CN118186066A

CN118186066A - Application and method of C2C9 nuclease in preparing gene detection product

Info

Publication number: CN118186066A
Application number: CN202211601878.1A
Authority: CN
Inventors: 季泉江; 陈未中; 马佳诚
Original assignee: ShanghaiTech University
Current assignee: ShanghaiTech University
Priority date: 2022-12-13
Filing date: 2022-12-13
Publication date: 2024-06-14

Abstract

The invention relates to the field of biotechnology, in particular to application of C2C9 nuclease in preparing a gene detection product and a method thereof, wherein the gene detection product comprises a fluorescent probe, and C2C9 nuclease and sgRNA or nucleotides for encoding the C2C9 nuclease and the sgRNA. The gene detection product can be used for identifying biological species, and the identification or detection method comprises the step of detecting a gene sequence to be detected by using the gene detection product. The gene detection method is simple and convenient to operate, has high detection efficiency, and can rapidly realize the detection of the specific gene to be detected.

Description

Application and method of C2C9 nuclease in preparing gene detection product

Technical Field

The invention relates to the field of biotechnology, in particular to application and a method of C2C9 nuclease in preparing a gene detection product.

Background

Gene detection, also known as nucleic acid detection, is an important component in the biotechnology field. Different nucleic acid detection methods have been developed based on the sequence specificity of the genetic material DNA and RNA of different organisms and species. Traditional nucleic acid detection comprises a sequencing technology, a isothermal amplification technology and the like, can detect DNA or RNA specific sequences of various pathogens, and can also analyze and detect SNP loci related to diseases.

CRISPR/Cas (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) is a novel gene editing system developed in recent years. The system is mainly composed of a Cas endonuclease and a corresponding guide RNA (sgRNA). Cas endonuclease is able to specifically bind to guide RNA, targeting recognition and cleavage of specific DNA sites with base complementary pairing, resulting in double strand breaks of DNA. Because of the simplicity and efficiency of CRISPR systems, the systems have been widely used in basic research, biotechnology development, and disease treatment research and development in the fields of life sciences, medicine, agronomy, etc. In addition to having a fairly wide range of applications in the field of gene editing, the system has also been developed as a gene detection tool.

The conventional gene detection technology has complex flow, high requirements on detection equipment and relatively long detection time. CRISPR system gene detection tools can greatly reduce this flow. Therefore, the detection tool which is simple, convenient, efficient and low in cost is developed, the requirements of the gene detection technical equipment and instruments can be reduced while the efficient gene detection is realized, and the detection tool has good practical application value and commercial value.

Disclosure of Invention

In view of the above-described drawbacks of the prior art, an object of the present invention is to provide a use and a method of C2C9 nuclease in the preparation of a gene detection product for solving the problems in the prior art.

To achieve the above and other related objects, the present invention discloses a novel gene detection system based on a very small CRISPR/C2C9 system and an application of C2C9 nuclease in preparing a gene detection product. The CRISPR/C2C9 detection system is used for detecting samples, and the specificity of the guide part in the sgRNA is used for detecting specific nucleic acid sequences, so that the detection of target genes can be rapidly and efficiently realized.

The invention provides an application of C2C9 nuclease in preparing a gene detection product.

The invention also provides a gene detection product, which comprises a fluorescent probe, and a C2C9 nuclease and an sgRNA, or a nucleotide encoding the C2C9 nuclease and the sgRNA.

The invention also provides application of the gene detection product in identifying biological species.

The invention also provides a gene detection method for the purpose of non-disease diagnosis, which comprises the step of detecting a gene sequence to be detected by using the gene detection product of the claim.

As described above, the use and method of the C2C9 nuclease of the present invention for preparing a gene detection product has the following advantageous effects: the gene detection method is simple and convenient to operate, high in detection efficiency and capable of rapidly detecting the specific gene to be detected.

Drawings

FIG. 1 is a schematic diagram of an experiment for detecting a target gene by using a CRISPR/C2C9 system, wherein the CRISPR system mainly used in the invention is a V-type CRISPR system, effector proteins are mainly C2C9 nuclease family, and the inventor finds that the nuclease generally has nonspecific ssDNA cleavage activity, and the nonspecific cleavage activity is activated only after a complex of the C2C9 effector proteins and sgRNA is correctly targeted to a target sequence. By adding a ssDNA fluorescent probe with fluorescent groups and fluorescence quenching groups such as FAM (F) and BHQ1 (Q), effector protein side cleavage activity is activated when a target sequence of interest exists in a sample, and the probe is cut off to release fluorescence, so that detection of the target gene sequence is realized.

FIG. 2 is a fluorescent signal of sgRNA targeting ssDNA fragments and non-targeting sgRNAs in a CRISPR/C2C9 detection system. The results show that targeting sgrnas can produce a distinct detection signal, whether or not PAM sequence is contained in the substrate ssDNA, but the substrate ssDNA containing PAM sequence produces a fluorescent signal at a faster rate. Whereas the non-targeted sgrnas produce substantially no fluorescent signal.

FIG. 3 is a fluorescent signal of sgRNA targeting dsDNA fragments and non-targeting sgRNA in a CRISPR/C2C9 detection system. The results show that targeting sgrnas can produce a distinct detection signal, whether or not PAM sequence is contained in the substrate dsDNA, but the substrate dsDNA containing PAM sequence produces a fluorescent signal at a faster rate. Whereas the non-targeted sgrnas produce substantially no fluorescent signal.

Detailed Description

The C2C9 nuclease belongs to an important effector protein in the V-type CRISPR system. The inventors found in experimental studies that the complex of C2C9 nuclease and sgRNA not only has directed double-stranded DNA (dsDNA) cleavage activity, but also has non-specific single-stranded DNA (ssDNA) cleavage activity. When the complex of C2C9-sgRNA binds to the target DNA substrate, the nonspecific collateral ssDNA cleavage activity is activated, and in vitro gene detection can be achieved by utilizing this special property. Meanwhile, C2C9 has unique PAM recognition properties of 5'aan, unlike that of most V-type Cas12 family proteins 5' T-rich PAM. Thus, C2C9 can greatly expand the recognition and detection range of double-stranded DNA substrates.

The invention firstly provides application of C2C9 nuclease in preparing a gene detection product.

The C2C9 nuclease has at least one of the following activities: regulate transcription within a target gene (e.g., target DNA), cleavage activity (endoribonuclease and/or endonuclease activity), gene editing activity, etc. The C2C9 nuclease may be derived from any biological species.

The C2C9 nucleases described in the present invention may be C2C9 nucleases conventional in the art.

The C2C9 nuclease is:

(I) A wild-type C2C9 nuclease or fragment thereof, having RNA-guided nucleic acid binding activity;

(II) a variant having at least 30% sequence homology with the amino acid sequence of (I) and having RNA-guided nucleic acid binding activity;

(III) according to (I) or (II), further comprising a nuclear localization signal fragment;

(IV) according to (I) or (II) or (III), further comprising:

(a) One or more modifications or mutations that result in a dna sequence having significantly reduced endonuclease activity, or a loss of endonuclease activity, compared to the endonuclease sequence prior to the modification or mutation; and/or

(B) A polypeptide or domain having other functional activity;

(V) the C2C9 nuclease according to (I) or (II) or (III) has endonuclease activity.

In the present invention, the amino acid sequence of the C2C9 nuclease is derived from any one or more ：Actinomadura craniellae、Candidatus Frankia meridionalis、Corynebacterium glutamicum、Dermacoccus sp.UBA1591、Kitasatospora sp.NA04385、Kocuria indica、Kocuria sp.cx-455、Mesorhizobium sp.B2-6-6、Mycobacterium heckeshornense、Micrococcus luteus、Mycobacterium sp.MS1601、Mycolicibacterium goodii、Nocardiopsis metallicus、Nocardiopsis synnemataformans DSM 44143、Planctomycetes bacterium、Propionimicrobium lymphophilum、Pseudactinotalea sp.HY160、Rothia dentocariosa、Rothia kristinae、Rothia mucilaginosa、Rothia nasimurium、Rothia sp.HMSC071C12、Saccharomonospora piscinae、Saccharopolyspora rectivirgula DSM 43747、Streptomyces albidus、Streptomyces bauhiniae、Streptomyces bauhiniae、Streptomyces griseocarneus、Streptomyces longispororuber、Streptomyces lydicus、Streptomyces netropsis、Streptomyces niveus NCIMB 11891、Streptomyces rimosus subsp.Rimosus、Streptomyces sp.CB04723、Streptomyces sp.CNH099、Streptomyces sp.CNS606、Streptomyces sp.IB2014 016-6、Streptomyces sp.man185、Streptomyces sp.S4、Streptomyces sp.SID2563、Streptomyces sp.UH6、Streptomyces spororaveus、Streptomyces xanthochromogenes、Streptosporangium violaceochromogenes、Cellulosimicrobium cellulans、Rhodococcus hoagii、Rhodococcus sp.OK519、Rothia sp.HMSC071C12、Nocardiopsis alba、Streptosporangium subroseum、Streptomyces agglomeratus、Murinocardiopsis flavida、Streptomyces sp.NRRL F-2664、Cellulomonas marina、Miniimonas sp.S16、Streptomyces sp.SPB074、Streptomyces yunnanensis、Mycobacteroides abscessus、Streptomyces sp.CB02009、Kitasatospora mediocidica、Streptomyces sp.、Quadrisphaera granulorum、Prauserella rugosa、Rothia sp.HMSC061D12、Mycobacterium grossiae、Mycobacterium avium、Corynebacterium maris、Streptomyces sp.NRRL S-378、Kitasatospora cheerisanensis KCTC 2395、Mycobacterium sp.SWH-M3、Streptomyces roseochromogenus、Mycobacterium intracellulare subsp.yongonense 05-1390、Mycobacterium sp.JS623、Mycolicibacterium fallax、Nocardiopsis sp.JB363、Rothia sp.HMSC069C03、Mycobacterium avium. of the following, and the amino acid sequence of the C2C9 nuclease is any one of the amino acid sequences shown in SEQ ID No.1-115, or has at least 50% sequence homology with any one of the amino acid sequences shown in SEQ ID No.1-115, and has RNA-guided nucleic acid binding activity and/or endonuclease activity.

In a preferred embodiment of the invention, the C2C9 nuclease is Actinomadura CRANIELLAE C C9 (AcC 2C 9). The nucleotide sequence encoding the AcC2C9 preferably comprises the sequence shown as SEQ ID NO:116 or a variant thereof having at least about 90% (such as at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to SEQ ID NO: 116. Actinomadura CRANIELLAE C C9 or "AcC2C9" recognizes the PAM sequence AAN.

In some embodiments, to take advantage of various enzymatic properties of different C2C9 nucleases (e.g., for different PAM sequence preferences), C2C9 nucleases of different species sources are advantageous for providing different applications, e.g., increasing or decreasing enzymatic activity; increasing or decreasing the level of cytotoxicity; altering the balance between NHEJ, homology directed repair, single strand breaks, double strand breaks, etc.

A C2C9 nuclease from a different species or a different C2C9 nuclease from the same species may require a different PAM sequence in the target DNA, and thus, the PAM sequence requirements may be different from the PAM sequences described previously for the particular C2C9 nuclease selected. For example, acC2C9 nuclease can recognize effectively PAM sequence 5' -AAN (N represents A, T, C or G, either base), cgC2C9 can recognize effectively PAM sequence 5' -AAS (S represents either C or G, either base), rdC C9 and MiC C9 can recognize effectively PAM sequence 5' -AAH (N represents A, T or C, either base).

In some embodiments, these small C2C9 s can be used in any of the systems, compositions, kits, and methods described herein below.

The C2C9 nuclease provided by the invention has a small number of amino acids. In a preferred embodiment, the C2C9 nuclease interacts with the PAM sequence AAN when the amino acid sequence is set forth in SEQ ID NO. 116. Cleavage sites are typically present within one to three base pairs upstream of the PAM sequence. A C2C9 nuclease from a different species or a different C2C9 nuclease from the same species may require a different PAM sequence in the target DNA, and thus, for a particular C2C9 nuclease selected, the PAM sequence requirements may be different from the PAM sequences described previously; C2C9 can be engineered to target PAM sequence "AAN" or other suitable PAM targeting sequences.

In the present invention, the C2C9 nuclease variants may also be formed by modification, mutation, DNA shuffling, or the like, such that the C2C9 nuclease variants have improved desired characteristics, such as function, activity, kinetics, half-life, or the like. The modification may be, for example, a deletion, insertion or substitution of an amino acid, and may be, for example, the replacement of the "cleavage domain" of a C2C9 nuclease with a homologous or heterologous cleavage domain from a different nuclease (e.g., the HNH domain of a CRISPR-associated nuclease); the DNA targeting of C2C9 nucleases can be altered, for example, by any modification method known in the art for DNA binding and/or DNA modification proteins, such as methylation, demethylation, acetylation, and the like. By DNA shuffling is meant the exchange of sequence fragments between DNA sequences of C2C9 nucleases of different origins to produce chimeric DNA sequences encoding synthetic proteins with RNA-guided endonuclease activity. The modification, mutation, DNA shuffling, etc. may be used singly or in combination.

The C2C9 nuclease variant may be:

(I) Variants having at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence homology to the amino acid sequence of a wild-type C2C9 nuclease and retaining activity of the wild-type C2C9 nuclease;

(II) a C2C9 nuclease according to (I) or variant thereof, further comprising additional components, such as a nuclear localization signal fragment, such that the constructed C2C9 CRISPR system has suitable activity in a cell-free reaction, a prokaryotic cell, or a eukaryotic cell environment;

(III) a codon optimized variant of the corresponding polynucleotide sequence encoding a wild-type C2C9 nuclease or variant according to (I) and (II);

(IV) a variant according to wild-type C2C9 nuclease and any one of (I) to (III), further comprising:

(a) One or more modifications or mutations that produce C2C9 with significantly reduced or undetectable nuclease activity; and

(B) Polypeptides or domains having other functional activities.

Suitable polypeptides under (IV) (b) are linked to the C-terminal domain of a C2C9 nuclease and variants thereof.

The C2C9 nuclease variant under (IV) can convert any base pair to any possible other base pair by modification or mutation, rather than introducing a double-strand break in the target DNA sequence.

The C2C9 nuclease variant under (IV) may be a fusion or chimeric polypeptide of the wild-type C2C9 nuclease or variants of (I) to (III) with a heterologous sequence. Such heterologous sequences include, but are not limited to, light-induced transcriptional modulators, small molecule/drug response transcriptional modulators, transcription factors, transcriptional repressors, and the like. In forming the fusion or chimeric polypeptide, the wild-type C2C9 nuclease or variant of (I) to (III) may be a complete or partially or completely deficient C2C9 nuclease. For example, a C2C9 nuclease containing a catalytically active endonuclease domain is fused to a Fokl domain to form a chimeric protein; or a C2C9 nuclease that has been fused to the Fokl domain by modification to lose endonuclease domain activity, to form a chimeric protein; or an epigenetic modifier, tag, imaging agent, transcription modulator, histone, other component that modulates gene structure or activity, or the like, is fused with a C2C9 nuclease to produce a chimeric protein. In some embodiments, the heterologous sequence may provide a tag (e.g., a fluorescent protein, such as Green Fluorescent Protein (GFP), YFP, RFP, CFP, etc., his tag, hemagglutinin (HA) tag, FLAG tag, myc tag, etc.) for ease of tracking or purification. In some embodiments, the heterologous sequence may provide subcellular localization of a C2C9 nuclease.

In some embodiments, the C2C9 nuclease variant is a modified form of a C2C9 nuclease. In some embodiments, the modified form of the C2C9 nuclease comprises an amino acid change that reduces the naturally occurring nuclease activity of the C2C9 nuclease. For example, in some embodiments, the modified form of the C2C9 nuclease has less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% nuclease activity of the corresponding wild-type C2C9 nuclease. In some embodiments, the modified form of the C2C9 nuclease does not have significant nuclease activity, but retains the ability to interact with the gRNA. In some embodiments, the modified form of the C2C9 nuclease lacks nuclease activity. In some embodiments, the endoribonuclease activity of a C2C9 nuclease or binding affinity to DNA has a mutant polypeptide with altered or deleted DNA endoribonuclease activity without substantial reduction or enhancement.

The C2C9 nuclease variants may have the following specific properties, including but not limited to:

has enhanced or reduced binding to the target site, or retains the ability to bind to the target site;

Has enhanced or reduced endoribonuclease and/or endonuclease activity, or retains endoribonuclease and/or endonuclease activity;

Has deaminase activity, which acts on cytosine, guanine or adenine bases and is then replicated through deamination sites and repaired in cells, producing guanine, thymine and guanine respectively;

Has the activity of regulating the transcription of the target DNA, and can be used for increasing or reducing the transcription of the target DNA at a specific position in the target DNA;

Has altered DNA targeting;

increased or decreased or maintained stability;

the complementary strand of the target DNA may be cleaved, but with reduced ability to cleave non-complementary strands of the target DNA;

non-complementary strands of the target DNA may be cleaved, but with reduced ability to cleave complementary strands of the target DNA;

Has a reduced ability to cleave both the complementary strand and the non-complementary strand of the target DNA;

the enzyme activity that has the ability to modify a polypeptide associated with DNA (e.g., histone) may be one or more of methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitination activity, ribosylation activity, etc. (covalent modification of the protein is catalyzed by these enzyme activities; e.g., C2C9 nuclease variants modify the histone by methylation, acetylation, ubiquitination, phosphorylation, etc., to cause structural changes in the histone-associated DNA, thereby controlling the structure and properties of the DNA).

In some embodiments, the C2C9 nuclease variant has no cleavage activity. In some embodiments, the C2C9 nuclease variant has single-stranded cleavage activity. In some embodiments, the C2C9 nuclease variant has double-strand cleavage activity.

By having enhanced activity or capacity is meant having an activity or capacity that is increased by at least 1%, 5%, 10%, 20%, 30%, 40%, 50% relative to the wild-type C2C9 nuclease.

With reduced activity and capacity is meant having less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5% or less than 1% activity or capacity relative to a wild-type C2C9 nuclease.

The terms "C2C9", "C2C9 nuclease" include wild-type C2C9 nuclease and all variants thereof, and the type of C2C9 nuclease variant can be determined by one of ordinary skill in the art by conventional means without being limited to those exemplified above, if not otherwise specified.

In some embodiments, the C2C9 nuclease may be used in combination with other enzyme components or other components to further function as a C2C9 nuclease. For example, binding to sgrnas or fluorescent probes for detection of genes.

The guide RNA (sgRNA) of the invention directs a nuclease, such as a C2C9 nuclease, to a target sequence within a gene to be tested.

In some embodiments, the sgRNA comprises:

A first segment of nucleotide sequence complementary to a target sequence in a target gene (also referred to as a "gene targeting sequence" or "gene targeting fragment"); and

A second fragment (also referred to as a "protein binding sequence" or "protein binding fragment") that interacts with a C2C9 nuclease.

In some embodiments, the sgrnas comprise an array of repeat spacer regions, wherein the spacer regions comprise nucleic acid sequences that are complementary to target sequences in genes.

In some embodiments, the sgRNA comprises:

i. A gene targeting segment (i.e., a targeting sequence, such as a DNA-targeting segment) capable of hybridizing to a target sequence,

Tracr mate sequence, and

Tracr RNA sequence;

The guide RNA is a strand formed by the DNA-targeting segment (i) linked in sequence to the tracr mate sequence (ii) and tracr RNA sequence (iii); alternatively, the guide RNA comprises two strands, one of which is formed by ligation of the gene targeting segment (i) with the tracr mate sequence (ii) and the other strand is the tracr RNA sequence (iii).

Wherein, the gene targeting segment (i) is preferably an RNA sequence corresponding to a nucleic acid fragment with the length of 20bp after the PAM sequence.

Wherein the tracr mate sequence (ii) hybridizes to the tracrRNA sequence (iii) and forms a stem-loop structure.

Wherein the tracr mate sequence (ii) and the tracr RNA sequence (iii) are capable of being joined together to form a single guide RNA backbone sequence.

Wherein the RNA sequence (crRNA) resulting from ligation of the gene targeting segment (i) hybridized to the target sequence and the tracr mate sequence (ii) and the tracr RNA sequence (iii) as two separate RNA sequences, in the presence of the same time, mediate C2C9 endonuclease activity. Or a complete guide RNA expression construct for the target sequence, obtained after ligation of the guide RNA backbone sequence and the DNA-targeting segment (i) hybridized to the target sequence, may also mediate C2C9 endonuclease activity.

The gene targeting segment of the gRNA comprises a nucleotide sequence complementary to a sequence in the target gene that interacts with the target gene in a sequence-specific manner by hybridization (i.e., base pairing). The gene targeting sequence of the gRNA can be modified, for example, by genetic engineering, so that the gRNA hybridizes to any desired sequence within the target gene. The gRNA directs the bound polypeptide to a specific nucleotide sequence within the target gene via the gene targeting sequence described above.

The stem-loop structure forms a protein binding structure that interacts with a C2C9 nuclease. In some embodiments, the protein binding structure of the gRNA comprises a 4-stem loop structure, and the tracr mate sequence (ii) is typically paired with the tracr RNA sequence (iii) by base complementarity. The activity of the C2C9 nuclease can be increased or nonspecific recognition reduced by engineering the base sequence of the gRNA.

In some embodiments, the target gene is a DNA sequence. In some embodiments, the target gene is an RNA sequence.

In some embodiments, the targeting sequence of the target gene may have a length of 12-40 nucleotides, for example, may be 13-20, 18-25, 22-32, 26-37, 30-38, 32-40 nucleotides in length. The percent complementarity between the targeting segment (i) of the guide RNA and the target sequence of the target gene can be at least 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%).

In some embodiments, the gRNA further comprises a transcription terminator.

In the present invention, the gRNA, or polynucleotide encoding the gRNA, is suitable for use in any biological or in vitro environment, including but not limited to bacterial, archaebacterial, fungal, protozoal, plant or animal. Accordingly, suitable target cells include, but are not limited to, bacterial cells, archaeal cells, fungal cells, protozoan cells, plant cells, or animal cells. Suitable target cells may be any type of cell, including stem cells, somatic cells, and the like.

The sgrnas comprise fixed sequences (i.e., tracr mate sequence and tracr RNA sequence) and variable region sequences (i.e., targeting sequences), wherein the targeting sequences can vary according to variations in the target sequence in the test sequence. The targeting sequence of the sgRNA comprises 15-40 ribonucleotides, preferably 20 ribonucleotides, and can be selectively combined with a site to be detected through base complementary pairing. For a particular gene fragment, sgrnas assist in targeting C2C9 nucleases to the target sequence, while activating a paralytic cleavage activity, such that ssDNA fluorescent probes are cut off to release fluorescence.

In one embodiment, the immobilization sequence is set forth in SEQ ID NO. 121. In one embodiment, the AcC2C9 sgRNA contains a sequence as shown in SEQ ID No. 117, comprising a targeting sequence as shown in SEQ ID No. 118.

One end of the fluorescent probe comprises a fluorescent group, and the other end of the fluorescent probe comprises a quenching group. Specifically, the 5 'end contains a fluorescent group and the 3' end contains a quenching group. The fluorescent group is selected from FAM, TET, JOE, HEX, CY, ROX, texas Red, LC RED640, CY5, LC RED705, etc. The quenching group is selected from TAMRA, MGBNFQ, BHQ-1, BHQ-2 and the like. The fluorescent group and the quenching group are linked by a single-stranded DNA of 4nt to 20 nt. The single-stranded DNA preferably contains the nucleotide sequence shown as SEQ ID NO. 120.

The invention also provides a gene detection product comprising a fluorescent probe, and a C2C9 nuclease and a sgRNA, or a nucleic acid encoding the C2C9 nuclease, the sgRNA.

The gene sequence to be detected by the gene detection product comprises a target sequence which can be in base complementary pairing with a target sequence in the sgRNA. In one embodiment, the nucleotide sequence of the test gene sequence is shown as SEQ ID NO. 119.

The gene detection product also comprises any one or more of a reducing agent, a buffer solution, metal ions and chloride ions.

The reducing agent is used to prevent nuclease oxidation. The reducing agent is, for example, beta-mercaptoethanol, DTT, tris (2-carboxyethyl) phosphine, SDS. The buffer solution is selected from phosphate buffer solution and Tris-HCl. The metal ion is, for example, sodium ion or magnesium ion. The metal ion is provided by a metal ion selected from NaCl and MgCl ₂.

Use of said gene detection product for the identification of biological species. The organism is a microorganism (e.g., a bacterium) or a cell. The use is for non-disease diagnostic purposes. In particular, the gene detection product is used to detect the presence or absence of a particular microorganism in a food or environment. Accordingly, based on the conserved region sequences of a specific microorganism as a target sequence, sgrnas comprising gene targeting segments capable of hybridizing to the target sequence are designed so that the presence or absence of the specific microorganism in a sample can be detected.

The invention also provides a gene detection method for the purpose of non-disease diagnosis, which comprises the step of detecting a gene sequence to be detected by using the gene detection product.

The gene sequence to be tested can be derived from a gene extracted from a sample or obtained by amplifying an existing gene. The sample is selected from an environmental sample, a food sample, or a biological sample. The environmental sample is, for example, soil. In the present invention, the amplification is a conventional technique in the art, such as one or more of PCR amplification, MDA amplification, LAMP amplification, RPA amplification, and RT-RPA amplification.

The gene sequence to be tested is selected from double-stranded DNA or single-stranded DNA.

In certain embodiments of the invention, the fluorescent probe operates at a concentration of 100-1000nM. The working concentration of the fluorescent probe is selected from any one of the following concentration ranges: 100nM-300nM, 300nM-500nM, 500nM-700nM, 700nM-1000nM. Preferably, the working concentration of the fluorescent probe is 300nM-700nM.

In certain embodiments of the invention, the C2C9 nuclease operates at a concentration of 10nM to 100. Mu.M. The working concentration of the C2C9 nuclease is selected from any one of the following concentration ranges: 10nM-100nM, 100 nM-1. Mu.M, 1. Mu.M-1.5. Mu.M, 1.5. Mu.M-2. Mu.M, 2. Mu.M-2.5. Mu.M, 2.5. Mu.M-5. Mu.M, 5. Mu.M-7. Mu.M, 7. Mu.M-10. Mu.M. Preferably, the working concentration of the C2C9 nuclease is 1-3 mu M. More preferably, the working concentration of the C2C9 nuclease is 1.5-2.5 mu M.

In certain embodiments of the invention, the sgRNA is present at a working concentration of 10nM to 100. Mu.M. The working concentration of the sgRNA is selected from any one of the following concentration ranges: 10nM-100nM, 100 nM-1. Mu.M, 1. Mu.M-1.5. Mu.M, 1.5. Mu.M-2. Mu.M, 2. Mu.M-2.5. Mu.M, 2.5. Mu.M-5. Mu.M, 5. Mu.M-7. Mu.M, 7. Mu.M-10. Mu.M. Preferably, the working concentration of the sgRNA is 1-3 mu M. More preferably, the working concentration of the sgRNA is 1.5-2.5. Mu.M.

In certain embodiments of the invention, the working concentration of the reducing agent in the reaction system is 100. Mu.M-2 mM based on the total volume of the reaction system. The working concentration of the reducing agent is selected from any one of the following concentration ranges: 100. Mu.M-500. Mu.M, 500. Mu.M-1 mM, 1mM-1.5mM, 1.5mM-2mM. Preferably, the working concentration of the reducing agent is 500 to 1.5mM.

In certain embodiments of the present invention, the working concentration of NaCl in the reaction system is 50 to 500mM, based on the total volume of the reaction system. The working concentration of NaCl is selected from any one of the following concentration ranges: 50-100 mM, 100-150 mM, 150-250mM, 250-350 mM, 350-500 mM. Preferably, the working concentration of NaCl is 50-150 mM.

In certain embodiments of the present invention, the working concentration of MgCl ₂ in the reaction system is from 1 to 20mM, based on the total volume of the reaction system. The working concentration of MgCl ₂ is selected from any one of the following concentration ranges: 1-5 mM, 5-10 mM, 10-15 mM, 15-20 mM. Preferably, the working concentration of MgCl ₂ -15 mM.

In certain embodiments of the invention, the working concentration of Tris-HCl is 1-20 mM. The working concentration of Tris-HCl is selected from any one of the following concentration ranges: 1-5 mM, 5-10 mM, 10-15 mM, 15-20 mM. Preferably, the working concentration of Tris-HCl is 5-15 mM.

In certain embodiments of the invention, the pH of the reaction system is from 7.0 to 8.0.

In the invention, the working concentration is the final concentration of the reaction system based on the total volume of the reaction system unless otherwise specified.

Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.

Before the embodiments of the invention are explained in further detail, it is to be understood that the invention is not limited in its scope to the particular embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention; in the description and claims of the invention, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.

EXAMPLE 1 preparation of AcC2C9 protein

Full Gene sequence Synthesis AcC2C9 nuclease Gene expression cassette (amino acid sequence SEQ ID NO: 122) and pET28a plasmid backbone (amino acid sequence SEQ ID NO: 123), these two DNA fragments were assembled into pET28a-sumo-AcC2C9 plasmid (sequence SEQ ID NO: 124) using the Gibson assembly technique. E.coli DH 5. Alpha. Commercial competent cells were transformed, plated on LB medium plates with 50. Mu.g/mL kanamycin, and screened. And (3) picking up the monoclonal, performing amplification culture, extracting plasmids, and sequencing to identify plasmid sequences to obtain the pET28a-sumo-AcC2C9 plasmids.

PET28a-sumo-AcC2C9 plasmid was introduced into E.coli expression strain BL21 (DE 3). The next day, the transformants were transferred to 1L of LB medium and shake-cultured at 37 ℃. When the OD600 reached 0.6, 0.5mL of 1MIPTG was added to the broth and the temperature was lowered to 16℃for further incubation overnight. The strain after overnight collection was sonicated and purified using HISTRAP NI-NTA column (GE HEALTHCARE). Further purification was then performed using HiLoad 16/600Superdex 200pg molecular sieves (GE HEALTHCARE). The purified protein was concentrated using ultrafiltration tubing and the concentration was determined and stored in 500mM NaCl,10mM Tris-HCl, ph=7.5, 1mm DTT buffer for subsequent experiments.

Example 2 preparation of DNA substrate, sgRNA and fluorescent Probe

Preparation of ssDNA substrate:

The ssDNA substrate PAM-ssDNA (SEQ ID NO: 125) containing the PAM sequence and the ssDNA substrate noPAM-ssDNA (SEQ ID NO: 126) containing NO PAM sequence were synthesized by Shanghai Biotechnology (Shanghai) Inc. Diluted with ultrapure water to a concentration of 10. Mu.M for fluorescent probe experiments.

Preparation of dsDNA substrate:

Synthesis of a reverse complement of the ssDNA substrate containing the PAM sequence, PAM-ssDNA-REV (SEQ ID NO: 127) and of the ssDNA substrate free of the PAM sequence, noPAM-ssDNA-REV (SEQ ID NO: 128) by Shanghai Biotechnology (Shanghai) Inc

The two primer sequences (PAM-ssDNA and PAM-ssDNA-REV, non-PAM-ssDNA and noPAM-ssDNA-REV) corresponding to the above were annealed, and the specific reaction system was as follows: 5 μl of 10×T4 DNA LIGASE Buffer (NEB Co.), 10 μl of ssDNA (50 μM), 10 μl of ssDNA-REV (50 μM), 25 μl of ddH ₂ O. The mixture is heated at 95℃for 5min and then slowly cooled to room temperature over 1-2 hours, so that the two single-stranded primers form double-stranded DNA by base pairing. Thus, dsDNA substrates with PAM and without PAM sequences were obtained at a final concentration of 10 μm.

Preparation of sgRNA:

The sequence of the sgRNA containing the targeting sequence is (SEQ ID NO: 117):

CAGUGCUGAUCGAUCGAAACGUCGCCUGCGAUAGGCGGGAGACGCUAAACGCCCGU

GGAGCAUCCAUAAGACCAACCACCUCUCGGGGCGGUAGGCACGACGCAUCGAAGCG

GGAAGGCUCCGGCGCUCGGCCUGAGUCACCUCAGCAGAGUGAUCUGCUGACGCUCG

AAAGAGCGAUCGcgguucaggugaaagugaaa

Wherein the underlined lower case letter moiety is the targeting sequence and the non-underlined moiety is the immobilization region.

An sgRNA transcription template containing a targeting sequence, the sequence of which is SEQ ID NO. 129, was synthesized at Suzhou gold intellectual Biotech Co. The sgrnas were prepared by transcription using HiScribe T7 HIGH YIELD RNA SYNTHESIS KIT (NEB) kit, and the experimental procedures were performed according to the kit instructions. The prepared sgRNA is purified by phenol chloroform extraction and ethanol precipitation.

The sequence of the sgRNA without targeting sequence is SEQ ID NO. 130. The transcription template sequence is SEQ ID NO. 131, and is synthesized by the Souzhou Jin Weizhi Biotechnology Co. The preparation of the sgrnas without targeting sequences is identical to the above-described sgrnas with targeting sequences.

Preparation of fluorescent probes:

The fluorescent probe contains a FAM fluorescent group at the 5 'end and a BHQ1 quenching group at the 3' end, and the fluorescent group and the quenching group are linked by a section of single-stranded DNA of 12 nt. The sequence of the single-stranded DNA is shown as SEQ ID NO. 120. Fluorescent probes were synthesized by the company Shanghai, inc. of biological engineering.

Example 3AcC2C9 detection of fluorescent Signal of ssDNA substrate

Fluorescence detection experiments were performed in 100mM NaCl,10mM MgCl ₂, 10mM Tris-HCl, pH=7.5, 1mM DTT. The total reaction volume was 20. Mu.L, which included 20nM ssDNA substrate, 2. Mu.M AcC2C9, 2. Mu.M sgRNA and 500nM fluorescent probe. In addition, one set of experiments did not add ssDNA substrate as a control.

The detection reactions were placed in a fluorescent quantitative PCR instrument CFX96 Real-TIME SYSTEM (Bio-Rad), the reaction temperature was 37 ℃, FAM fluorescent signals were recorded every two minutes apart for a total of 120 times, and each reaction was repeated three times in parallel. The detection results obtained by recording the detection signals using a Bio-Rad CFX Manager are shown in FIG. 2.

The results show that targeting sgrnas can produce a distinct detection signal, whether or not PAM sequence is contained in ssDNA, but ssDNA containing PAM sequence produces a fluorescent signal at a faster rate. Whereas the non-targeted sgrnas produce substantially no fluorescent signal. Thus, acC2C9 can be used to detect ssDNA containing a specific target sequence.

Example 4AcC2C9 detection of fluorescent Signal of dsDNA substrates

The procedure for fluorescence detection of dsDNA substrates was identical to ssDNA. Fluorescence detection experiments were performed in 100mM NaCl,10mM MgCl ₂, 10mM Tris-HCl, pH=7.5, 1mM DTT. The total reaction volume was 20. Mu.L, which included 20nM dsDNA substrate, 2. Mu.M AcC2C9, 2. Mu.M sgRNA and 500nM fluorescent probe. In addition, one set of experiments did not add dsDNA substrate as a control.

The detection reactions were placed in a fluorescent quantitative PCR instrument CFX96 Real-TIME SYSTEM (Bio-Rad), the reaction temperature was 37 ℃, FAM fluorescent signals were recorded every two minutes apart for a total of 120 times, and each reaction was repeated three times in parallel. The detection results obtained by recording the detection signals using a Bio-Rad CFX Manager are shown in FIG. 3.

The results show that targeting sgrnas can produce a distinct detection signal, whether or not PAM sequence is contained in dsDNA, but dsDNA containing PAM sequence produces a fluorescent signal at a faster rate. Whereas the non-targeted sgrnas produce substantially no fluorescent signal. Thus, acC2C9 can be used to detect dsDNA containing a specific target sequence.

In the invention, the CRISPR/C2C9 system has paralytic cleavage activity through experiments, so that the system is developed into a novel gene detection tool. The amplification product containing the target sequence is obtained through a proper amplification method, and the specificity of the target sequence part in the sgRNA is utilized to detect the specific nucleic acid sequence by combining with a CRISPR/C2C9 detection system, so that the detection of the target gene can be realized rapidly and efficiently.

The above examples are provided to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. Further, various modifications of the methods set forth herein, as well as variations of the methods of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the present invention.

Claims

Use of a c2c9 nuclease for the preparation of a gene detection product.
2. The use according to claim 1, wherein the C2C9 nuclease is:

(I) A wild-type C2C9 nuclease or fragment thereof, having RNA-guided nucleic acid binding activity;

(II) a variant having at least 30% sequence homology with the amino acid sequence of (I) and having RNA-guided nucleic acid binding activity;

(III) according to (I) or (II), further comprising a nuclear localization signal fragment;

(IV) according to (I) or (II) or (III), further comprising:

(a) One or more modifications or mutations that result in a dna sequence having significantly reduced endonuclease activity, or a loss of endonuclease activity, compared to the endonuclease sequence prior to the modification or mutation; and/or

(B) A polypeptide or domain having other functional activity;

(V) the C2C9 nuclease according to (I) or (II) or (III) has endonuclease activity.
3. The use of claim 1, wherein the C2C9 nuclease does not exceed 800 amino acids; and/or, the C2C9 nuclease has RNA-guided nucleic acid binding activity; preferably, the C2C9 nuclease is Actinomadura CRANIELLAE C C9.
4. The use according to claim 1, wherein the amino acid sequence of the C2C9 nuclease is SEQ ID NO.

1-115, Or any amino acid sequence shown in SEQ ID No.1-115, has at least 50% sequence homology with any amino acid sequence shown in SEQ ID No.1-115, and has RNA-guided nucleic acid binding activity and/or endonuclease activity.
5. The use according to claim 1, wherein the C2C9 nuclease and sgRNA, fluorescent probe are used in combination for the preparation of a gene detection product.
6. The use according to claim 5, wherein the sgRNA comprises a fixed sequence and a variable region sequence, the variable region sequence being a fragment capable of hybridizing to a target sequence.
7. The use according to claim 5, wherein the nucleotide sequence of the fixed sequence is shown as SEQ ID NO. 121; preferably, the nucleotide sequence of the sgRNA that matches the AcC2C9 nuclease is shown in SEQ ID NO. 117.
8. The use according to claim 5, wherein the fluorescent probe comprises a fluorescent group at one end and a quenching group at the other end; preferably, the 5 'end contains a fluorescent group and the 3' end contains a quenching group; more preferably, the fluorophore is selected from FAM, TET, JOE, HEX, CY, ROX, texas Red, LC Red640, CY5, or LC Red705;

and/or the quenching group is selected from TAMRA, MGBNFQ, BHQ-1 or BHQ-2.
9. The use according to claim 8, wherein between the fluorescent group and the quenching group is a stretch of 4nt to 20nt single-stranded DNA; preferably, the single stranded DNA comprises the nucleotide sequence shown as SEQ ID NO. 120.
10. A gene detection product comprising a fluorescent probe, and a C2C9 nuclease and a sgRNA, or nucleotides encoding the C2C9 nuclease, the sgRNA.
11. The product according to claim 10, wherein the gene test product comprises a target sequence capable of base complementary pairing with the variable region sequence in the sgRNA.
12. The product of claim 10, wherein the gene detection product further comprises any one or more of a reducing agent, a buffer, a metal ion, and a chloride ion; preferably, the reducing agent is any one or more of beta-mercaptoethanol, DTT, tris (2-carboxyethyl) phosphine or SDS; preferably, the buffer is selected from phosphate buffer or Tris-HCl; preferably, the metal ion is sodium ion or magnesium ion.
13. Use of a gene assaying product according to any one of claims 10-12 for the identification of biological species.
14. The use according to claim 13, wherein the organism is a microorganism or a cell; and/or, the use is for non-disease diagnostic purposes; preferably, the gene detection product is used to detect the presence or absence of a specific microorganism in a food or environment.
15. A method for detecting a gene for the purpose of non-disease diagnosis, which comprises detecting a gene sequence to be detected using the gene detection product according to any one of claims 10 to 12.
16. The method according to claim 15, wherein the gene sequence to be tested is derived from a gene extracted from a sample or obtained by amplifying an existing gene; preferably, the sample is selected from an environmental sample, a food sample or a biological sample.
17. The method of claim 15, further comprising one or more of the following features:

1) The gene sequence to be detected is selected from double-stranded DNA or single-stranded DNA;

2) The working concentration of the fluorescent probe is 100-1000nM based on the total volume of the reaction system;

3) The working concentration of the C2C9 nuclease is 10nM-100 mu M based on the total volume of the reaction system;

4) The working concentration of the sgRNA is 10nM-100 mu M based on the total volume of the reaction system;

5) The working concentration of the reducing agent in the reaction system is 100 mu M-2mM based on the total volume of the reaction system;

6) The metal ions are provided by NaCl and MgCl ₂, the working concentration of NaCl in the reaction system is 50-500 mM based on the total volume of the reaction system, and/or the working concentration of MgCl ₂ in the reaction system is 1-20 mM;

7) The working concentration of the Tris-HCl is 1-20 mM by taking the total volume of the reaction system as a reference;

8) The pH of the reaction system is 7.0-8.0.