CN113234795A - Method for detecting nucleic acid by using Cas protein - Google Patents

Abstract

The invention provides a method for detecting nucleic acid by using Cas protein, in particular to a method, a system and a kit for detecting target nucleic acid based on CRISPR technology.

Description

Method for detecting nucleic acid by using Cas protein

Technical Field

The invention relates to the field of nucleic acid detection, relates to a method for detecting nucleic acid by using a Cas protein, in particular to a method, a system and a kit for detecting target nucleic acid based on a CRISPR technology, and particularly relates to a method for detecting multiple target nucleic acid based on the CRISPR technology.

Background

The method for specifically detecting Nucleic acid molecules (Nucleic acid detection) has important application values, such as pathogen detection, genetic disease detection and the like. In the aspect of pathogen detection, each pathogenic microorganism has a unique characteristic nucleic acid molecule sequence, so that nucleic acid molecule detection for a specific species, also called Nucleic Acid Diagnostics (NADs), can be developed, and is important in the fields of food safety, detection of environmental microbial contamination, infection of human pathogenic bacteria, and the like. Another aspect is the detection of Single Nucleotide Polymorphisms (SNPs) in humans or other species. Understanding the relationship between genetic variation and biological functions at the genomic level provides a new perspective for modern molecular biology, and SNPs are closely related to biological functions, evolution, diseases and the like, so the development of detection and analysis techniques of SNPs is particularly important.

The detection of specific nucleic acid molecules established today 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. The existing detection technologies include restriction endonuclease methods, Southern, Northern, dot blot, fluorescent PCR detection technologies, LAMP loop-mediated isothermal amplification technologies, recombinase polymerase amplification technologies (RPA) and the like. After 2012, CRISPR gene editing technology arose, a new nucleic acid diagnosis technology (SHERLOCK technology) of targeted RNA with Cas13 as a core was developed by the zhanfeng team based on RPA technology, a diagnosis technology (DETECTR technology) with Cas12 enzyme as a core was developed by the Doudna team, and a new nucleic acid detection technology (HOLMES technology) based on Cas12 was also developed by the royal doctor of the institute of physiology and ecology of plants in the shanghai of the chinese academy of sciences. Nucleic acid detection techniques developed based on CRISPR technology are playing an increasingly important role.

Although the existing nucleic acid detection technologies are numerous, how to detect more quickly, simply, cheaply and accurately is still an important direction for improving the detection technology, and especially how to perform multiple detection on nucleic acid is a problem to be solved urgently.

Disclosure of Invention

The invention provides a method for detecting nucleic acid based on CRISPR technology, in particular to a method, a system and a kit for carrying out multiple detection on nucleic acid.

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 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 cleavage single-stranded nucleic acid detector, thereby detecting the target nucleic acid;

the nucleic acid detecting composition is selected from any one or two of a first nucleic acid detecting composition and a second nucleic acid detecting composition;

the first nucleic acid detection composition includes Cas12j12, a first gRNA that can bind Cas12j12 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 includes Cas12j22, a second gRNA that can bind Cas12j22 and hybridize to a second target sequence on the target nucleic acid, and a second single-stranded nucleic acid detector.

The Cas12j12 is selected from the following i-iii:

i. a Cas protein having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to SEQ ID No.1 and having trans cleavage activity;

ii. A Cas protein having an amino acid sequence with substitution, deletion or addition of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acids compared to SEQ ID No.1, and having trans cleavage activity;

iii, a Cas protein comprising the amino acid sequence shown in SEQ ID No. 1.

The Cas12j22 is selected from the following i-iii:

i. a Cas protein having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to SEQ ID No.2 and having trans cleavage activity;

ii. A Cas protein having an amino acid sequence with one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions, deletions, or additions compared to SEQ ID No.2, and having trans cleavage activity;

iii, a Cas protein comprising the amino acid sequence shown in SEQ ID No. 2.

In one embodiment, the region sequence of the first gRNA that binds Cas12j12 is set forth in SEQ ID No. 3. The region sequence of the second gRNA that binds Cas12j22 is shown in SEQ ID No. 4.

The nucleotides of the first single-stranded nucleic acid detector are composed of polyT or polyC, preferably, polyT.

The nucleotides of the second single-stranded nucleic acid detector are composed of polyA or polyC, preferably, polyA.

In one embodiment, the polyA, polyT, polyC consists of a number of A, T or C, respectively, preferably the number is at least 2, e.g. 2-25, as well as 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20.

In a preferred embodiment, in the gRNA, the region that binds to the Cas protein is placed 5' to the guide sequence that hybridizes to the target sequence on the target nucleic acid.

In the present invention, the Cas12j12 can specifically cleave the first single-stranded nucleic acid detector compared to the Cas12j22 protein, thereby generating a first detectable signal; the Cas12j22 can specifically cleave the second single-stranded nucleic acid detector compared to the Cas12j12 protein, thereby generating a second detectable signal.

The specific cleavage means that a certain protein has higher cleavage efficiency and exhibits a better detectable signal than other proteins with respect to a single-stranded nucleic acid detector to which the protein is directed.

The detectable signal is achieved by: vision-based detection, sensor-based detection, color detection, gold nanoparticle-based detection, fluorescence polarization, fluorescence signal-based detection, colloidal phase transition/dispersion, electrochemical detection, and semiconductor-based detection.

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, detection based on fluorescence signals, 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 first detectable signal and the second detectable signal are different detectable signals from each other.

Preferably, the single-stranded nucleic acid detector has a fluorescent group and a quencher group disposed at both ends thereof, respectively, and can exhibit a detectable fluorescent signal when the single-stranded nucleic acid detector is cleaved. The fluorescent group is selected from one or more 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 one embodiment, the first single-stranded nucleic acid detector and the second single-stranded nucleic acid detector are respectively provided with a first fluorescent group, a first quenching group, a second fluorescent group and a second quenching group at two ends; the first and second fluorophores may be the same or different fluorophores; the first quencher and the second quencher may be the same or different quenchers from each other.

In other embodiments, different labeled molecules are respectively disposed at the 5 'end and the 3' end of the single-stranded nucleic acid detector, and the results of the colloidal gold test before and after cleavage by the Cas protein of the single-stranded nucleic acid detector are detected by means of colloidal gold detection; the single-stranded nucleic acid detector shows different color development results on a colloidal gold detection line and a quality control line before and after being cut by the Cas protein.

In the present invention, the first target sequence and the second target sequence may be the same target sequence or different target sequences.

The skilled person can select the first target sequence and the second target sequence to be the same or different according to actual needs.

Preferably, the target sequences are different from each other, and thus the method for detecting a target nucleic acid of the present invention can realize multiplex detection of a nucleic acid in a sample; in one embodiment, the first target sequence, the second target sequence can be target sequences designed for the same target nucleic acid or different sites of the same gene, or target sequences designed for different target nucleic acids or different genes. In one embodiment, different target sequences may be designed for a particular bacterial, viral or disease-associated nucleic acid; in other embodiments, different target sequences may be designed for different species of bacterial, viral or disease-associated nucleic acids.

In one embodiment, the first nucleic acid detection composition and the second nucleic acid detection composition can be used in combination to achieve dual detection of the target nucleic acid.

For example, when performing a duplex detection using the first nucleic acid detecting composition and the second nucleic acid detecting composition, a different target sequence can be designed for SARS-CoV2(COVID-19) virus, and a duplex detection can be performed for two target nucleic acids of SARS-CoV2 (COVID-19); alternatively, the first and second target sequences may be designed for SARS-CoV2(COVID-19) and SARS virus, respectively, for dual detection of both SARS-CoV2(COVID-19) and SARS virus.

In another aspect, the invention provides a method of multiplex detection of a target nucleic acid in a sample, the method comprising 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 cleavage single-stranded nucleic acid detector, thereby detecting the target nucleic acid; the nucleic acid detecting composition is selected from any one or two of the first nucleic acid detecting composition and the second nucleic acid detecting composition.

In another aspect, the present invention provides a nucleic acid detecting composition selected from any one or two of the first nucleic acid detecting composition and the second nucleic acid detecting composition described above.

In another aspect, the invention also provides a system for detecting a target nucleic acid in a sample, the system comprising 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; the nucleic acid detecting composition is selected from any one or two of the first nucleic acid detecting composition and the second nucleic acid detecting composition.

In another aspect, the invention also provides a kit for detecting a target nucleic acid in a sample, the kit comprising 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; the nucleic acid detecting composition is selected from any one or two of the first nucleic acid detecting composition and the second nucleic acid detecting composition.

In another aspect, the invention also provides the use of the above system or kit for detecting a target nucleic acid in a sample. As described above, when the system or the kit of the present invention detects a target nucleic acid in a sample, one or more of the first nucleic acid detecting composition and the second nucleic acid detecting composition may be used to detect the same target sequence, or different target sequences may be used to detect, thereby achieving a dual detection effect.

In another aspect, the invention also provides the use of a nucleic acid detection composition for detecting a target nucleic acid in a sample, or in the preparation of a system or kit for detecting a target nucleic acid in a sample. The nucleic acid detecting composition is selected from any one or two of the first nucleic acid detecting composition and the second nucleic acid detecting composition.

In the present invention, the target nucleic acid includes ribonucleotide or deoxyribonucleotide, and includes single-stranded nucleic acid, double-stranded nucleic acid, such as single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA.

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 acid, thereby cleaving the single-stranded nucleic acid detector and thereby generating a detectable signal.

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 of the invention 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.

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

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.

In the invention, the guide sequence comprises 10-40 bp; preferably, 12-25 bp; preferably, 15-23 bp; preferably, 16-18 bp.

In the present invention, the gRNA has at least 50% match to a target sequence on a target nucleic acid, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%.

In one embodiment, when the target sequence contains one or more characteristic sites (e.g., a particular mutation site or SNP), the characteristic site is a perfect match to the gRNA.

In one embodiment, one or more grnas with targeting sequences different from each other can be included in the detection method, targeting different target sequences.

In one embodiment, the amino acid sequence of Cas12j12 is shown as SEQ ID No.1, or a derivative protein formed by substitution, deletion or addition of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid residues of the amino acid sequence shown as SEQ ID No.1 or an active fragment thereof and having substantially the same function.

In one embodiment, the amino acid sequence of Cas12j22 is shown as SEQ ID No.2, or a derivative protein formed by substituting, deleting or adding one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid residues of the amino acid sequence shown as SEQ ID No.2 or an active fragment thereof, and having substantially the same function.

The terms "hybridize" or "complementary" or "substantially complementary" refer to a nucleic acid (e.g., RNA, DNA) that comprises a nucleotide sequence that enables it to bind non-covalently, i.e., to form base pairs and/or G/U base pairs with another nucleic acid in a sequence-specific, antiparallel manner (i.e., the nucleic acid binds specifically to the complementary nucleic acid), "anneal" or "hybridize". Hybridization requires that the two nucleic acids contain complementary sequences, although mismatches between bases are possible. Suitable conditions for hybridization between two nucleic acids depend on the length and degree of complementarity of the nucleic acids, variables well known in the art. Typically, the length of the hybridizable nucleic acid is 8 nucleotides or more (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 is understood that the sequence of a polynucleotide need not be 100% complementary to the sequence of its target nucleic acid to specifically hybridize. A polynucleotide 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 a target region that hybridizes thereto has 100% sequence complementarity of the target region.

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. Each protein in an organism is 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, whether double-stranded or single-stranded.

The term "oligonucleotide" refers to a sequence of 3 to 100 nucleotides, preferably 3 to 30 nucleotides, preferably 4 to 20 nucleotides, more preferably 5 to 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 of the 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 the two sequences are aligned to yield maximum identity. Such an alignment can be determined by using, for example, the identity of the amino acid sequences by conventional methods, as taught by, for example, Smith and Waterman,1981, adv.Appl.Math.2:482 Pearson & Lipma n,1988, Proc.Natl.Acad.Sci.USA 85:2444, Thompson.1994, Nucleic Acids Res 22:467380, etc., by computerized operational algorithms (GAP, BESTFIT, TA, and TFASTA, Genetics Computer group p in the Wisconsin Genet ics software package). The BLAST algorithm, available from the national center for Biotechnology information (NCBI www.ncbi.nlm.ni h. gov /), can also be used, determined using default parameters.

As used herein, the "CRISPR" refers to Clustered, regularly interspaced short palindromic repeats (Clustered regular interspersed short palindromic repeats) derived from the immune system of a microorganism.

As used herein, "biotin", also known as vitamin H, is a small molecule vitamin with a molecular weight of 244 Da. "avidin", also called avidin, is a basic glycoprotein having 4 binding sites with extremely high affinity to biotin, and streptavidin is a commonly used avidin. The very strong affinity of biotin to avidin can be used to amplify or enhance the detection signal in the detection system. For example, biotin is easily bonded to a protein (such as an antibody) by a covalent bond, and an avidin molecule bonded to an enzyme reacts with a biotin molecule bonded to a specific antibody, so that not only is a multi-stage amplification effect achieved, but also color is developed due to the catalytic effect of the enzyme when the enzyme meets a corresponding substrate, and the purpose of detecting an unknown antigen (or antibody) molecule is achieved.

Target nucleic acid

As used herein, the "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-celled organisms such as bacteria, yeasts, protozoa and amoebae and the like, multicellular organisms (e.g. plants or animals, including samples from healthy or superficially healthy human subjects or human patients affected by a condition or disease to be diagnosed or investigated, 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, feces, sputum, mucus, lymph, synovial fluid, bile, ascites, pleural effusion, seroma, saliva, cerebrospinal fluid, aqueous or vitreous humor, or any bodily secretion, exudate (e.g., 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 biopsies or autopsy specimens, e.g., tumor biopsies) or may comprise cells (primary cells or cultured cells) or culture medium conditioned by any cell, tissue or organ. Exemplary samples include, but are not limited to, cells, cell lysates, blood smears, cytocentrifuge 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, callus, tissue or organ (e.g., root, stem, leaf, flower, seed, fruit), and the like.

In the present invention, the target nucleic acid also includes a DNA molecule formed by reverse transcription of RNA, and further, the target nucleic acid can be amplified by a technique known in the art, such as isothermal amplification techniques, such as 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 (NEAR), and non-isothermal amplification techniques. 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 reagents for amplification include one or more of the following: DNA polymerase, strand displacing enzyme, helicase, recombinase, single-strand binding protein, and the like.

Cas protein

The Cas protein is a protein at least having trans cleavage activity, and 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 a PAM site and specifically cut a target sequence under the action of the gRNA.

In embodiments, a Cas protein, as referred to herein, such as Cas12, also encompasses a functional variant of Cas or a homolog or ortholog thereof. As used herein, a "functional variant" of a protein refers to a variant of such a protein that at least partially retains the activity of the protein. Functional variants may include mutants (which may be insertion, deletion or substitution mutants), including polymorphs and the like. Also included in functional variants are fusion products of such proteins with another, usually unrelated, nucleic acid, protein, polypeptide or peptide. 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 orthologs or homologs thereof, may be codon optimized for expression in a eukaryotic cell. Eukaryotes can be as described herein. One or more nucleic acid molecules may be engineered or non-naturally occurring.

In one embodiment, the Cas12 protein or ortholog or homolog thereof may comprise one or more mutations (and thus the nucleic acid molecule encoding it may have one or more mutations.

In one embodiment, the Cas protein may be from: cilium, listeria, corynebacterium, satrapia, legionella, treponema, Proteus, eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flavivivola, Flavobacterium, Azospirillum, Sphaerochaeta, gluconacetobacter, Neisseria, Rochelia, Parvibaculum, Staphylococcus, Nitrarefactor, Mycoplasma, Campylobacter, and Muspirillum.

In one embodiment, the Cas protein is selected from the group consisting of proteins consisting of:

(1) a protein shown as SEQ ID No. 1-2;

(2) derived proteins which are formed by substituting, deleting or adding one or more (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid residues in the amino acid sequence shown in SEQ ID No.1-2 or active fragments thereof and have basically the same functions.

In one embodiment, the Cas protein further includes proteins having 50%, preferably 55%, preferably 60%, preferably 65%, preferably 70%, preferably 75%, preferably 80%, preferably 85%, preferably 90%, preferably 95%, sequence identity to the above sequences and having trans activity.

The Cas protein can be obtained by recombinant expression vector technology, namely, a nucleic acid molecule 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 cells, or the protein can be obtained by breaking cells through a conventional extraction technology. The encoding 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 which facilitate sequence integration, or self-replication. The vector may be, for example, of the plasmid, virus, cosmid, phage, etc. type, which are well known to those skilled in the art, and preferably, the expression vector of 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 an appropriate vector and host cell.

gRNA

As used herein, the "gRNA" is also referred to as guide RNA or guide RNA and has a meaning commonly understood by those skilled in the art. In general, the guide RNA may comprise, or consist essentially of, a direct repeat and a guide sequence (guide sequence). grnas may include crRNA and tracrRNA or only crRNA depending on Cas protein on which they depend in different CRISPR systems. The crRNA and tracrRNA may be artificially engineered to fuse to form single guide RNA (sgRNA). In certain instances, the guide sequence is any polynucleotide sequence that is sufficiently complementary to the target sequence (the signature sequence described in the present invention) to hybridize to the target sequence and direct specific binding of the CRISPR/Cas complex to the target sequence, typically having a sequence length of 12-25 nt. The direct repeat sequence can fold to form a specific structure (such as a stem-loop structure) for recognition by the Cas protein to form a complex. The targeting sequence need not be 100% complementary to the signature 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 match) 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. 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.

The gRNA of the invention can be natural, and can also be artificially modified or designed and synthesized.

Single-stranded nucleic acid detector

The single-stranded nucleic acid detector of the present invention comprises different reporter groups or marker molecules at both ends, and exhibits no reporter signal when in an initial state (i.e., non-cleaved state), and a detectable signal when the single-stranded nucleic acid detector is cleaved, i.e., exhibits 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.

In one embodiment, the reporter group or the marker molecule comprises a fluorescent group and a quenching group, wherein the fluorescent group is selected from one or any several 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 one embodiment, the single stranded 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 single-stranded 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 that binds to a first molecule (i.e. a first molecular antibody) at the sample contacting end (colloidal gold), an antibody that binds to the first molecular antibody at the first line (control line), and an antibody that binds to a second molecule (i.e. a second molecular antibody, such as avidin) at the second line (test line). As 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 reporters are 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 (side) flow test or a (side) flow immunochromatographic assay. In some aspects, the molecules in the single-stranded nucleic acid detector may be replaced with each other, or the positions of the molecules may be changed, and the modified form is also included in the present invention as long as the reporting principle is the same as or similar to that of the present invention.

Drawings

Figure 1 is a graph of the fluorescence results of Cas12j12 for different single stranded nucleic acid detectors when used for in vitro nucleic acid detection.

Figure 2 is a graph of the fluorescence results of Cas12j22 for different single stranded nucleic acid detectors when used for in vitro nucleic acid detection.

Detailed description of the preferred embodiments

The present invention will be further described with reference to the following examples, which are intended to be illustrative only and not to be limiting of the invention in any way, and any person skilled in the art can modify the present invention by applying the teachings disclosed above and applying them to equivalent embodiments with equivalent modifications. Any simple modification or equivalent changes made to the following embodiments according to the technical essence of the present invention, without departing from the technical spirit of the present invention, fall within the scope of the present invention.

The technical scheme of the invention is based on the following principle, the nucleic acid of a sample to be detected is obtained, for example, a target nucleic acid can be obtained by an amplification method, and the gRNA which can be paired with the target nucleic acid is used for guiding the Cas protein to be identified and combined on the target nucleic acid; subsequently, the Cas protein activates the cleavage activity of the single-stranded nucleic acid detector, thereby cleaving the single-stranded nucleic acid detector in the system; the two ends of the single-stranded nucleic acid detector are respectively provided with a fluorescent group and a quenching group, and if the single-stranded nucleic acid detector is cut, fluorescence can be excited; in other embodiments, both ends of the single-stranded nucleic acid detector may be provided with a label capable of being detected by colloidal gold.

In this embodiment, the Cas proteins used are Cas12j12 and Cas12j22, and the amino acid sequences thereof are respectively shown as SEQ ID nos. 1 and 2. The direct repeats (the region binding to the Cas protein, also called DR region) of the grnas corresponding to Cas12j12 and Cas12j22 are shown as SEQ ID nos. 3 and 4, respectively.

Example 1 nucleic acid detection Using Cas12j12 and Cas12j22

In this embodiment, different single-stranded nucleic acid detectors are designed and used for detection with Cas12j12 and Cas12j 22. The different single-stranded nucleic acid detectors are respectively Reporter-FB-T (polyT), Reporter-FB-A (polyA) and Reporter-FB-C (polyC), the sequences are respectively: 5 '6-FAM-TTTTT-3' BHQ1, 5 '6-FAM-AAAAA-3' BHQ1 and 5 '6-FAM-CCCCC-3' BHQ 1.

Target nucleic acids targeted by different Cas proteins and designed grnas are shown in the following tables, respectively:

the following reaction system is adopted: cas enzyme final concentration is 50nM, gRNA final concentration is 50nM, target nucleic acid final concentration is 500nM, single-stranded nucleic acid detector final concentration is 200 nM. Incubation at 37 ℃ and reading FAM fluorescence/1 min. The control group had no target nucleic acid added.

As shown in fig. 1-2, Cas12j12 was able to significantly exhibit a fluorescent signal when using single stranded nucleic acid detectors of polyC and polyT, but did not substantially cleave polyA; cas12j22 was able to significantly exhibit a fluorescent signal when using a single-stranded nucleic acid detector of polyA and polyC, but the fluorescent signal was weak when using polyT. The above experiments reflect that Cas12j12 and Cas12j22 can be used for detection of target nucleic acids in conjunction with single-stranded nucleic acid detectors.

As described above, it is surprising that Cas12j12 and Cas12j22 exhibit different base preferences for the nucleotides of a single-stranded nucleic acid detector. Specifically, both can exhibit significant cleavage activity to polyC; however, Cas12j12 and Cas12j22 exhibit different preferences for polyA and polyT. Cas12j22 showed significant cleavage activity for polyA single-stranded nucleic acid detector, but Cas12j12 did not substantially cleave polyA; cas12j12 showed significant cleavage activity for a single stranded nucleic acid detector of polyT, but Cas12j22 did not substantially cleave polyT; this means that using a combination of Cas12j12 and polyT and Cas12j22 and polyA, it can be used for multiplex detection of nucleic acids.

SEQUENCE LISTING

<110> Shunheng Biotech Co., Ltd

<120> method for nucleic acid detection using Cas protein

<130> SF065

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 1013

<212> PRT

<213> Artificial Sequence

<220>

<223> Cas12j12

<400> 1

Met Ala Ser Ser Asp Ala Gln Lys Phe Pro Gln Thr His Asn Lys Val

1 5 10 15

Met Ser Phe Arg Leu Thr Ala Ser Asn Ile Gly Ser Val Leu Ser Leu

20 25 30

His Ser Asn Leu His Asp Ala Ala Glu Ile Gly Ile Asn Glu Cys Arg

35 40 45

Trp Trp Ile Gly Asp Gly Glu Ile Tyr Glu Arg Asp Pro Ala Cys Arg

50 55 60

Ser Ile Lys Lys Gly Asn Asp Ile Arg Thr Val Thr Ser Glu Lys Ile

65 70 75 80

Lys Glu Leu Trp Thr Lys His Thr Asp His Ser Val Pro Leu Val Asp

85 90 95

Phe Ile Asp Met Leu Lys Phe Val Ala Gln Cys Ala Ile Tyr Gly Asp

100 105 110

Ser Arg Ala Leu Ala Ser Thr Leu Phe Gly Lys Ser Lys Ala Glu Thr

115 120 125

Arg Gly Val Ser Thr Glu Asp Met Thr Val Ile Arg Ala Trp Ile Ala

130 135 140

Glu Thr Asp Ala Val Leu Ala Ser Gly Leu Ser Pro Lys Lys Lys Lys

145 150 155 160

Lys Lys Glu Lys Glu Ala Gly Lys Lys Glu Arg Lys Pro Asp Val Lys

165 170 175

Met Glu Met Cys Arg Arg Ile Arg Cys Thr Met Val Gln Cys Gly Tyr

180 185 190

Phe Arg Arg Phe Pro Phe Glu Ala Lys Ile Asp Asn Gly Gly Glu Arg

195 200 205

Gly Lys Met Asp Ser Glu Leu Ser Tyr Val Ser Ala Arg Asn Leu Leu

210 215 220

Arg Cys Leu Ser Thr Trp Arg Ala Ser Ser Val Met Arg Arg Asp Ser

225 230 235 240

Tyr Leu Ile Glu Glu Glu Arg Ile Lys Glu Ala Glu Ser Lys Met Thr

245 250 255

Pro Glu Ile Ile Asp Gly Leu Arg Arg Leu Tyr Arg Tyr Cys Ala Val

260 265 270

Asp His Asp Phe Leu Lys Trp Phe Gly Gly Arg Ile Ile Arg His Ile

275 280 285

Asp Ser Cys Leu Ala Pro Ala Ile Ala Gly Asn Thr Gly Arg Pro Thr

290 295 300

Gly Gly Glu Ser Phe Thr Val Ile Tyr Asp Arg Arg Lys Lys Arg Asp

305 310 315 320

Val Lys Ile Thr Tyr Ser Val Pro Glu Glu Ile Tyr Gly Tyr Leu Ser

325 330 335

Ser His Pro Glu Leu Val Ala Ile Gly Lys Asp Gly Met Thr Pro Ile

340 345 350

Ser Arg His Ala Asp Tyr Leu Glu Met Ile Ala Ser His Glu Lys His

355 360 365

Arg Trp Tyr Ala Thr Phe Pro Thr Val Gly Lys Glu Asp Gly Tyr Arg

370 375 380

Thr Ser Val Leu Leu Gly Lys Asn Tyr Leu Thr Tyr Asp Leu Ser Tyr

385 390 395 400

Asp Gly Glu Ser Val Pro Asp Lys Lys Ile Asn Val Ile Ser Lys Gly

405 410 415

Gln Pro Val Cys Leu Asp Leu His Asp Gly Arg Arg Val Ser Ser Leu

420 425 430

Tyr Leu Thr Val Gly Glu Ser Ala Ala Tyr Asp Ile Ala Val Arg Lys

435 440 445

Asn Lys Arg His His Gly Lys Pro Ala Asp Tyr Cys Arg Met Arg Val

450 455 460

His Leu Thr Gln Glu Arg Glu Asp Lys Thr Tyr Asn Asp Pro Tyr Phe

465 470 475 480

Ser Asn Met Glu Ile Trp Arg Ala Gly Asp Gln Val Tyr Ala Ile Glu

485 490 495

Phe Asp Arg His Gly Ala Arg Tyr Thr Ala Ile Val Lys Glu Pro Ser

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Val Glu Tyr Arg Asn Lys Lys Leu Tyr Leu Arg Val Asn Met Val Leu

515 520 525

Asp Ser Pro Ser Arg Gln Asp Asp Lys Asp Met Tyr Tyr Ala Tyr Met

530 535 540

Thr Ala Tyr Pro Ser Ser Asn Pro Pro Val Glu Thr Ser Asp Asn Lys

545 550 555 560

Lys Arg Phe Glu Arg Leu Gly Pro Gly Arg Arg Ala Ile Gly Gly Ile

565 570 575

Asp Ile Gly Ile Gly Arg Pro Tyr Val Ala Val Val Ala Ser Tyr Glu

580 585 590

Val Gly Pro Ala Gly Thr Glu Gln Lys Phe Gln Ile Glu Asp Arg Leu

595 600 605

Ile Glu Asp Asp Gly Ser Ser Pro Tyr Asp Ser Leu Tyr Asn Asp Phe

610 615 620

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

625 630 635 640

Ile Ser Glu Gly Asp Leu Glu Asp Ile Pro Ser Asp Met Ser Val Asp

645 650 655

Glu Asp Gly Ser Ile Ala Ala Thr Met Lys Arg Met Ser Ala Arg Ile

660 665 670

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

675 680 685

Thr Phe Leu Lys Met Asn His Lys Gln Arg Leu Asp Ile Leu Leu Thr

690 695 700

Gln Lys Ala Ser Asn Ala Thr Leu Lys Gln Leu Val Glu Glu Asp Pro

705 710 715 720

Ser Phe Leu Pro Arg Ile Cys Val Tyr Tyr Val Ile Ser Val Glu Arg

725 730 735

Glu Leu Lys Asn Lys His Arg Asn Ala Tyr Leu Asp Gly Leu Thr Val

740 745 750

Asp Glu Lys Tyr Ser Gly Glu Thr Lys Arg Gly Tyr Ala Gln Lys Arg

755 760 765

Leu Asn Ser Met Leu Arg Ala Tyr Ser Ala Leu Gly Glu Glu Glu Thr

770 775 780

Asp Glu Val Arg Thr Phe Ser Thr Arg Ser Glu Lys Val Arg Asn Met

785 790 795 800

Ala Lys Asn Ala Ile Lys Arg Asn Ala Arg Lys Leu Val Asn Phe Tyr

805 810 815

Val Gly Lys Gly Ile Arg Thr Ile Val Ala Glu Asp Thr Asp Pro Thr

820 825 830

Lys Ser Arg Asn Asp Gly Lys Lys Ser Asn Arg Ile Lys Ala Ala Trp

835 840 845

Ser Pro Lys Gln Phe Leu Ala Ala Val Lys Asn Ala Ala Gln Trp His

850 855 860

Gly Leu Glu Ile Ala Glu Val Asp Pro Arg Met Thr Ser Gln Val His

865 870 875 880

Pro Glu Thr Gly Leu Ile Gly Tyr Arg Asp Gly Asp Thr Leu His Cys

885 890 895

Pro Asp Gly Ser Lys Ile Asp Ala Asp Val Ala Gly Ala Ala Asn Val

900 905 910

Cys Arg Val Phe Ala Gly Arg Gly Leu Trp Arg Phe Ser Ile Asn Thr

915 920 925

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

930 935 940

Ile Val His His Phe Gly Ser Glu Ser Asn Trp Glu Lys Phe Arg Lys

945 950 955 960

Gln Tyr Pro Ser Gly Thr Thr Leu Tyr Leu His Gly Arg Glu Trp Leu

965 970 975

Thr Ala Glu Glu His Lys Ser Ala Ile Asp Arg Ile Arg Asp Asp Val

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

995 1000 1005

Lys Val Glu Ile Phe

1010

<210> 2

<211> 968

<212> PRT

<213> Artificial Sequence

<220>

<223> Cas12j22

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Met Ser Lys Ala Thr Arg Lys Thr Lys Thr Thr Val Pro Glu Ser Thr

1 5 10 15

Asp Thr Glu Ser Pro Ala Ala Asp Thr Gln Val Arg Val His Trp Leu

20 25 30

Ala Ala Ser His Arg Ala Ser Pro Gly Leu Gln Gln Val Lys Glu Met

35 40 45

Ile Gln Gln His Ala Asp Val Ala Ser Val Leu Phe Gln Gly Leu Val

50 55 60

Arg Thr Ala Pro Ile Val Phe Arg Asn Asp Asp Gly Ser Pro Val Lys

65 70 75 80

Pro Leu Asp Leu Leu Leu Ala Ser Leu Arg Pro Thr Tyr Lys Val Gln

85 90 95

Arg Asp Thr Glu Thr Val Leu Val Thr Lys Asp Asp Val Ile Arg Cys

100 105 110

Leu Thr Leu Ala Thr Thr Ala Val Asn Gly Gly Gln Ala Thr Asn Val

115 120 125

Ala Val Phe Ala Ser Ala Asp Pro Ala Leu Ser Ala Pro Leu Ala Thr

130 135 140

Leu Leu Ala Gln Leu Arg Ala Leu Glu Ser Val Asp Ser Ser Trp Ser

145 150 155 160

Val Val Gly Lys Leu Asp Ile Asn Leu Arg Lys Phe Val Trp Leu Val

165 170 175

Leu Ser Ala Ala Gly Val Leu Pro Ala Leu Ala Asp Leu Glu Gly Tyr

180 185 190

Ala Ala Lys Ser Val Leu Ala Asn Val Gln Gly Lys Tyr Lys Ser Leu

195 200 205

Gln Ala Cys Ala Asp Thr His Ala Ala Leu Tyr Lys Gln His Gln Thr

210 215 220

Asn Lys Glu Gln Leu Glu Lys Leu Ile Ala Asp Pro Gly Phe Val Ala

225 230 235 240

Leu Cys Ser Ala Leu Leu Gln Asp Pro Asp Leu Arg Ser Val Asp Ser

245 250 255

Arg Arg Leu Ala Ala Leu Glu Glu Met Leu Gly Phe Val Ala Ala Asp

260 265 270

Lys Asn Tyr Ser Glu Tyr Thr Ser Thr Arg Lys Cys Asp Gly Trp Ala

275 280 285

Pro Pro Ala Asn Met Phe Asp Leu Leu Cys Glu His Lys Glu Ala Val

290 295 300

Arg Arg Asn Ile Val Val Asp Asn Ser Lys Cys Leu Ser Arg Arg Ile

305 310 315 320

Ser Leu Val Ala Asp Gly Asp Val Asn Glu Val Ser Val Phe Glu Leu

325 330 335

Leu Asn Glu Met Arg Trp Leu Ser Val His Ser Ser Gly Ile Arg Met

340 345 350

Pro Asn Tyr Pro Lys His Ala Tyr Ala Leu Lys Phe Gly Asp Asn Tyr

355 360 365

Ile Ser Val Lys Ser Phe Glu Thr Val Val Asp Gly Gly Cys Ser Leu

370 375 380

Leu Arg Met Thr Ala Arg Val Gly Lys Asn Asp Leu Val Cys Asp Phe

385 390 395 400

Val Leu Gly Arg Gly Asn Glu Tyr Trp Asn Asn Leu Lys Ile Thr Pro

405 410 415

Met Gly Lys Gly Ile Phe Ala Val Val Lys Thr Val Arg Arg Phe Thr

420 425 430

Ala Thr Gly Ala Lys Leu Val Glu Leu Arg Gly Val Cys Lys Glu Pro

435 440 445

Glu Ile Arg Tyr Glu Arg Gly Val Leu Gly Leu Arg Leu Pro Ile Ser

450 455 460

Phe Asp Val Tyr Gly Lys Val Glu Glu Asp Ser Ile Ala Phe Gly Lys

465 470 475 480

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

485 490 495

Phe Gln Gly Leu Leu Asp Tyr Arg Asn Thr Thr Ala Arg Asp Gly Tyr

500 505 510

Ile Tyr Tyr Ala Gly Phe Asp Gln Gly Glu Asn Asp Gln Val Val Gly

515 520 525

Ile Tyr Arg Thr Arg Thr Tyr Lys Asn Ala Thr Met Leu Glu Phe Phe

530 535 540

Asn Val Ser Asp Thr Leu Glu Glu Val Ala Ser Cys Arg Phe Ser Asp

545 550 555 560

Tyr Gln Glu Arg Lys Arg Arg Leu Arg Gly Asp Thr Gly Val Leu Asp

565 570 575

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

580 585 590

Leu Ile Ser Thr Leu Arg Ala Cys Ala Ser His Thr Asp Trp Tyr Pro

595 600 605

Lys Leu Lys Glu Arg Arg Arg Leu Glu Trp Ala Val Leu Ala Gln Gly

610 615 620

Val Gly Val Ser Asp Phe Asp Thr Glu Ile Glu Arg Ala Glu Thr Ala

625 630 635 640

Leu Ser Ala Val Ala Ala Val Asp Phe Val Arg Asp Pro Thr Cys Ile

645 650 655

Ile Asn Val Met Asp Lys His Ile Tyr Ala Gln Phe Lys Gln Leu Arg

660 665 670

Ser Glu Arg Asn Glu Lys Tyr Arg Ser Gln His Gln His Asp Tyr Lys

675 680 685

Trp Leu Gln Leu Val Asp Ser Val Ile Ser Leu Arg Lys Ser Ile Tyr

690 695 700

Arg Phe Gly Lys Ala Pro Glu Pro Arg Gly Ala Gly Glu Leu Tyr Pro

705 710 715 720

Gln Asn Leu Tyr Thr Tyr Arg Asp Asn Leu Met Gln Gln Tyr Arg Lys

725 730 735

Glu Val Ala Ala Phe Ile Arg Asp Val Cys Leu Glu His Gly Val Arg

740 745 750

Gln Leu Ala Val Glu Ala Leu Asn Pro Thr Ser Tyr Ile Gly Glu Asp

755 760 765

Ser Asp Ala Asn Arg Lys Arg Ala Leu Phe Ala Pro Ser Glu Leu His

770 775 780

Asn Asp Ile Val Leu Ala Cys Ser Leu His Ser Ile Ala Val Val Ala

785 790 795 800

Val Asp Glu Thr Met Thr Ser Arg Val Ala Pro Asn Asn Arg Leu Gly

805 810 815

Phe Arg Ser His Gly Asp Tyr Gln Lys Phe Ser Glu Thr Ala Gln Gly

820 825 830

Arg Phe Asn Trp Lys His Leu His Tyr Phe Gly Asp Asn Asp Val Ser

835 840 845

Glu His Cys Asp Ala Asp Glu Asn Ala Cys Arg Asn Ile Val Leu Arg

850 855 860

Ala Leu Thr Cys Gly Ala Ser Lys Pro Arg Phe Ser Arg Gln Ser Leu

865 870 875 880

Leu Gly Lys Ile Lys Gly Pro Val Leu Arg Thr Gln Leu Ala Tyr Leu

885 890 895

Ala His Lys Arg Gly Leu Leu Thr Ala Ser Thr Glu Pro Lys Lys Ala

900 905 910

Ala Glu Thr Gly Phe Glu Leu Val Glu Ala Asp Leu Gly Gly Ala Leu

915 920 925

Arg Val Gly Lys Gly Phe Ile Tyr Val Asp Ala Gly Ile Cys Ile Asn

930 935 940

Ala Thr Thr Arg Lys Glu Arg Ser His Lys Val Gly Glu Ala Val Val

945 950 955 960

Ser Arg Ser Leu Ala Ser Pro Phe

965

<210> 3

<211> 37

<212> RNA

<213> Artificial Sequence

<220>

<223> Cas12j12-DR

<400> 3

gagguagugu ggaaguccag cagggcuucg uugacac 37

<210> 4

<211> 36

<212> RNA

<213> Artificial Sequence

<220>

<223> Cas12j22-DR

<400> 4

auuucagugc uggccugugg aagcaggcuc ugucac 36

Claims (10)

1. A method of detecting a target nucleic acid in a sample, the method comprising 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 cleavage single-stranded nucleic acid detector, thereby detecting the target nucleic acid;

the nucleic acid detecting composition is selected from any one or two of a first nucleic acid detecting composition and a second nucleic acid detecting composition;

the first nucleic acid detection composition includes Cas12j12, a first gRNA that can bind Cas12j12 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 includes Cas12j22, a second gRNA that can bind Cas12j22 and hybridize to a second target sequence on the target nucleic acid, and a second single-stranded nucleic acid detector;

the Cas12j12 is selected from Cas proteins shown as any one of i-iii below:

i. a Cas protein having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to SEQ ID No.1 and having trans cleavage activity;

ii. A Cas protein having an amino acid sequence with substitution, deletion or addition of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acids compared to SEQ ID No.1, and having trans cleavage activity;

iii, a Cas protein comprising the amino acid sequence shown in SEQ ID No. 1;

the Cas12j22 is selected from Cas proteins shown as any one of i-iii below:

i. a Cas protein having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to SEQ ID No.2 and having trans cleavage activity;

ii. A Cas protein having an amino acid sequence with one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions, deletions, or additions compared to SEQ ID No.2, and having trans cleavage activity;

iii, a Cas protein comprising the amino acid sequence shown in SEQ ID No. 2.

2. A method of multiplex detection of a target nucleic acid in a sample, the method comprising 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 cleavage single-stranded nucleic acid detector, thereby detecting the target nucleic acid;

the nucleic acid detection composition is selected from the group consisting of the first nucleic acid detection composition and the second nucleic acid detection composition of claim 1.

3. The method according to claim 1 or 2,

the nucleotides of the first single-stranded nucleic acid detector consist of polyT or polyC, preferably, polyT;

the nucleotides of the second single-stranded nucleic acid detector are composed of polyA or polyC, preferably, polyA.

4. The method of claim 1 or 2, wherein the detectable signal is detected by: vision-based detection, sensor-based detection, color detection, gold nanoparticle-based detection, fluorescence polarization, fluorescence signal-based detection, colloidal phase transition/dispersion, electrochemical detection, and semiconductor-based detection.

5. The method of claim 1 or 2, wherein the target nucleic acid comprises a ribonucleotide or a deoxyribonucleotide; preferably, it includes single-stranded nucleic acids, double-stranded nucleic acids, for example, single-stranded DNA, double-stranded DNA, single-stranded RNA.

6. The method according to claim 1 or 2, wherein the 5 'end and the 3' end of the single-stranded nucleic acid detector are provided with different reporter groups, respectively; alternatively, different marker molecules are provided at the 5 'end and the 3' end of the single-stranded nucleic acid detector, respectively.

7. The method according to claim 1 or 2, wherein the target nucleic acid is derived from a sample of a virus, a bacterium, a microorganism, soil, a water source, a human body, an animal, a plant, or the like.

8. The method of claim 1 or 2, further comprising the step of obtaining the target nucleic acid from the sample.

9. A system or composition or kit for detecting a target nucleic acid in a sample, the system or composition or kit comprising 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; the nucleic acid detecting composition is selected from any one or two of the first nucleic acid detecting composition and the second nucleic acid detecting composition described in claim 1.

10. Use of the system or composition or kit of claim 9 for detecting a target nucleic acid in a sample.

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