu Ser Leu Leu Arg Gln Glu Asn Leu
35 40 45
Tyr Arg Arg Ser Pro Asn Gly Asp Gly Glu Gln Glu Cys Asp Lys Thr
50 55 60
Ala Glu Glu Cys Lys Ala Glu Leu Leu Glu Arg Leu Arg Ala Arg Gln
65 70 75 80
Val Glu Asn Gly His Arg Gly Pro Ala Gly Ser Asp Asp Glu Leu Leu
85 90 95
Gln Leu Ala Arg Gln Leu Tyr Glu Leu Leu Val Pro Gln Ala Ile Gly
100 105 110
Ala Lys Gly Asp Ala Gln Gln Ile Ala Arg Lys Phe Leu Ser Pro Leu
115 120 125
Ala Asp Lys Asp Ala Val Gly Gly Leu Gly Ile Ala Lys Ala Gly Asn
130 135 140
Lys Pro Arg Trp Val Arg Met Arg Glu Ala Gly Glu Pro Gly Trp Glu
145 150 155 160
Glu Glu Lys Glu Lys Ala Glu Thr Arg Lys Ser Ala Asp Arg Thr Ala
165 170 175
Asp Val Leu Arg Ala Leu Ala Asp Phe Gly Leu Lys Pro Leu Met Arg
180 185 190
Val Tyr Thr Asp Ser Glu Met Ser Ser Val Glu Trp Lys Pro Leu Arg
195 200 205
Lys Gly Gln Ala Val Arg Thr Trp Asp Arg Asp Met Phe Gln Gln Ala
210 215 220
Ile Glu Arg Met Met Ser Trp Glu Ser Trp Asn Gln Arg Val Gly Gln
225 230 235 240
Glu Tyr Ala Lys Leu Val Glu Gln Lys Asn Arg Phe Glu Gln Lys Asn
245 250 255
Phe Val Gly Gln Glu His Leu Val His Leu Val Asn Gln Leu Gln Gln
260 265 270
Asp Met Lys Glu Ala Ser Pro Gly Leu Glu Ser Lys Glu Gln Thr Ala
275 280 285
His Tyr Val Thr Gly Arg Ala Leu Arg Gly Ser Asp Lys Val Phe Glu
290 295 300
Lys Trp Gly Lys Leu Ala Pro Asp Ala Pro Phe Asp Leu Tyr Asp Ala
305 310 315 320
Glu Ile Lys Asn Val Gln Arg Arg Asn Thr Arg Arg Phe Gly Ser His
325 330 335
Asp Leu Phe Ala Lys Leu Ala Glu Pro Glu Tyr Gln Ala Leu Trp Arg
340 345 350
Glu Asp Ala Ser Phe Leu Thr Arg Tyr Ala Val Tyr Asn Ser Ile Leu
355 360 365
Arg Lys Leu Asn His Ala Lys Met Phe Ala Thr Phe Thr Leu Pro Asp
370 375 380
Ala Thr Ala His Pro Ile Trp Thr Arg Phe Asp Lys Leu Gly Gly Asn
385 390 395 400
Leu His Gln Tyr Thr Phe Leu Phe Asn Glu Phe Gly Glu Arg Arg His
405 410 415
Ala Ile Arg Phe His Lys Leu Leu Lys Val Glu Asn Gly Val Ala Arg
420 425 430
Glu Val Asp Asp Val Thr Val Pro Ile Ser Met Ser Glu Gln Leu Asp
435 440 445
Asn Leu Leu Pro Arg Asp Pro Asn Glu Pro Ile Ala Leu Tyr Phe Arg
450 455 460
Asp Tyr Gly Ala Glu Gln His Phe Thr Gly Glu Phe Gly Gly Ala Lys
465 470 475 480
Ile Gln Cys Arg Arg Asp Gln Leu Ala His Met His Arg Arg Arg Gly
485 490 495
Ala Arg Asp Val Tyr Leu Asn Val Ser Val Arg Val Gln Ser Gln Ser
500 505 510
Glu Ala Arg Gly Glu Arg Arg Pro Pro Tyr Ala Ala Val Phe Arg Leu
515 520 525
Val Gly Asp Asn His Arg Ala Phe Val His Phe Asp Lys Leu Ser Asp
530 535 540
Tyr Leu Ala Glu His Pro Asp Asp Gly Lys Leu Gly Ser Glu Gly Leu
545 550 555 560
Leu Ser Gly Leu Arg Val Met Ser Val Asp Leu Gly Leu Arg Thr Ser
565 570 575
Ala Ser Ile Ser Val Phe Arg Val Ala Arg Lys Asp Glu Leu Lys Pro
580 585 590
Asn Ser Lys Gly Arg Val Pro Phe Phe Phe Pro Ile Lys Gly Asn Asp
595 600 605
Asn Leu Val Ala Val His Glu Arg Ser Gln Leu Leu Lys Leu Pro Gly
610 615 620
Glu Thr Glu Ser Lys Asp Leu Arg Ala Ile Arg Glu Glu Arg Gln Arg
625 630 635 640
Thr Leu Arg Gln Leu Arg Thr Gln Leu Ala Tyr Leu Arg Leu Leu Val
645 650 655
Arg Cys Gly Ser Glu Asp Val Gly Arg Arg Glu Arg Ser Trp Ala Lys
660 665 670
Leu Ile Glu Gln Pro Val Asp Ala Ala Asn His Met Thr Pro Asp Trp
675 680 685
Arg Glu Ala Phe Glu Asn Glu Leu Gln Lys Leu Lys Ser Leu His Gly
690 695 700
Ile Cys Ser Asp Lys Glu Trp Met Asp Ala Val Tyr Glu Ser Val Arg
705 710 715 720
Arg Val Trp Arg His Met Gly Lys Gln Val Arg Asp Trp Arg Lys Asp
725 730 735
Val Arg Ser Gly Glu Arg Pro Lys Ile Arg Gly Tyr Ala Lys Asp Val
740 745 750
Val Gly Gly Asn Ser Ile Glu Gln Ile Glu Tyr Leu Glu Arg Gln Tyr
755 760 765
Lys Phe Leu Lys Ser Trp Ser Phe Phe Gly Lys Val Ser Gly Gln Val
770 775 780
Ile Arg Ala Glu Lys Gly Ser Arg Phe Ala Ile Thr Leu Arg Glu His
785 790 795 800
Ile Asp His Ala Lys Glu Asp Arg Leu Lys Lys Leu Ala Asp Arg Ile
805 810 815
Ile Met Glu Ala Leu Gly Tyr Val Tyr Ala Leu Asp Glu Arg Gly Lys
820 825 830
Gly Lys Trp Val Ala Lys Tyr Pro Pro Cys Gln Leu Ile Leu Leu Glu
835 840 845
Glu Leu Ser Glu Tyr Gln Phe Asn Asn Asp Arg Pro Pro Ser Glu Asn
850 855 860
Asn Gln Leu Met Gln Trp Ser His Arg Gly Val Phe Gln Glu Leu Ile
865 870 875 880
Asn Gln Ala Gln Val His Asp Leu Leu Val Gly Thr Met Tyr Ala Ala
885 890 895
Phe Ser Ser Arg Phe Asp Ala Arg Thr Gly Ala Pro Gly Ile Arg Cys
900 905 910
Arg Arg Val Pro Ala Arg Cys Thr Gln Glu His Asn Pro Glu Pro Phe
915 920 925
Pro Trp Trp Leu Asn Lys Phe Val Val Glu His Thr Leu Asp Ala Cys
930 935 940
Pro Leu Arg Ala Asp Asp Leu Ile Pro Thr Gly Glu Gly Glu Ile Phe
945 950 955 960
Val Ser Pro Phe Ser Ala Glu Glu Gly Asp Phe His Gln Ile His Ala
965 970 975
Asp Leu Asn Ala Ala Gln Asn Leu Gln Gln Arg Leu Trp Ser Asp Phe
980 985 990
Asp Ile Ser Gln Ile Arg Leu Arg Cys Asp Trp Gly Glu Val Asp Gly
995 1000 1005
Glu Leu Val Leu Ile Pro Arg Leu Thr Gly Lys Arg Thr Ala Asp
1010 1015 1020
Ser Tyr Ser Asn Lys Val Phe Tyr Thr Asn Thr Gly Val Thr Tyr
1025 1030 1035
Tyr Glu Arg Glu Arg Gly Lys Lys Arg Arg Lys Val Phe Ala Gln
1040 1045 1050
Glu Lys Leu Ser Glu Glu Glu Ala Glu Leu Leu Val Glu Ala Asp
1055 1060 1065
Glu Ala Arg Glu Lys Ser Val Val Leu Met Arg Asp Pro Ser Gly
1070 1075 1080
Ile Ile Asn Arg Gly Asn Trp Thr Arg Gln Lys Glu Phe Trp Ser
1085 1090 1095
Met Val Asn Gln Arg Ile Glu Gly Tyr Leu Val Lys Gln Ile Arg
1100 1105 1110
Ser Arg Val Pro Leu Gln Asp Ser Ala Cys Glu Asn Thr Gly Asp
1115 1120 1125
Ile
<210> 5
<211> 1045
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> Cas12i
<400> 5
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
<210> 6
<211> 908
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> Cas12j
<400> 6
Met Pro Ser Tyr Lys Ser Ser Arg Val Leu Val Arg Asp Val Pro Glu
1 5 10 15
Glu Leu Val Asp His Tyr Glu Arg Ser His Arg Val Ala Ala Phe Phe
20 25 30
Met Arg Leu Leu Leu Ala Met Arg Arg Glu Pro Tyr Ser Leu Arg Met
35 40 45
Arg Asp Gly Thr Glu Arg Glu Val Asp Leu Asp Glu Thr Asp Asp Phe
50 55 60
Leu Arg Ser Ala Gly Cys Glu Glu Pro Asp Ala Val Ser Asp Asp Leu
65 70 75 80
Arg Ser Phe Ala Leu Ala Val Leu His Gln Asp Asn Pro Lys Lys Arg
85 90 95
Ala Phe Leu Glu Ser Glu Asn Cys Val Ser Ile Leu Cys Leu Glu Lys
100 105 110
Ser Ala Ser Gly Thr Arg Tyr Tyr Lys Arg Pro Gly Tyr Gln Leu Leu
115 120 125
Lys Lys Ala Ile Glu Glu Glu Trp Gly Trp Asp Lys Phe Glu Ala Ser
130 135 140
Leu Leu Asp Glu Arg Thr Gly Glu Val Ala Glu Lys Phe Ala Ala Leu
145 150 155 160
Ser Met Glu Asp Trp Arg Arg Phe Phe Ala Ala Arg Asp Pro Asp Asp
165 170 175
Leu Gly Arg Glu Leu Leu Lys Thr Asp Thr Arg Glu Gly Met Ala Ala
180 185 190
Ala Leu Arg Leu Arg Glu Arg Gly Val Phe Pro Val Ser Val Pro Glu
195 200 205
His Leu Asp Leu Asp Ser Leu Lys Ala Ala Met Ala Ser Ala Ala Glu
210 215 220
Arg Leu Lys Ser Trp Leu Ala Cys Asn Gln Arg Ala Val Asp Glu Lys
225 230 235 240
Ser Glu Leu Arg Lys Arg Phe Glu Glu Ala Leu Asp Gly Val Asp Pro
245 250 255
Glu Lys Tyr Ala Leu Phe Glu Lys Phe Ala Ala Glu Leu Gln Gln Ala
260 265 270
Asp Tyr Asn Val Thr Lys Lys Leu Val Leu Ala Val Ser Ala Lys Phe
275 280 285
Pro Ala Thr Glu Pro Ser Glu Phe Lys Arg Gly Val Glu Ile Leu Lys
290 295 300
Glu Asp Gly Tyr Lys Pro Leu Trp Glu Asp Phe Arg Glu Leu Gly Phe
305 310 315 320
Val Tyr Leu Ala Glu Arg Lys Trp Glu Arg Arg Arg Gly Gly Ala Ala
325 330 335
Val Thr Leu Cys Asp Ala Asp Asp Ser Pro Ile Lys Val Arg Phe Gly
340 345 350
Leu Thr Gly Arg Gly Arg Lys Phe Val Leu Ser Ala Ala Gly Ser Arg
355 360 365
Phe Leu Ile Thr Val Lys Leu Pro Cys Gly Asp Val Gly Leu Thr Ala
370 375 380
Val Pro Ser Arg Tyr Phe Trp Asn Pro Ser Val Gly Arg Thr Thr Ser
385 390 395 400
Asn Ser Phe Arg Ile Glu Phe Thr Lys Arg Thr Thr Glu Asn Arg Arg
405 410 415
Tyr Val Gly Glu Val Lys Glu Ile Gly Leu Val Arg Gln Arg Gly Arg
420 425 430
Tyr Tyr Phe Phe Ile Asp Tyr Asn Phe Asp Pro Glu Glu Val Ser Asp
435 440 445
Glu Thr Lys Val Gly Arg Ala Phe Phe Arg Ala Pro Leu Asn Glu Ser
450 455 460
Arg Pro Lys Pro Lys Asp Lys Leu Thr Val Met Gly Ile Asp Leu Gly
465 470 475 480
Ile Asn Pro Ala Phe Ala Phe Ala Val Cys Thr Leu Gly Glu Cys Gln
485 490 495
Asp Gly Ile Arg Ser Pro Val Ala Lys Met Glu Asp Val Ser Phe Asp
500 505 510
Ser Thr Gly Leu Arg Gly Gly Ile Gly Ser Gln Lys Leu His Arg Glu
515 520 525
Met His Asn Leu Ser Asp Arg Cys Phe Tyr Gly Ala Arg Tyr Ile Arg
530 535 540
Leu Ser Lys Lys Leu Arg Asp Arg Gly Ala Leu Asn Asp Ile Glu Ala
545 550 555 560
Arg Leu Leu Glu Glu Lys Tyr Ile Pro Gly Phe Arg Ile Val His Ile
565 570 575
Glu Asp Ala Asp Glu Arg Arg Arg Thr Val Gly Arg Thr Val Lys Glu
580 585 590
Ile Lys Gln Glu Tyr Lys Arg Ile Arg His Gln Phe Tyr Leu Arg Tyr
595 600 605
His Thr Ser Lys Arg Asp Arg Thr Glu Leu Ile Ser Ala Glu Tyr Phe
610 615 620
Arg Met Leu Phe Leu Val Lys Asn Leu Arg Asn Leu Leu Lys Ser Trp
625 630 635 640
Asn Arg Tyr His Trp Thr Thr Gly Asp Arg Glu Arg Arg Gly Gly Asn
645 650 655
Pro Asp Glu Leu Lys Ser Tyr Val Arg Tyr Tyr Asn Asn Leu Arg Met
660 665 670
Asp Thr Leu Lys Lys Leu Thr Cys Ala Ile Val Arg Thr Ala Lys Glu
675 680 685
His Gly Ala Thr Leu Val Ala Met Glu Asn Ile Gln Arg Val Asp Arg
690 695 700
Asp Asp Glu Val Lys Arg Arg Lys Glu Asn Ser Leu Leu Ser Leu Trp
705 710 715 720
Ala Pro Gly Met Val Leu Glu Arg Val Glu Gln Glu Leu Lys Asn Glu
725 730 735
Gly Ile Leu Ala Trp Glu Val Asp Pro Arg His Thr Ser Gln Thr Ser
740 745 750
Cys Ile Thr Asp Glu Phe Gly Tyr Arg Ser Leu Val Ala Lys Asp Thr
755 760 765
Phe Tyr Phe Glu Gln Asp Arg Lys Ile His Arg Ile Asp Ala Asp Val
770 775 780
Asn Ala Ala Ile Asn Ile Ala Arg Arg Phe Leu Thr Arg Tyr Arg Ser
785 790 795 800
Leu Thr Gln Leu Trp Ala Ser Leu Leu Asp Asp Gly Arg Tyr Leu Val
805 810 815
Asn Val Thr Arg Gln His Glu Arg Ala Tyr Leu Glu Leu Gln Thr Gly
820 825 830
Ala Pro Ala Ala Thr Leu Asn Pro Thr Ala Glu Ala Ser Tyr Glu Leu
835 840 845
Val Gly Leu Ser Pro Glu Glu Glu Glu Leu Ala Gln Thr Arg Ile Lys
850 855 860
Arg Lys Lys Arg Glu Pro Phe Tyr Arg His Glu Gly Val Trp Leu Thr
865 870 875 880
Arg Glu Lys His Arg Glu Gln Val His Glu Leu Arg Asn Gln Val Leu
885 890 895
Ala Leu Gly Asn Ala Lys Ile Pro Glu Ile Arg Thr
900 905

Claims (15)

1. A method of detecting a target nucleic acid in a sample, the method being a method for non-disease diagnosis and treatment purposes, the method comprising contacting the sample with a CRISPR effector protein, a gRNA comprising a region that binds to the CRISPR effector protein and a targeting sequence that hybridizes to the target nucleic acid, and a nucleic acid detector; detecting a detectable signal resulting from cleavage of the nucleic acid detector by a CRISPR effect protein, thereby detecting a target nucleic acid, the nucleic acid detector not hybridizing to the gRNA; the CRISPR effect protein is Mad7, and the amino acid sequence of the Mad7 is shown as SEQ ID No. 1; the nucleic acid of the nucleic acid detector is a single-stranded nucleic acid having an inverted repeat sequence that forms a double-stranded complementary pairing structure by base-complementary pairing, or the nucleic acid of the nucleic acid detector is a double-stranded nucleic acid.
2. The method of claim 1, further comprising contacting the sample with a nucleic acid detection composition comprising Cas protein, gRNA, and single-stranded nucleic acid detector; the gRNA includes a region that binds to the Cas protein and a guide sequence that hybridizes to a target sequence on a target nucleic acid; detecting a detectable signal generated by the Cas protein cleaving the single-stranded nucleic acid detector, thereby detecting the target nucleic acid; the single stranded nucleic acid detector does not hybridize to the gRNA;
the nucleic acid detection composition is selected from any one, any two or three of a first nucleic acid detection composition, a second nucleic acid detection composition and a third nucleic acid detection composition;
the first nucleic acid detection composition comprises Cas12i, a first gRNA that can bind Cas12i and hybridize to a first target sequence on a target nucleic acid, and a first single-stranded nucleic acid detector;
the second nucleic acid detection composition comprises Cas12b, a second gRNA that can bind Cas12b and hybridize to a second target sequence on a target nucleic acid, and a second single-stranded nucleic acid detector;
the third nucleic acid detection composition comprises Cas12j, a third gRNA that can bind Cas12j and hybridize to a third target sequence on a target nucleic acid, and a third single-stranded nucleic acid detector;
The nucleic acid of the first single-stranded nucleic acid detector consists of two consecutive nucleotides;
the nucleic acid structure of the second single-stranded nucleic acid detector is a nucleic acid analog, which is a Locked Nucleic Acid (LNA);
the nucleic acid structure of the third single stranded nucleic acid detector is a nucleic acid analog that is a 2' oxymethyl RNA.
3. The method of claim 1, wherein the detectable signal is achieved by: detection based on gel electrophoresis, detection based on sensors, detection based on gold nanoparticles, detection based on fluorescent signals, electrochemical detection or detection based on semiconductors.
4. The method of claim 1, wherein the detectable signal is achieved by: and (5) color detection.
5. The method of claim 1, wherein the detectable signal is achieved by: visual-based detection.
6. A system or composition or kit for detecting a target nucleic acid in a sample, the system or composition or kit comprising the CRISPR effector protein, gRNA, and nucleic acid detector of claim 1.
7. Use of the system or composition or kit of claim 6 for detecting a target nucleic acid in a sample for non-disease diagnostic and therapeutic purposes.
8. A reagent or system or kit for detecting a target nucleic acid in a sample, the reagent or system or kit comprising the CRISPR-effect protein, gRNA and nucleic acid detector of claim 1; the reagent or system or kit further comprises any one, any two or three selected from the group consisting of the first nucleic acid detecting composition, the second nucleic acid detecting composition and the third nucleic acid detecting composition of claim 2.
9. Use of the reagent, system or kit of claim 8 for detecting a target nucleic acid in a sample for non-disease diagnostic and therapeutic purposes.
10. The method of claim 1, or the system or composition or kit of claim 6, or the agent or system or kit of claim 8, or the use of claim 7 or 9; characterized in that the sample comprises a sample derived from a microorganism, an animal, a plant, soil, or a water source.
11. The method of claim 1, or the system or composition or kit of claim 6, or the agent or system or kit of claim 8, or the use of claim 7 or 9; characterized in that the sample comprises a sample derived from a virus, a bacterium, or a human.
12. The method of claim 1, or the system or composition or kit of claim 6, or the agent or system or kit of claim 8, or the use of claim 7 or 9; wherein the target nucleic acid is derived from a microbial, animal, plant, soil, or water source sample.
13. The method of claim 1, or the system or composition or kit of claim 6, or the agent or system or kit of claim 8, or the use of claim 7 or 9; wherein the target nucleic acid is derived from a virus, bacteria, or human sample.
14. A method of cleaving a non-target nucleic acid, the method being a method of non-disease diagnosis and treatment, the method comprising contacting a population of nucleic acids with a CRISPR effector protein and a gRNA, the population of nucleic acids comprising a target nucleic acid and a plurality of non-target nucleic acids, the gRNA comprising a region that binds to the CRISPR effector protein and a targeting sequence that hybridizes to the target nucleic acid; the CRISPR effector protein cleaves the non-target nucleic acid; the non-target nucleic acid is a single-stranded nucleic acid having an inverted repeat sequence that forms a double-stranded complementary pairing structure by base-complementary pairing, or the non-target nucleic acid is a double-stranded nucleic acid; the non-target nucleic acid does not hybridize to the gRNA; the CRISPR effect protein is Mad7, and the amino acid sequence of the Mad7 is shown as SEQ ID No. 1.
15. Use of the CRISPR effector protein and the gRNA of claim 14 for non-specific cleavage of a non-target nucleic acid for non-disease diagnosis and treatment purposes, or use of the CRISPR effector protein, the gRNA and the non-target nucleic acid of claim 14 for the preparation of a reagent or kit for non-specific cleavage of a non-target nucleic acid; the non-target nucleic acid is a single-stranded nucleic acid having an inverted repeat sequence that forms a double-stranded complementary pairing structure by base-complementary pairing, or the non-target nucleic acid is a double-stranded nucleic acid; the non-target nucleic acid does not hybridize to the gRNA.
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Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116732003A (en) * 2022-03-10 2023-09-12 青岛清原化合物有限公司 Engineered nucleases and uses thereof
CN114634972B (en) * 2022-05-19 2022-08-26 舜丰生物科技(海南)有限公司 Method for detecting nucleic acid by using Cas enzyme
CN116004573B (en) * 2022-10-25 2023-09-12 山东舜丰生物科技有限公司 Cas protein with improved editing activity and application thereof

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019104058A1 (en) * 2017-11-22 2019-05-31 The Regents Of The University Of California Type v crispr/cas effector proteins for cleaving ssdnas and detecting target dnas
WO2020142739A1 (en) * 2019-01-04 2020-07-09 Mammoth Biosciences, Inc. COMPOSITIONS AND METHODS FOR DETECTING MODIFIED NUCLEIC ACIDS AND AMPLIFYING ssDNA
CN111630162A (en) * 2017-10-04 2020-09-04 博德研究所 CRISPR-Effector System-based diagnostics
CN111690717A (en) * 2020-04-30 2020-09-22 山东舜丰生物科技有限公司 Method and system for detecting target nucleic acid based on CRISPR technology
CN111690773A (en) * 2020-06-17 2020-09-22 山东舜丰生物科技有限公司 Method and system for detecting target nucleic acid by using novel Cas enzyme
CN111876525A (en) * 2020-07-08 2020-11-03 广州再生医学与健康广东省实验室 gRNA, primer and kit for detecting SARS-CoV-2
WO2020257356A2 (en) * 2019-06-18 2020-12-24 Mammoth Biosciences, Inc. Assays and methods for detection of nucleic acids
CN112391446A (en) * 2020-05-29 2021-02-23 山东舜丰生物科技有限公司 Method for detecting target nucleic acid based on CRISPR technology
CN113166798A (en) * 2018-11-28 2021-07-23 主基因有限公司 Targeted enrichment by endonuclease protection

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3931313A2 (en) * 2019-01-04 2022-01-05 Mammoth Biosciences, Inc. Programmable nuclease improvements and compositions and methods for nucleic acid amplification and detection

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111630162A (en) * 2017-10-04 2020-09-04 博德研究所 CRISPR-Effector System-based diagnostics
WO2019104058A1 (en) * 2017-11-22 2019-05-31 The Regents Of The University Of California Type v crispr/cas effector proteins for cleaving ssdnas and detecting target dnas
CN111630163A (en) * 2017-11-22 2020-09-04 加利福尼亚大学董事会 V-type CRISPR/CAS effector proteins for cleaving SSDNA and detecting target DNA
CN113166798A (en) * 2018-11-28 2021-07-23 主基因有限公司 Targeted enrichment by endonuclease protection
WO2020142739A1 (en) * 2019-01-04 2020-07-09 Mammoth Biosciences, Inc. COMPOSITIONS AND METHODS FOR DETECTING MODIFIED NUCLEIC ACIDS AND AMPLIFYING ssDNA
WO2020257356A2 (en) * 2019-06-18 2020-12-24 Mammoth Biosciences, Inc. Assays and methods for detection of nucleic acids
CN111690717A (en) * 2020-04-30 2020-09-22 山东舜丰生物科技有限公司 Method and system for detecting target nucleic acid based on CRISPR technology
CN112391446A (en) * 2020-05-29 2021-02-23 山东舜丰生物科技有限公司 Method for detecting target nucleic acid based on CRISPR technology
CN111690773A (en) * 2020-06-17 2020-09-22 山东舜丰生物科技有限公司 Method and system for detecting target nucleic acid by using novel Cas enzyme
CN111876525A (en) * 2020-07-08 2020-11-03 广州再生医学与健康广东省实验室 gRNA, primer and kit for detecting SARS-CoV-2

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
Cas12a trans-cleavage can be modulated in vitro and is active on ssDNA, dsDNA, and RNA;Ryan T. Fuchs等;bioRxiv;第1-23页 *
Cas12a-based on-site, rapid detection of genetically modified crops;Zhiqiang Duan等;Journal of Integrative Plant Biology;第64卷(第10期);第1856-1859页 *
CRISPR/Cas12a-mediated Interfacial Cleaving of Hairpin DNA Reporter for Electrochemical Nucleic Acid Sensing;Decai Zhang等;ACS Sens;第1-26页 *
CRISPR/Cas技术在核酸检测中的应用进展;史铠等;分析测试学报;第37卷(第10期);第1217-1220页 *
CRISPR-Cas 技术用于核酸检测的现状和前景;李凯强等;中国预防兽医学报;第43卷(第7期);第791-795页 *
CRISPR-Cas12a has widespread off-target and dsDNA-nicking effects;Karthik Murugan等;J. Biol. Chem.;第295卷(第17期);第5538-5553页 *
CRISPR-Cas12a trans-cleaves DNA G-quadruplexes;Ying Li等;Chem. Commun.(第83期);第1-4页 *
G-triplex: A new type of CRISPR-Cas12a reporter enabling highly sensitive nucleic acid detection;Tao Li等;Biosensors and Bioelectronics;第187卷;第1-9页 *
Probing CRISPR-Cas12a Nuclease Activity Using Double-Stranded DNA-Templated Fluorescent Substrates;Christopher W. Smith等;Biochemistry;第59卷(第15期);第1474–1481页 *
Recognition of DNA target formulations by CRISPR-Cas12a using a dsDNA reporter;Christopher W. Smith等;ACS Synth. Biol.;第10卷(第7期);第1785−1791页 *

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