CN118186066A - Use and method of C2C9 nuclease in preparing gene detection products - Google Patents

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

Use and method of C2C9 nuclease in preparing gene detection products

Technical Field

The present invention relates to the field of biotechnology, and in particular to the use and method of C2C9 nuclease in preparing gene detection products.

Background technique

Genetic testing, also known as nucleic acid testing, is an important part of the biotechnology field. Different nucleic acid testing methods have been developed based on the sequence specificity of DNA and RNA, the genetic materials of different organisms and species. Traditional nucleic acid testing includes sequencing technology, constant temperature amplification technology, etc., which can detect DNA or RNA specific sequences of various pathogens, and can also analyze and detect disease-related SNP sites.

CRISPR/Cas (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) is a new gene editing system developed in recent years. The system is mainly composed of Cas endonuclease and corresponding guide RNA (sgRNA). Cas endonuclease can specifically bind to guide RNA, use base complementary pairing to target and cut specific DNA sites, causing double-strand breaks in DNA. Due to the simplicity and efficiency of the CRISPR system, the system has been widely used in basic research, biotechnology development, and disease treatment research and development in the fields of life sciences, medicine, and agronomy. In addition to its wide application in the field of gene editing, the system has also been developed as a genetic testing tool.

Conventional genetic testing technology has a complex process, has high requirements for testing instruments and equipment, and the testing time is relatively long. CRISPR system genetic testing tools can greatly shorten this process. Therefore, the development of a simple, efficient and low-cost testing tool can achieve efficient genetic testing while reducing the demand for genetic testing technology equipment and instruments, which has good practical application value and commercial value.

Summary of the invention

In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide the use and method of C2C9 nuclease in preparing gene detection products, so as to solve the problems in the prior art.

To achieve the above objectives and other related objectives, the present invention discloses a novel gene detection system based on an extremely small CRISPR/C2C9 system and the application of C2C9 nuclease in the preparation of gene detection products. By detecting samples through the CRISPR/C2C9 detection system and using the specificity of the guide part in the sgRNA to detect specific nucleic acid sequences, the target gene can be detected quickly and efficiently.

The present invention provides use of C2C9 nuclease in preparing gene detection products.

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

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

The present invention also provides a gene detection method for non-disease diagnosis purposes, the detection method comprising using the gene detection product described in the claims to detect a gene sequence to be detected.

As described above, the use and method of the C2C9 nuclease of the present invention in preparing a gene detection product has the following beneficial effects: the gene detection method is simple to operate, has high detection efficiency, and can quickly detect a specific gene to be detected.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is an experimental schematic diagram of the CRISPR/C2C9 system for detecting target genes. The CRISPR system mainly used in the present invention is a V-type CRISPR system, in which the effector protein is mainly a C2C9 nuclease family. The inventors found that this type of nuclease generally has non-specific ssDNA cleavage activity, and the non-specific cleavage activity is activated only when the C2C9 effector protein and sgRNA complex are correctly targeted to the target sequence. By adding an ssDNA fluorescent probe, the fluorescent probe carries a fluorescent group and a fluorescent quenching group such as FAM (F in the figure) and BHQ1 (Q in the figure), when the target targeting sequence is present in the sample, the effector protein collateral cleavage activity is activated, and the probe is cut off to release fluorescence, thereby realizing the detection of the target gene sequence.

Figure 2 shows the fluorescence signals of sgRNA targeting ssDNA fragments and non-targeting sgRNA in the CRISPR/C2C9 detection system. The results show that regardless of whether the substrate ssDNA contains a PAM sequence, the targeting sgRNA can generate obvious detection signals, but the substrate ssDNA containing a PAM sequence generates fluorescence signals faster. Non-targeting sgRNA basically does not generate fluorescence signals.

Figure 3 shows the fluorescence signals of sgRNA targeting dsDNA fragments and non-targeting sgRNA in the CRISPR/C2C9 detection system. The results show that regardless of whether the substrate dsDNA contains a PAM sequence, the targeting sgRNA can generate obvious detection signals, but the substrate dsDNA containing the PAM sequence generates fluorescence signals faster. Non-targeting sgRNA basically does not generate fluorescence signals.

Detailed ways

C2C9 nuclease is 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 directional double-stranded DNA (dsDNA) cutting activity, but also has non-specific single-stranded DNA (ssDNA) cutting activity. When the C2C9-sgRNA complex binds to the target DNA substrate, the non-specific paralogous ssDNA cutting activity is activated, and this special property can be used to achieve in vitro gene detection. At the same time, C2C9 has a unique 5'AAN PAM recognition property, which is different from the recognition properties of the 5'T-rich PAM of most V-type Cas12 family proteins. Therefore, C2C9 can greatly expand the recognition and detection range of double-stranded DNA substrates.

The present invention first provides the use of C2C9 nuclease in preparing gene detection products.

The C2C9 nuclease has at least one of the following activities: regulating transcription in a target gene (eg, 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 nuclease described in the present invention may be a conventional C2C9 nuclease in the art.

The C2C9 nuclease is:

(I) a wild-type C2C9 nuclease or a 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 significantly reduced endonuclease activity compared to before the modification or mutation, or a loss of endonuclease activity; and/or

(b) polypeptides or domains having other functional activities;

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

In the present invention, the amino acid sequence of the C2C9 nuclease is derived from any one or more of the following: 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, Pseudactinotalesp.HY160, Rothia dentocariosa, Rothia kristinae, Rothia mucilaginosa, Rothianasimurium, Rothia sp.HMSC071C12, Saccharomonospora piscinae, Saccharopolysporarectivirgula 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 cessp.CB04723, Streptomyces sp.CNH099, Streptomyces sp.CNS606, Streptomyces cessp.IB2014 016-6, Streptomyces sp.man185, Streptomyces sp.S4, Streptomyces cessp.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, Corynebacteriummaris, Streptomyces sp.NRRL S-378, Kitasatospora cheerisanensis KCTC 2395, Mycobacterium sp.SWH-M3, Streptomyces roseochromogenus, Mycobacteriumintracellulare subsp.yongonense 05-1390, Mycobacterium sp.JS623, Mycolicibacterium fallax, Nocardiopsis sp.JB363, Rothia sp.HMSC069C03, Mycobacterium avium. In the present invention, 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 nuclease activity.

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

In some embodiments, in order to utilize the various enzymatic properties of different C2C9 nucleases (e.g., for different PAM sequence preferences), C2C9 nucleases from different species are advantageously used to provide different applications, for example, to increase or decrease enzymatic activity; to increase or decrease the level of cytotoxicity; to change the balance between NHEJ, homology-directed repair, single-strand breaks, double-strand breaks, etc.

C2C9 nucleases from different species or different C2C9 nucleases from the same species may require different PAM sequences in the target DNA, and therefore, for a particular C2C9 nuclease selected, the PAM sequence requirement may be different from the aforementioned PAM sequence. For example, the PAM sequence that AcC2C9 nuclease can effectively recognize is 5'-AAN (N represents any base of A, T, C or G), the PAM sequence that CgC2C9 can effectively recognize is 5'-AAS (S represents any base of C or G), and the PAM sequence that RdC2C9 and MiC2C9 can effectively recognize is 5'-AAH (N represents any base of A, T or C).

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

The C2C9 nuclease provided by the present invention has a small number of amino acids. In a preferred embodiment, when the amino acid sequence of the C2C9 nuclease is as shown in SEQ ID NO: 116, it interacts with the PAM sequence AAN. The cleavage site is usually present within one to three base pairs upstream of the PAM sequence. C2C9 nucleases from different species or different C2C9 nucleases from the same species may require different PAM sequences in the target DNA. Therefore, for the specific C2C9 nuclease selected, the PAM sequence requirement may be different from the aforementioned PAM sequence; C2C9 can be engineered to target the PAM sequence "AAN" or other suitable targeting PAM sequences.

In the present invention, C2C9 nuclease variants can also be formed by modification, mutation, DNA shuffling, etc., so that the C2C9 nuclease variant has improved desired characteristics, such as function, activity, kinetics, half-life, etc. The modification can be, for example, the deletion, insertion or substitution of amino acids, and another example can be the replacement of the "cleavage domain" of the C2C9 nuclease with a homologous or heterologous cleavage domain from a different nuclease (e.g., the HNH domain of a CRISPR-associated nuclease); by any modification method of DNA binding and/or DNA modification protein known in the art, such as methylation, demethylation, acetylation, etc., for example, the DNA targeting of the C2C9 nuclease can be changed. The DNA shuffling refers to exchanging sequence fragments between DNA sequences of C2C9 nucleases from different sources to produce a chimeric DNA sequence encoding a synthetic protein with RNA-guided endonuclease activity. The modification, mutation, DNA shuffling, etc. can be used alone or in combination.

The C2C9 nuclease variant may be:

(I) a variant 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 wild-type C2C9 nuclease and retaining the activity of wild-type C2C9 nuclease;

(II) The C2C9 nuclease or variant thereof according to (I), further comprising other components, such as a nuclear localization signal fragment, so that the constructed C2C9 CRISPR system has appropriate 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 the wild-type C2C9 nuclease or a 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 result in a C2C9 with significantly reduced or undetectable nuclease activity; and

(b) Polypeptides or domains having other functional activities.

A suitable polypeptide under (IV)(b) is linked to the C-terminal domain of C2C9 nuclease and variants thereof.

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

The C2C9 nuclease variant under (IV) can be a fusion or chimeric polypeptide formed by a wild-type C2C9 nuclease or a variant of (I) to (III) and a heterologous sequence. The heterologous sequence includes, but is not limited to, a light-induced transcriptional regulator, a small molecule/drug response transcriptional regulator, a transcription factor, a transcriptional repressor protein, etc. When forming a fusion or chimeric polypeptide, the wild-type C2C9 nuclease or the variant of (I) to (III) can be a complete or partially or completely defective C2C9 nuclease. For example, a C2C9 nuclease containing a catalytically active endonuclease domain is fused with a Fok1 domain to form a chimeric protein; or a C2C9 nuclease that has lost the activity of the endonuclease domain through modification is fused with a Fok1 domain to form a chimeric protein; or, an epigenetic modifier, a label, an imaging agent, a transcriptional regulator, a histone, other components that regulate gene structure or activity, etc. are fused with a C2C9 nuclease to prepare a chimeric protein. In some embodiments, the heterologous sequence can provide a tag for easy tracking or purification (e.g., a fluorescent protein, such as green fluorescent protein (GFP), YFP, RFP, CFP, etc.; a His tag; a hemagglutinin (HA) tag; a FLAG tag; a Myc tag, etc.). In some embodiments, the heterologous sequence can provide subcellular localization of the C2C9 nuclease.

In some embodiments, the C2C9 nuclease variant is a modified form of the 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% of the nuclease activity of the corresponding wild-type C2C9 nuclease. In some embodiments, the modified form of the C2C9 nuclease has no significant nuclease activity, but retains the ability to interact with gRNA. In some embodiments, the modified form of the C2C9 nuclease has no nuclease activity. In some embodiments, the endoribonuclease activity of the C2C9 nuclease or the binding affinity to DNA has a mutant polypeptide with a changed or cancelled DNA endonuclease activity without being substantially reduced or enhanced.

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

having an enhanced or reduced ability to bind to a target, or retaining the ability to bind to a target;

having enhanced or reduced endoribonuclease and/or endonuclease activity, or retaining endoribonuclease and/or endonuclease activity;

Possessing deaminase activity, which can act on cytosine, guanine, or adenine bases and subsequently replicate through the deamination site and repair within the cell to produce guanine, thymine, and guanine, respectively;

It has the activity of regulating the transcription of the target DNA, which can be to increase or decrease the transcription of the target DNA at a specific position in the target DNA;

with altered DNA targeting;

increase or decrease or maintain stability;

Can cleave the complementary strand of the target DNA, but has a reduced ability to cleave the non-complementary strand of the target DNA;

Can cleave the non-complementary strand of the target DNA, but has a reduced ability to cleave the complementary strand of the target DNA;

has a reduced ability to cleave both complementary and non-complementary strands of target DNA;

It has an enzymatic activity for modifying polypeptides associated with DNA (e.g., histones), and the enzymatic activity may be one or more of methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, ribosylation activity, etc. (these enzymatic activities catalyze the covalent modification of proteins; for example, the C2C9 nuclease variant modifies histones through methylation, acetylation, ubiquitination, phosphorylation, etc., to cause structural changes in histone-associated DNA, thereby controlling the structure and properties of 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-stranded cleavage activity.

Having enhanced activity or ability means that the activity or ability is increased by at least 1%, 5%, 10%, 20%, 30%, 40%, or 50% relative to the wild-type C2C9 nuclease.

Having reduced activity and capacity means having less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5% or less than 1% of the activity or capacity relative to the wild-type C2C9 nuclease.

If not otherwise specified, the terms "C2C9" and "C2C9 nuclease" include wild-type C2C9 nuclease and all variants thereof. Those skilled in the art can determine the type of C2C9 nuclease variant by conventional means, and are not limited to those exemplified above.

In some embodiments, the C2C9 nuclease can be used in combination with other enzyme components or other components to further exert the effect of the C2C9 nuclease, such as in combination with sgRNA or fluorescent probes for gene detection.

The guide RNA (sgRNA) of the present invention guides a nuclease such as C2C9 nuclease to a target sequence within a gene to be detected.

In some embodiments, the sgRNA comprises:

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

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

In some embodiments, the sgRNA comprises an array of repeat sequence spacers, wherein the spacers comprise nucleic acid sequences complementary to target sequences in a gene.

In some embodiments, the sgRNA comprises:

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

ii. tracr partner sequence, and

iii. tracr RNA sequence;

The guide RNA is a single chain, which is formed by sequentially connecting the DNA-targeting segment (i) to the tracr partner sequence (ii) and the tracr RNA sequence (iii); or, the guide RNA comprises two chains, one of which is formed by connecting the gene targeting segment (i) to the tracr partner sequence (ii), and the other chain is the tracrRNA sequence (iii).

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

Wherein, the tracr partner sequence (ii) hybridizes with the tracrRNA sequence (iii) to form a stem-loop structure.

Wherein, the tracr partner sequence (ii) and the tracrRNA sequence (iii) can be connected together to form a single guide RNA backbone sequence.

Among them, the RNA sequence (crRNA) obtained by connecting the gene targeting segment (i) hybridizing with the target sequence and the tracr partner sequence (ii), as two separate RNA sequences, can mediate C2C9 endonuclease activity when present at the same time. Or the complete guide RNA expression construct for the target sequence obtained by connecting the guide RNA backbone sequence and the DNA-targeting segment (i) hybridizing with the target sequence can also mediate C2C9 endonuclease activity.

The gene targeting segment of gRNA contains a nucleotide sequence complementary to the sequence in the target gene, and the gene targeting sequence interacts with the target gene in a sequence-specific manner by hybridization (i.e., base pairing). The gene targeting sequence of gRNA can be modified by, for example, genetic engineering to hybridize gRNA with any desired sequence in the target gene. gRNA guides the bound polypeptide to a specific nucleotide sequence in the target gene through the above-mentioned gene targeting sequence.

The stem-loop structure forms a protein binding structure that interacts with the C2C9 nuclease. In some embodiments, the protein binding structure of the gRNA comprises four stem-loop structures, and the tracr partner sequence (ii) is usually paired with the tracr RNA sequence (iii) through base complementarity. The activity of the C2C9 nuclease can be improved or non-specific recognition can be reduced by modifying 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 can have a length of 12-40 nucleotides, for example, 13-20, 18-25, 22-32, 26-37, 30-38, 32-40 nucleotides. The complementarity percentage 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 the polynucleotide encoding the gRNA, is applicable to any organism or in vitro environment, including but not limited to bacteria, archaea, fungi, protists, plants or animals. Accordingly, applicable target cells include but are not limited to bacterial cells, archaea cells, fungal cells, protist cells, plant cells or animal cells. Applicable target cells can be any type of cells, including stem cells, somatic cells, etc.

The sgRNA comprises a fixed sequence (i.e., a tracr partner sequence and a tracr RNA sequence) and a variable region sequence (i.e., a targeting sequence), wherein the targeting sequence can vary according to the change of the target sequence in the sequence to be tested. The targeting sequence of the sgRNA of the present invention comprises 15-40 ribonucleotides, preferably 20 ribonucleotides, and the targeting sequence can selectively bind to the site to be detected through base complementary pairing. For a specific gene fragment, the sgRNA assists the C2C9 nuclease in targeting the target sequence and activates the collateral cleavage activity, so that the ssDNA fluorescent probe is cut off and releases fluorescence.

In one embodiment, the fixed sequence is as shown in SEQ ID NO: 121. In one embodiment, the sgRNA of AcC2C9 contains the sequence as shown in SEQ ID NO: 117, wherein the targeting sequence contained therein is the sequence as shown in SEQ ID NO: 118.

One end of the fluorescent probe contains a fluorescent group and the other end contains 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, CY3, ROX, Texas Red, LCRED640, CY5 or LC RED705, etc. The quenching group is selected from TAMRA, MGBNFQ, BHQ-1 or BHQ-2, etc. A single-stranded DNA link of 4nt to 20nt is provided between the fluorescent group and the quenching group. The single-stranded DNA preferably contains a nucleotide sequence as shown in SEQ ID NO:120.

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

The gene sequence to be tested by the gene detection product contains a target sequence that can be complementary to the target sequence base in the sgRNA. In one embodiment, the nucleotide sequence of the gene sequence to be tested is shown in SEQ ID NO:119.

The gene detection product also includes any one or more of a reducing agent, a buffer, a metal ion, and a chloride ion.

The reducing agent is used to prevent nuclease oxidation. The reducing agent is, for example, β-mercaptoethanol, DTT, tris(2-carboxyethyl)phosphine, SDS. The buffer is selected from phosphate buffer and Tris-HCl. The metal ion is, for example, sodium ion or magnesium ion. The metal ion is provided by a selected from NaCl, _MgCl2 .

The use of the gene detection product in identifying biological species. The organism is a microorganism (such as bacteria) or a cell. The use is for non-disease diagnosis purposes. Specifically, the gene detection product is used to detect whether a specific microorganism exists in food or the environment. Accordingly, based on the conserved region sequence of a specific microorganism as a target sequence, an sgRNA containing a gene targeting segment that can hybridize with the target sequence is designed so that the presence of the specific microorganism in the sample can be detected.

The present invention also provides a gene detection method for non-disease diagnosis purposes, the detection method comprising using the gene detection product to detect a gene sequence to be detected.

The gene sequence to be tested may 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, 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 present invention, the working concentration of the fluorescent probe is 100-1000 nM. The working concentration of the fluorescent probe is selected from any of the following concentration ranges: 100 nM-300 nM, 300 nM-500 nM, 500 nM-700 nM, 700 nM-1000 nM. Preferably, the working concentration of the fluorescent probe is 300 nM-700 nM.

In certain embodiments of the present invention, the working concentration of the C2C9 nuclease is 10nM-100μM. The working concentration of the C2C9 nuclease is selected from any of the following concentration ranges: 10nM-100nM, 100nM-1μM, 1μM-1.5μM, 1.5μM-2μM, 2μM-2.5μM, 2.5μM-5μM, 5μM-7μM, 7μM-10μM. Preferably, the working concentration of the C2C9 nuclease is 1 to 3μM. More preferably, the working concentration of the C2C9 nuclease is 1.5 to 2.5μM.

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

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

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

In certain embodiments of the present invention, based on the total volume of the reaction system, the working concentration of _MgCl2 in the reaction system is 1-20 mM. The working concentration of _MgCl2 is selected from any of the following concentration ranges: 1-5 mM, 5-10 mM, 10-15 mM, 15-20 mM. Preferably, the working concentration of _MgCl2 is 5-15 mM.

In certain embodiments of the present invention, the working concentration of Tris-HCl is 1-20 mM. The working concentration of Tris-HCl is selected from any 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 present invention, the pH of the reaction system is 7.0-8.0.

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

The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

Example 1 Preparation of AcC2C9 protein

The AcC2C9 nuclease gene expression cassette (amino acid sequence is SEQ ID NO: 122) and the pET28a plasmid backbone (amino acid sequence is SEQ ID NO: 123) were synthesized by the whole gene sequence, and the two DNA fragments were assembled into the pET28a-sumo-AcC2C9 plasmid (whose sequence is SEQ ID NO: 124) using the Gibson assembly technique. The commercial competent cells of Escherichia coli DH5α were transformed and plated on LB medium plates with 50 μg/mL kanamycin for screening. After picking a single clone, the culture was expanded, the plasmid was extracted, and the plasmid sequence was sequenced to obtain the pET28a-sumo-AcC2C9 plasmid.

The pET28a-sumo-AcC2C9 plasmid was transferred to the Escherichia coli expression strain BL21 (DE3). The next day, the transformant was transferred to 1L of LB medium and cultured at 37°C. When OD600 reached 0.6, 0.5mL1MIPTG was added to the culture solution, and the temperature was lowered to 16°C and cultured overnight. The strains were collected overnight, ultrasonically broken, and purified using a HisTrapNi-NTA column (GE Healthcare). Then HiLoad 16/600Superdex 200pg molecular sieve (GE Healthcare) was used for further purification. The purified protein was concentrated using an ultrafiltration tube and the concentration was determined, and stored in a buffer of 500mM NaCl, 10mM Tris-HCl, pH=7.5, and 1mM DTT 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 a PAM sequence and the ssDNA substrate noPAM-ssDNA (SEQ ID NO: 126) not containing a PAM sequence were synthesized by Shanghai Sangon Biotechnology Co., Ltd. and diluted to a concentration of 10 μM with ultrapure water for use in fluorescent probe experiments.

Preparation of dsDNA substrate:

The reverse complement sequence PAM-ssDNA-REV (SEQ ID NO: 127) of the ssDNA substrate containing the PAM sequence and the reverse complement sequence noPAM-ssDNA-REV (SEQ ID NO: 128) of the ssDNA substrate not containing the PAM sequence were synthesized at Shanghai Sangon Biotechnology (Shanghai) Co., Ltd.

The two corresponding primer sequences (PAM-ssDNA and PAM-ssDNA-REV, noPAM-ssDNA and noPAM-ssDNA-REV) were annealed, and the specific reaction system was as follows: 5μl 10×T4 DNA ligase Buffer (NEB), 10μL ssDNA (50μM), 10μL ssDNA-REV (50μM), 25μL ddH ₂ O. Heat at 95°C for 5 minutes, and then slowly cool to room temperature within 1 to 2 hours to allow the two single-stranded primers to form double-stranded DNA through base pairing. Thus, dsDNA substrates containing PAM and dsDNA without PAM sequences were obtained, with a final concentration of 10μM.

Preparation of sgRNA:

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

CAGUGCUGAUCGAUCGAAACGUCGCCUGCGAUAGGCGGGAGACGCUAAACGCCCGU

GGAGCAUCCAUAAGACCAACCACCUCUCGGGGCGGUAGGCACGACGCAUCGAAGCG

GGAAGGCUCCGGCGCUCGGCCUGAGUCACCUCAGCAGAGUGAUCUGCUGACGCUCG

AAAGAGCGAUCGcgguucaggugaaagugaaa

The underlined lowercase letters are the target sequence, and the ununderlined part is the fixed region.

The sgRNA transcription template containing the target sequence was synthesized at Suzhou Jinweizhi Biotechnology Co., Ltd., and its sequence was SEQ ID NO: 129. The sgRNA was prepared by transcription using the HiScribe T7 High Yield RNA Synthesis Kit (NEB), and the experimental steps were carried out according to the instructions of the kit. The prepared sgRNA was purified by phenol chloroform extraction and ethanol precipitation.

The sequence of the sgRNA without a targeting sequence is SEQ ID NO: 130. Its transcription template sequence is SEQ ID NO: 131, which was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. The preparation steps of the sgRNA without a targeting sequence are the same as those of the sgRNA with a targeting sequence.

Preparation of fluorescent probes:

The 5' end of the fluorescent probe contains a FAM fluorescent group, and the 3' end contains a BHQ1 quenching group, and the fluorescent group and the quenching group are linked by a 12nt single-stranded DNA. The sequence of the single-stranded DNA is shown in SEQ ID NO: 120. The fluorescent probe was synthesized by Sangon Biotech (Shanghai) Co., Ltd.

Example 3: Fluorescence signal of AcC2C9 detecting ssDNA substrate

Fluorescence detection experiments were performed in 100 mM NaCl, 10 mM MgCl ₂ , 10 mM Tris-HCl, pH=7.5, 1 mM DTT. The total reaction volume was 20 μL, including 20 nM ssDNA substrate, 2 μM AcC2C9, 2 μM sgRNA and 500 nM fluorescent probe. In addition, a group of experiments without ssDNA substrate was used as a control group.

The detection reaction was placed in a fluorescent quantitative PCR instrument CFX96 Real-Time System (Bio-Rad), the reaction temperature was 37°C, and the FAM fluorescence signal was recorded every two minutes, for a total of 120 times, and each reaction was repeated three times in parallel. The detection signal was recorded using Bio-Rad CFX Manager to obtain the detection results shown in Figure 2.

The results showed that regardless of whether the ssDNA contained a PAM sequence, the targeted sgRNA could generate a significant detection signal, but the ssDNA containing the PAM sequence generated a fluorescent signal at a faster rate. The non-targeted sgRNA basically did not generate a fluorescent signal. Therefore, AcC2C9 can be used to detect ssDNA containing a specific target sequence.

Example 4: Fluorescence signal of AcC2C9 detecting dsDNA substrate

The fluorescence detection experimental steps of dsDNA substrate are the same as those of ssDNA. The fluorescence detection experiment was carried out under the conditions of 100mM NaCl, 10mM MgCl ₂ , 10mM Tris-HCl, pH=7.5, 1mM DTT. The total reaction volume was 20μL, including 20nM dsDNA substrate, 2μM AcC2C9, 2μM sgRNA and 500nM fluorescent probe. In addition, a group of experiments without dsDNA substrate was used as the control group.

The detection reaction was placed in a fluorescent quantitative PCR instrument CFX96 Real-Time System (Bio-Rad), the reaction temperature was 37°C, and the FAM fluorescence signal was recorded every two minutes, for a total of 120 times, and each reaction was repeated three times in parallel. The detection signal was recorded using Bio-Rad CFX Manager to obtain the detection results shown in Figure 3.

The results showed that regardless of whether the dsDNA contained a PAM sequence, the targeted sgRNA could generate a significant detection signal, but the dsDNA containing the PAM sequence generated a fluorescent signal at a faster rate. The non-targeted sgRNA basically did not generate a fluorescent signal. Therefore, AcC2C9 can be used to detect dsDNA containing a specific target sequence.

In the present invention, the inventors discovered through experiments that the CRISPR/C2C9 system has collateral cleavage activity, and thus developed the system into a new type of gene detection tool. By obtaining an amplification product containing a target sequence through an appropriate amplification method, combined with the CRISPR/C2C9 detection system, the specificity of the targeting sequence part in the sgRNA is used to detect a specific nucleic acid sequence, which can quickly and efficiently detect the target gene.

The above examples are for the purpose of illustrating the embodiments disclosed by the present invention and are not to be construed as limitations of the present invention. In addition, the various modifications listed herein and the variations of the methods in the invention are obvious to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of the present invention.

Claims (17)

1. Use of C2C9 nuclease in the preparation of genetic testing products. 2. The use according to claim 1, characterized in that the C2C9 nuclease is: (I) a wild-type C2C9 nuclease or a 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 significantly reduced endonuclease activity compared to before the modification or mutation, or a loss of endonuclease activity; and/or (b) polypeptides or domains having other functional activities; (V) According to (I) or (II) or (III), the C2C9 nuclease has endonuclease activity. 3. The use according to claim 1, characterized in that 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 Actinomaduracraniellae C2C9. 4. The use according to claim 1, characterized in that the amino acid sequence of the C2C9 nuclease is SEQ ID NO. Any one of the amino acid sequences shown in SEQ ID NO. 1-115, or having at least 50% sequence homology with any one of the amino acid sequences shown in SEQ ID NO. 1-115, and having RNA-guided nucleic acid binding activity and/or nuclease activity. 5. The use according to claim 1, characterized in that the C2C9 nuclease, sgRNA and fluorescent probe are used in combination to prepare a gene detection product. 6. The use according to claim 5, characterized in that the sgRNA comprises a fixed sequence and a variable region sequence, and the variable region sequence is a fragment that can hybridize with the target sequence. 7. The use according to claim 5, characterized in that the nucleotide sequence of the fixed sequence is as shown in SEQ ID NO: 121; preferably, the nucleotide sequence of the sgRNA matching the AcC2C9 nuclease is as shown in SEQ ID NO: 117. 8. The use according to claim 5, characterized in that one end of the fluorescent probe comprises a fluorescent group, and the other end comprises a quenching group; preferably, the 5' end comprises a fluorescent group, and the 3' end comprises a quenching group; more preferably, the fluorescent group is selected from FAM, TET, JOE, HEX, CY3, 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, characterized in that there is a single-stranded DNA of 4 nt to 20 nt between the fluorescent group and the quenching group; preferably, the single-stranded DNA contains the nucleotide sequence shown in SEQ ID NO: 120. 10. A gene detection product, characterized in that the gene detection product comprises a fluorescent probe, a C2C9 nuclease and a sgRNA, or nucleotides encoding the C2C9 nuclease and the sgRNA. 11. The product according to claim 10, characterized in that the gene sequence to be tested by the gene detection product contains a target sequence that can be complementary to the variable region sequence base in the sgRNA. 12. The product according to claim 10, characterized in that the gene detection product also includes 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 β-mercaptoethanol, DTT, tris(2-carboxyethyl)phosphine or SDS; preferably, the buffer is selected from phosphate buffer or Tris-HCl; preferably, the metal ion is a sodium ion or a magnesium ion. 13. Use of the gene detection product according to any one of claims 10 to 12 in identifying biological species. 14. The use according to claim 13, characterized in that the organism is a microorganism or a cell; and/or the use is for non-disease diagnosis purposes; preferably, the gene detection product is used to detect whether specific microorganisms exist in food or the environment. 15. A gene detection method for non-disease diagnosis purposes, characterized in that the detection method comprises using the gene detection product described in any one of claims 10-12 to detect the gene sequence to be detected. 16. The method according to claim 15, characterized in that 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 according to claim 15, further comprising one or more of the following features: 1) The gene sequence to be tested is selected from double-stranded DNA or single-stranded DNA; 2) Based on the total volume of the reaction system, the working concentration of the fluorescent probe is 100-1000 nM; 3) Based on the total volume of the reaction system, the working concentration of the C2C9 nuclease is 10 nM-100 μM; 4) Based on the total volume of the reaction system, the working concentration of the sgRNA is 10 nM-100 μM; 5) Based on the total volume of the reaction system, the working concentration of the reducing agent in the reaction system is 100 μM-2 mM; 6) The metal ions are provided by NaCl and _MgCl2 , and based on the total volume of the reaction system, the working concentration of NaCl in the reaction system is 50-500 mM, and/or the working concentration of _MgCl2 in the reaction system is 1-20 mM; 7) Based on the total volume of the reaction system, the working concentration of Tris-HCl is 1 to 20 mM; 8) The pH of the reaction system is 7.0-8.0.