WO2021155775A1 - Procédé et kit de détection d'acide nucléique cible - Google Patents

Procédé et kit de détection d'acide nucléique cible Download PDF

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WO2021155775A1
WO2021155775A1 PCT/CN2021/074772 CN2021074772W WO2021155775A1 WO 2021155775 A1 WO2021155775 A1 WO 2021155775A1 CN 2021074772 W CN2021074772 W CN 2021074772W WO 2021155775 A1 WO2021155775 A1 WO 2021155775A1
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nucleic acid
amplification
sequence
target nucleic
crispr
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Chinese (zh)
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吴尧
史才雪
李秋实
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苏州克睿基因生物科技有限公司
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • This application relates to the field of biomedicine, in particular to a method and kit for detecting target nucleic acid in a sample.
  • Nucleic acids contain the universal information characteristics of organisms. The ability to quickly detect nucleic acids with high sensitivity and single-base specificity on a portable platform is of great significance for the diagnosis and monitoring of many diseases, providing valuable epidemiological information, and can be used as a general scientific tool. Although many nucleic acid detection methods have been developed (Du et al. 2017; Green et al. 2014; Kumar et al. 2014; Pardee et al. 2014; Pardee et al. 2016; Urdea et al. 2006), all There will inevitably be pros and cons between sensitivity, specificity, simplicity and speed. For example, the qPCR method is sensitive but expensive, and relies on complex instruments, thus limiting its usability in non-laboratory environments.
  • Zhang Feng s team used Cas13a and RPA isothermal amplification technology to develop the SHERLOCK system to detect Zika and Dengue viruses (Zhang et al. Science. 2017 Apr 28; 356( 6336):438–442); Jennifer’s research group found that Cas12a and other V-type Cas effector proteins have the characteristics of target-activated non-specific ssDNase cleavage.
  • the DETECTR nucleic acid detection method was created by combining Cas12a with isothermal amplification, which can Detection of human papillomavirus in patient samples (Doudna JA et al. Science. 2018 Apr 27; 360(6387):436-439).
  • the target nucleic acid targeted by the SHERLOCK system is RNA.
  • the amplified product must be transcribed into RNA to trigger the detection module of the SHERLOCK system for detection. This step introduces unnecessary non-specificity, increases the reaction time, aggravates the complexity of the reaction system, and also increases the detection cost.
  • the DETECTR system does not need to transcribe the amplified product into RNA, the cleavage of the nucleic acid to be detected by Cas12a will greatly affect the amplification reaction of the target nucleic acid.
  • the amplification of the nucleic acid to be detected The detection using V-type Cas effector protein needs to be performed separately to avoid the reduction of amplification efficiency due to the cleavage of the amplification template by the V-type Cas effector protein, which increases the complexity of the operation and inevitably brings expansion. Increase the risk of product contamination and cross-contamination; and if the amplification of the nucleic acid to be detected in the DETECTR system is carried out at the same time as the detection using the V-type Cas effector protein in the same system, it will be due to the V-type Cas effector protein.
  • the cleavage of the enhanced template thus affects the sensitivity and specificity of the detection.
  • This application provides a method for detecting target nucleic acid in a sample, which includes:
  • the single-stranded amplification product with i) a type V CRISPR/Cas effector protein, ii) gRNA, and iii) an indicator nucleic acid, wherein the gRNA includes a region that binds to the type V CRISPR/Cas effector protein and A guide sequence that hybridizes with the target sequence in the target nucleic acid, the sequence adjacent to the 5'end or 3'end of the target sequence in the target nucleic acid does not contain a PAM sequence, and the indicator nucleic acid is a single-stranded nucleic acid molecule and does not interact with The guide sequence of the gRNA hybridizes; and
  • the a) and the b) are carried out in the same reaction system.
  • the V-type CRISPR/Cas effector protein, the gRNA and the indicator nucleic acid are added to the reaction system containing the single-stranded amplification product.
  • the amplification includes asymmetric amplification.
  • the target nucleic acid comprises double-stranded DNA and/or single-stranded DNA.
  • a forward amplification primer and a reverse amplification primer are used in the amplification, and the concentration ratio of the forward amplification primer and the reverse amplification primer is 1:10-1 :40.
  • a forward amplification primer and a reverse amplification primer are used in the amplification, and the ratio of the reverse amplification primer and the forward amplification primer is 1:10-1:40. .
  • the amplification comprises isothermal amplification.
  • the amplification comprises recombinase polymerase amplification (RPA).
  • RPA recombinase polymerase amplification
  • the sample contains target nucleic acids derived from one or more of the following: cells of an organism, body fluids of an organism, and/or nucleic acid molecules of an organism.
  • the sample contains target nucleic acid derived from a virus, and the virus is selected from the group consisting of African swine fever virus, HCV and HIV.
  • the contacting occurs under in vivo, in vitro, or ex vivo conditions.
  • the type V CRISPR/Cas effector protein includes Cas12 protease.
  • the Cas12 protease is selected from the group consisting of Cas12a protease and Cas12b protease.
  • the type V CRISPR/Cas effector protein comprises the amino acid sequence set forth in any one of SEQ ID NO. 1-11.
  • the indicator nucleic acid comprises a detectable label.
  • the detectable label includes a fluorescent label.
  • the detectable signal includes a fluorescent signal.
  • the concentration of the target nucleic acid is at least 1*1E2 copies or more.
  • the present application also provides a kit for detecting target nucleic acid in a sample, which comprises i) type V CRISPR/Cas effector protein, ii) gRNA and iii) indicator nucleic acid; wherein the gRNA contains the V The region where the type CRISPR/Cas effector protein binds and the guide sequence that hybridizes to the target sequence in the target nucleic acid; and the guide sequence is designed to be in the 5'end or 3'end sequence of the target sequence to which it hybridizes No PAM sequence; the indicator nucleic acid is a single-stranded nucleic acid molecule and does not hybridize with the guide sequence of the gRNA.
  • the kit further includes reagents for obtaining a single-stranded amplification product containing the target nucleic acid.
  • the reagents for obtaining single-stranded amplification products include reagents required for asymmetric amplification.
  • the reagent for asymmetric amplification includes an upstream primer that amplifies the target nucleic acid and/or a downstream primer that amplifies the target nucleic acid.
  • the reagents for obtaining single-stranded amplification products include reagents required for isothermal amplification.
  • the reagents required for the isothermal amplification include RPA recombinase, RPA polymerase and/or RPA buffer.
  • the reagent for the single-stranded amplification product of the target nucleic acid and the V-type CRISPR/Cas effector protein and the gRNA are located in the same container.
  • the type V CRISPR/Cas effector protein includes Cas12 protease.
  • the Cas12 protease is selected from the group consisting of Cas12a protease and Cas12b protease.
  • the type V CRISPR/Cas effector protein comprises the amino acid sequence set forth in any one of SEQ ID NO. 1-11.
  • the indicator nucleic acid comprises a detectable label.
  • the detectable label includes a fluorescent label.
  • Figure 1 shows the VP72-gRNA/cas12a target nucleic acid detection result in this application.
  • FIG. 2 shows the K205R-gRNA/cas12a target nucleic acid detection result in this application.
  • Figure 3 shows the target nucleic acid detection results of the combination of primer-F/primer-R upstream and downstream in different ratios in this application.
  • Figure 4 shows the target nucleic acid detection results of the primer-F1/primer-R1 upstream and downstream combinations in different ratios in this application.
  • Figure 5 shows the detection results of different concentrations of target nucleic acid when the upstream and downstream ratio of primer-F/primer-R in the present application is 20:1.
  • Figure 6 shows the detection results of different concentrations of target nucleic acid when the upstream and downstream ratio of primer-F/primer-R is 1:1 in the present application.
  • Figure 7 shows the comparison results of detection of target nucleic acids at different concentrations when the upstream and downstream ratios of primer-F/primer-R in the present application are 1:1 and 20:1.
  • Figure 8 shows the detection results of different concentrations of target nucleic acid when the upstream and downstream ratio of primer-F1/primer-R1 in the present application is 20:1.
  • Fig. 9 shows the detection results of different concentrations of target nucleic acid when the upstream and downstream ratio of primer-F1/primer-R1 is 1:1 in the present application.
  • Figure 10 shows the comparison results of the detection of target nucleic acids at different concentrations when the upstream and downstream ratios of primer-F1/primer-R1 in the present application are 1:1 and 20:1.
  • CRISPR/Cas system CRISPR/Cas system
  • CRISPR/Cas system CRISPR/Cas system
  • CRISPR system CRISPR system
  • the molecule can direct and realize that the RNA-guided nuclease or other effector molecules modify the nucleic acid at the target nucleic acid, for example, create a gap in the target nucleic acid or cause the target nucleic acid to degrade.
  • CRISPR generally refers to clustered regularly interspaced short palindromic repeats. This sequence usually refers to the first gene sequence found in prokaryotes, which contains the gene fragments of viruses that have attacked the prokaryotes. The organism uses these gene fragments to recognize and resist the same virus attack and destroy it. Nucleic acid molecule. This type of genome constitutes a key part of the prokaryotic immune system. CRISPR usually includes multiple highly conserved repeats and spacers that are different from each other, and the two appear alternately. The length of the repetitive sequence is generally 23 to 50 bp, and the average length is about 31 bp.
  • the repetitive sequence is highly conserved in the same CRISPR site, and there can be a difference of 1 to 5 bases; but between microorganisms, or between CRISPR sites at different positions on the genome of the same microorganism, the conserved sequence of the repetitive sequence big difference.
  • the spacers distributed between the repeats generally consist of 17 to 84 bp, with an average length of about 36 bp. Spacers are poorly conserved. Even in the same CRISPR site, there are basically no identical spacers.
  • Cas Cas protein
  • CRISPR/Cas effector protein CRISPR-associated protein.
  • the Cas protein is the main executor to realize the functions of the CRISPR system, such as the acquisition of spacers or the shearing of DNA molecules.
  • CRISPR/Cas effector protein can be an enzyme with DNA cleavage activity in the CRISPR/Cas system, which can cut double-stranded DNA molecules or/and single-stranded DNA molecules.
  • the Cas protein is a larger Polymorphism family proteins. Genes encoding Cas proteins are generally located downstream of the CRISPR sequence, and sometimes scattered in the genome.
  • the CRISPR-Cas system mainly includes two categories, including multi-subunit protein effectors Type 1 of the complex and type 2 of the single-subunit protein effector complex.
  • the CRISPR-Cas system of type 1 is more common in bacteria and archaea (including all hyperthermophiles), and this type of protein accounts for about all 90% of the identified Cas proteins (Makarova KS, et al. An updated evolutionary classification of CRISPR-Cas systems. Nat. Rev. Microbiol. 2015; 13:722-736).
  • the Cas proteins available for this system mainly include I, Type III and IV effector proteins.
  • the type 2 CRISPR-Cas system is almost exclusively found in bacteria.
  • the Cas proteins available for this system mainly include type II, V, and VI effector proteins, which account for about 10% of the Cas protein.
  • Commonly used Cas9 protein (type II), and Cas12 (type V), Cas13 (VI) type, and Cas14 (type V) proteins Choylinski K. et al., Nucleic Acids Res. 2014; 42:6091-6105; Shmakov S, etc.) , Mol.Cell.2015,60:385–397; Sergey Shmakov et al., Nat Rev Microbiol.2017 March; 15(3):169–182; Doudna J.
  • V-type CRISPR/Cas system and V-type effector protein can be found in Shmakov et al., Nat Rev Microbiol. 2017 March; 15(3): 169-182, Koonin et al., CurrOpin Microbiol .2017 June; 37:67-78.
  • gRNA molecule or “guide RNA”, “guide RNA molecule”, and “gRNA” are used interchangeably, and generally refer to nucleases or other effector molecules that can promote specific guidance of RNA guidance (generally with gRNA molecule complex) to the nucleic acid molecule on the target nucleic acid.
  • the guidance is achieved by hybridizing a portion of the gRNA to DNA (eg, via a gRNA steering domain or guide sequence) and binding a portion of the gRNA molecule to an RNA-guided nuclease or other effector molecule.
  • the gRNA molecule is composed of a single continuous polynucleotide molecule, such as crRNA; in some embodiments, the gRNA molecule may be composed of multiple (such as two ) Polynucleotide molecular composition.
  • the "leader sequence” generally refers to a nucleic acid sequence in the gRNA molecule that can bind to the target sequence in whole or in part by Watson-Crick base pairing and/or G/U base pairing.
  • the "PAM sequence” generally refers to a nucleic acid sequence in the target nucleic acid that can be recognized by the Cas protein in the CRISPR system, and can usually be located at the 3'end or the gRNA complementary sequence in the target sequence according to the difference of the Cas protein. 5'end.
  • the PAM sequence for Cas12 can be located at the 5'end of the target sequence.
  • the PAM sequence can usually consist of 2-6 nucleotides.
  • target nucleic acid and “target polynucleotide” generally refer to a target nucleotide sequence that needs to be identified, detected or located.
  • the target nucleic acid usually contains a continuous or discontinuous sequence that is complementary to the guide sequence, which can become the target sequence. The hybridization between the target sequence and the guide sequence can promote the formation of the CRISPR complex.
  • the target nucleic acid may be double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA).
  • the target nucleic acid can be a polynucleotide from any source.
  • the target polynucleotide can be an exogenous polynucleotide that resides in the nucleus of a eukaryotic cell (for example, the target polynucleotide can be a viral genome sequence).
  • the target polynucleotide may be a sequence encoding a gene product (for example, a protein) or a non-coding sequence (for example, a regulatory polynucleotide or useless DNA).
  • the CRISPR complex generally refers to a complex formed by gRNA and Cas protein.
  • the term "amplification" generally refers to a process in which the copy number of the target nucleic acid is selectively increased while the copy numbers of other genes are not increased proportionally. That is to say, the amplification usually refers to the purposeful amplification of the target nucleic acid, and the non-specific increase in the copy number of sequences other than the target nucleic acid accompanying the process cannot be ruled out. Normally, the non-specific increase is not enough to prevent the detection, recognition or localization of the target nucleic acid.
  • single-stranded amplification product generally refers to a single-stranded DNA sequence obtained by amplification of a target nucleic acid.
  • the product obtained by amplifying the target nucleic acid contains single-stranded DNA sequence and double-stranded DNA sequence, and the single-stranded DNA sequence can be used as a target for recognition and detection of the CRISPR complex.
  • the single-stranded amplification product may be obtained by asymmetric amplification.
  • the term "indicator nucleic acid” generally refers to a single-stranded DNA fragment connected with a pair of fluorescent signal molecules.
  • the emission spectrum of one signal molecule overlaps the region of the absorption spectrum of the other signal molecule in the pair.
  • the fragmentation of the DNA fragment causes the connected fluorescent semi-colon molecules to emit fluorescence or to diminish the fluorescence emitted by the fluorescent signal molecules, thereby achieving the purpose of detection.
  • the fluorescent signal molecule may be a fluorescence resonance energy transfer (FRET) pair or a quencher/fluorescer pair.
  • hybridization generally refers to the nucleus contained in nucleic acid (e.g., RNA, DNA) under conditions in vitro and/or in vivo at a suitable temperature and ionic strength of the solution.
  • nucleic acid e.g., RNA, DNA
  • the nucleotide sequence enables it to specifically non-covalently bind (ie form Watson-Crick base pairs and/or G/U base pairs) to another nucleic acid sequence.
  • Watson-Crick base pairing includes: adenine/adenosine (A) paired with thymidine/thymine (T), A paired with uracil/uridine (U), and guanine/guanosine (G) paired with cytos. Pyrimidine/cytidine (C) pairing.
  • the hybridization between two RNA molecules for example, dsRNA
  • the hybridization between a DNA molecule and an RNA molecule for example, when a DNA target nucleic acid base is paired with a guide RNA, etc.
  • G can also be U base pairing.
  • Hybridization requires that the two nucleic acids contain complementary sequences, but possible mismatches between bases cannot be ruled out.
  • the conditions suitable for hybridization between two nucleic acids depend on the length and degree of complementarity of the nucleic acids, which are well known in the art. The greater the degree of complementarity between two nucleotide sequences, the greater the value of melting temperature (Tm) of hybrids of nucleic acids having these complementary sequences.
  • the length of a hybridizable nucleic acid is 8 nucleotides or more (e.g., 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 20 nucleotides Acid or more, 22 nucleotides or more, 25 nucleotides or more or 30 nucleotides or more).
  • sequence of a polynucleotide need not be 100% complementary to the sequence of its target nucleic acid. For example, it can be 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more More, 98% or more, 99% or more, 99.5% or more complementary.
  • the remaining non-complementary nucleotides can be clustered or interspersed with complementary nucleotides and need not be adjacent to each other or complementary nucleotides.
  • a polynucleotide can hybridize on one or more segments so that no intermediate or adjacent segments are involved in the hybridization event (e.g., loop structure or hairpin structure, "bulge", etc.).
  • BLAST https://blast.ncbi.nlm.nih.gov/Blast.cgi
  • PowerBLAST program Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res .,1997,7,649-656
  • Gap program Gap program (Wisconsin sequence analysis software package, Unix version 8, Genetics Computer Group, University Research Park, Madison Wis).
  • Complementarity can also be expressed by identity, for example, the Needle program of the EMBOSS software package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, Trends in Genetics 16:276-277), version 3.0.0 or more The Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) executed in the higher version is determined.
  • the optional parameters used are gap penalty 10, gap extension penalty 0.5 and EBLOSUM62 replacement matrix (EMBOSS version of BLOSUM62).
  • the term "asymmetric amplification” generally refers to a method of setting the concentration or length of upstream and downstream primers to be different to obtain single-stranded nucleic acid (single-stranded DNA or single-stranded RNA) amplification products.
  • the asymmetric amplification can include the following types: 1) Asymmetric amplification is performed using upstream and downstream primers of different concentrations. As the circulation increases, the primers with a small amount are gradually depleted, and the primers with an excessive amount can continue to amplify to generate single-stranded DNA (Gyllensten and Erlich, Proc, Natl. Acad Sci. USA, 1988, 85: 7652-7656) .
  • the term "isothermal amplification” generally refers to a method of performing nucleic acid molecule amplification under constant temperature conditions.
  • the traditional polymerase chain reaction (PCR) usually involves denaturation (about 95°C), annealing (about 5°C lower than the primer Tm value, generally 45-55°C), and extension (about 72°C).
  • PCR polymerase chain reaction
  • a specific reaction instrument such as a PCR machine
  • isothermal amplification does not require the above-mentioned temperature change process, and is completed under a certain constant temperature condition, so there is no need for a PCR machine, and the operation is simple. Isothermal amplification was first published in 2000 by Japanese scholar Notomi on Nucleic Acids Res.
  • Loop-mediated isothermal amplification technology (Loop-mediated isothermal amplification) was developed. Later, a variety of methods for amplifying nucleic acid molecules under isothermal conditions have been developed. For example, Recombinase Polymerase Amplification (RPA) and so on.
  • RPA Recombinase Polymerase Amplification
  • RPA recombinase polymerase amplification
  • SSB single-stranded DNA-binding protein
  • SSB single-stranded DNA-binding protein
  • Enzyme strand-displacing polymerase
  • RPA was developed and released by the British biotechnology company TwistDx Ltd. (formerly known as ASM Scientific Ltd.). The above-mentioned recombinase, single-stranded DNA binding protein, and single-stranded displacement polymerase are used as the core factors in the RPA process.
  • the recombinase can combine with the primer to form a protein-DNA complex, find homologous sequences in the double-stranded DNA and make the primer pair with the homologous sequence in the double-stranded DNA.
  • the primer When the primer is positioned to the homologous sequence, stranding will occur. Exchange reaction and start DNA synthesis under the action of single-strand displacement polymerase, and exponentially amplify the target region on the template. The replaced DNA strand binds to the SSB to prevent further replacement.
  • the target nucleic acid is amplified by using two opposite primers.
  • RPA can be performed under suitable temperature conditions (e.g.
  • RNA and DNA can be achieved by adding reverse transcriptase to the RPA reaction system without the need for a separate step to produce cDNA; for example, by adding a CRISPR system and a fluorescent indicator to achieve the detection of target nucleic acids.
  • Cas12 protease is also referred to as “Cas12”, “Cas12 protein”, “Cas12 nuclease”, and generally refers to a type of CRISPR-related protein that can detect single-stranded DNA or double-stranded DNA under the guidance of gRNA. DNA is cut.
  • This protein was originally called Cpf1 and was first proposed by Zhang Feng's team (see “Cpf1Is a Single RNA-Guided Endonuclease of a Class 2CRISPR-Cas System", Cell, 2015).
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value. Variation within the range of 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • the present application provides a method for detecting target nucleic acid in a sample, which includes:
  • the single-stranded amplification product with i) a type V CRISPR/Cas effector protein, ii) gRNA, and iii) an indicator nucleic acid, wherein the gRNA includes a region that binds to the type V CRISPR/Cas effector protein and A guide sequence that hybridizes with the target sequence in the target nucleic acid, the sequence adjacent to the 5'end or 3'end of the target sequence in the target nucleic acid does not contain a PAM sequence, and the indicator nucleic acid is a single-stranded nucleic acid molecule and does not interact with The guide sequence of the gRNA hybridizes; and
  • the above a) and the above b) are carried out in the same reaction system.
  • V-type CRISPR/Cas effector protein, the gRNA and the indicator nucleic acid are added to the system for amplifying the target nucleic acid in the sample.
  • V-type CRISPR/Cas effector protein, the gRNA and the indicator nucleic acid are added while preparing the system for the amplification of the target nucleic acid in the sample.
  • V-type CRISPR/Cas effector protein, the gRNA and the indicator nucleic acid are added to the reaction system containing the single-stranded amplification product.
  • gRNA guide RNA or guide RNA
  • PAM protospacer proximity motif
  • the type V CRISPR/Cas effector protein can specifically bind and/or cleave the target nucleic acid including: 1) Type V CRISPR/Cas effect
  • the protein recognizes the protospacer-adjacent motif (PAM) in the target nucleic acid and/or 2) guides the base pairing between the RNA and the target sequence.
  • PAM protospacer-adjacent motif
  • the gRNA may include a type V CRISPR/Cas effector protein (such as Cas12 protein, such as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e) combined to form a ribonucleoprotein complex (RNP), and the complex A single-stranded nucleic acid molecule that targets a specific target sequence within the target nucleic acid.
  • the gRNA may include RNA molecules, DNA/RNA hybrid molecules, that is, the gRNA may include DNA bases in addition to RNA bases.
  • the guide RNA may include a guide sequence and a region that binds to the V-type CRISPR/Cas effector protein (also referred to as a protein binding region or a constant region), and the guide sequence hybridizes with the target sequence of the target DNA.
  • the constant region can bind to the V-type CRISPR/Cas effector protein.
  • the gRNA can reduce the cleavage/degradation effect of the V-type CRISPR/Cas effector protein on the double-stranded target nucleic acid, and realize the amplification of the double-stranded target nucleic acid and the recognition/cutting of the single-stranded amplification product by the CRISPR/Cas effector protein In the same system.
  • the ribonucleoprotein complex formed by the gRNA and the V-type CRISPR/Cas effector protein cannot cleave a double-stranded nucleotide sequence, and at the same time can cleave a single-stranded nucleotide sequence.
  • the guide sequence in the gRNA is complementary to the target sequence (target DNA fragment), and the sequence adjacent to the 5'end or 3'end of the target sequence does not contain the PAM sequence in the target nucleic acid.
  • no PAM sequence in the 5'end or 3'end of the target sequence may include a PAM sequence that recognizes a double-stranded target nucleic acid by the ribonucleoprotein complex formed by the V-type CRISPR/Cas effector protein and the gRNA,
  • the guide sequence in the gRNA cannot interact with the sequence adjacent to the PAM sequence to initiate the cleavage of the double-stranded target nucleic acid.
  • the ribonucleoprotein complex can realize the cleavage of the single-stranded amplification product through the recognition of the gRNA and the target sequence in the single-stranded nucleotide that is complementary to the gRNA, without relying on PAM sequence that recognizes single-stranded nucleotides.
  • the absence of a PAM sequence in the sequence adjacent to the 5'end or 3'end of the target sequence may include: between the PAM sequence and the first nucleotide at the 5'end or 3'end of the target sequence at least There is 1 nucleotide, such as at least 2 nucleotides, such as at least 3 nucleotides, such as at least 4 nucleotides, such as at least 5 nucleotides, such as at least 6 nucleosides Acid, for example, at least 7 nucleotides, for example, at least 8 nucleotides, for example, at least 9 nucleotides, for example, at least 10 nucleotides, for example, at least 11 nucleotides, for example, at least 12 nucleotides, such as at least 13 nucleotides, such as at least 14 nucleotides, such as at least 15 nucleotides, such as at least 16 nucleotides, such as at least 17 nucleotides , Such as at least 1
  • Type V CRISPR/Cas effector proteins may require different PAM sequences in the target nucleic acid. Therefore, for the selected specific V-type CRISPR/Cas effector protein, the corresponding PAM sequence is selected.
  • Various methods including computer methods and/or experimental methods) for identifying suitable PAM sequences are known and conventional in the art, and any convenient method.
  • the PAM sequence may include: 5'-TTTN-3' (when the V-type CRISPR/Cas effector protein is LbCas12a and/or AsCas12a), 5'-TTN-3' (when the V When the type CRISPR/Cas effector protein is FnCas12a, PmCas12a, MbCas12a, Mb2Cas12a, Mb3Cas12a, TsCas12a, BsCas12a and/or AacCas12a).
  • the length of the guide sequence is 15-28 nucleotides (nt), such as 15-26 nucleotides, 15-24 nucleotides, 15-22 nucleotides, 15-20 nuclei.
  • the length of the leader sequence is 18-24 nucleotides.
  • the leader sequence is at least 15 nucleotides, for example, at least 16, 18, 20, or 22 nucleotides.
  • the guide sequence and the target sequence of the target nucleic acid have 80% or more (eg, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more , 98% or more, 99% or more, or 100%) complementarity.
  • the leader sequence may include at least 15 nucleotides that are complementary to the target sequence.
  • the constant region may include a V-type CRISPR/Cas effector protein (such as Cas12 protein, such as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, for example, LbCas12a, AsCasi2a, Frcas12a, PoCas12a, MbCas12a, Mb2Cas12a, Mb3Cas12a, IsCas12a, BsCas12a) are used together with the nucleotide sequence.
  • Cas12 protein such as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e
  • the constant region may be 15 or more nucleotides (nt) in length, for example, 18 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more. More, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 or more, 31 or more, 32 or more, 33 or more, 34 or More, or 35 or more.
  • nt nucleotides
  • the length of the constant region may range from 12 to 100 nucleotides, for example, 12 to 90, 12 to 80, 12 to 70, 12 to 60, 12 to 50, 12 to 40, 15 to 100, 15 To 90, 15 to 80, 15 to 70, 15 to 60, 15 to 50, 15 to 40, 20 to 100, 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40 , 25 to 100, 25 to 90, 25 to 80, 25 to 70, 25 to 60, 25 to 50, 25 to 40, 28 to 100, 28 to 90, 28 to 80, 28 to 70, 28 to 60, 28 To 50, 28 to 40, 28 to 100, 29 to 100, 29 to 90, 29 to 80, 29 to 70, 29 to 60, 29 to 50 or 29 to 40 nucleotides.
  • 12 to 90, 12 to 80, 12 to 70, 12 to 60, 12 to 50, 12 to 40 15 to 100, 15 To 90, 15 to 80, 15 to 70, 15 to 60, 15 to 50, 15 to 40, 20 to 100, 20 to
  • the constant region of the gRNA may be truncated relative to the corresponding region of the corresponding wild-type gRNA.
  • the constant region of the gRNA may be extended relative to the corresponding region of the corresponding wild-type gRNA.
  • the constant region may be located at the 5'end or 3'end of the guide sequence.
  • the constant region may comprise a complementary RNA sequence, which forms an RNA duplex (dsRNA) by self-folding.
  • dsRNA RNA duplex
  • the length of the RNA duplex can be 2 to 12 base pairs, for example, 2 to 10, 2 to 8, 2 to 6, 2 to, 2 to 4, 2 to 3 base pairs ;
  • the length of the RNA duplex (dsRNA) can be 3 to 12 base pairs, 3 to 10 base pairs, 3 to 8 base pairs, 3 to 6 base pairs, 3 to Base pairs, 3 to 4 base pairs, 4 to 12 base pairs, 4 to 10 base pairs, 4 to 8 base pairs, 4 to 6 base pairs or 4 to 5 bases Base pair.
  • the constant region may include a length of 2 or more base pairs, such as 3 or more, 4 or more, 5 or more, 6 or more or 7 DsRNA duplexes of two or more base pairs.
  • the constant region of the guide RNA may include a dsRNA duplex that is longer than the dsRNA duplex of the corresponding wild-type guide RNA.
  • the constant region of the guide RNA may include a dsRNA duplex that is shorter than the dsRNA duplex of the corresponding wild-type guide RNA.
  • the length of the constant region of the guide RNA can be 12 to 100 nucleotides, such as 12 to 90, 12 to 80, 12 to 70, 12 to 60, 12 to 50, 12 to 40, 15 to 100, 15 to 90, 15 to 80, 15 to 70, 15 to 60, 15 to 50, 15 to 40, 20 to 100, 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40, 25 to 100, 25 to 90, 25 to 80, 25 to 70, 25 to 60, 25 to 50, 25 to 40, 28 to 100, 28 to 90, 28 to 80, 28 to 70, 28 to 60, 28 to 50, 28 to 40, 29 to 100, 29 to 90, 29 to 80, 29 to 70, 29 to 60, 29 to 50, or 29 to 40 nucleotides and the constant region of the guide RNA
  • the length of can be in the range of 28 to 100 nucleotides, for example, the length of the constant region of the guide RNA is in the range of 28 to 40 nucleotides.
  • the constant region sequence of the guide RNA may include the nucleotide sequence shown in any one of SEQ ID NOs. 12-18.
  • the constant region of the gRNA may also include the constant region sequence shown in any one of SEQ ID NO. 12-18 having 70% or higher identity (for example, 80% or higher, 85% Or higher, 90% or higher, 95% or higher, 98% or higher, 99% or higher, or 100% identity).
  • the guide RNA may include a nucleotide sequence targeting African swine fever virus VP72 and/or African swine fever virus K205R.
  • the guide RNA may include a nucleotide sequence as shown in any one of SEQ ID NOs. 19-26.
  • type V CRISPR/Cas effector protein is a subtype of the 2 types of CRISPR/Cas effector protein, including any non-target ssDNA cleavage once activated (by hybridizing with its related guide RNA and target DNA) Active V-type CRISPR/Cas effector protein (see Doudna JA et al., Science. 2018 Apr 27; 360(6387):436-439).
  • the V-type CRISPR/Cas effector protein may be derived from different bacterial genera, and its enzyme activity may also be different.
  • the V-type CRISPR/Cas effector protein may include Cas12 effector protein, for example, Cas12a, Cas12b (C2c1), Cas12c (C2c3), C2c4, C2c8, C2c5, C2c10, C2c9, and CasX (Cas12e), CasY (Cas12d )Wait.
  • Cas12 effector protein for example, Cas12a, Cas12b (C2c1), Cas12c (C2c3), C2c4, C2c8, C2c5, C2c10, C2c9, and CasX (Cas12e), CasY (Cas12d )Wait.
  • the Cas12 effector protein may include Cas12a, Cas12b, Cas12c, Cas12d and/or Cas12e.
  • the Cas12 effector protein may include Cas12a, Cas12b, and Cas12c.
  • the type V CRISPR/Cas effector protein includes Cas12 protease.
  • the Cas12 protease is selected from the group consisting of Cas12a protease and Cas12b protease.
  • the Cas12 protease is selected from the group consisting of LbCas12a, AsCasi2a, Frcas12a, PoCas12a, MbCas12a, Mb2Cas12a, Mb3Cas12a, IsCas12a, BsCas12a.
  • V-type CRISPR/Cas effector protein comprises the amino acid sequence described in any one of SEQ ID NOs 1-11.
  • the type V CRISPR/Cas effector protein is a naturally occurring protein (e.g., naturally occurring in prokaryotic cells).
  • the type V CRISPR/Cas effector protein is a non-naturally-occurring protein (for example, a variant protein, a chimeric protein, a fusion protein, etc. of a naturally-occurring protein).
  • the non-naturally-occurring protein and its corresponding naturally-occurring protein have functional conservation, and the functional conservation means that the non-naturally-occurring protein maintains a certain degree of function of its corresponding naturally-occurring protein (e.g. 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, 100%).
  • the function can be detected by conventional technical means in the art, for example, the activity of the V-type CRISPR/Cas effector protein to cleave the target nucleic acid and/or non-specifically cleave ssDNA as described in this application.
  • the naturally-occurring protein may be from different genus, or from the same genus (for example, the same bacterial species), and the naturally-occurring protein has the function (for example, the type V CRISPR described in this application). /Cas effector protein activity) conservative (e.g.
  • the naturally occurring protein has 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more , 95% or more, 98% or more, 100%), the naturally occurring protein has 20% or more amino acid sequence identity (for example, 30% or more, 40% or more, 50% or more High, 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher,) amino acid sequence.
  • amino acid sequence identity for example, 30% or more, 40% or more, 50% or more High, 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, amino acid sequence.
  • the type V CRISPR/Cas effector protein may include the Cas12a, the Cas12b (C2c1), the Cas12c (C2c3), the C2c4, the C2c8, the C2c5, the C2c10, the The amino acid sequence of C2c9, the CasX (Cas12e) or the CasY (Cas12d) has at least 80%, 83%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical polypeptide sequences.
  • the Cas12a protein may be Cas12a proteins derived from different species, such as FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a and/or Lb4Cas12a.
  • Cas12a proteins derived from different species such as FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a and/or Lb4Cas12a.
  • the Cas12a protein may include an amino acid sequence as shown in any one of SEQ ID NO. 1-11.
  • the Cas12a protein may include at least 80%, 83%, 85%, 86%, 87%, 88%, 89%, 90% of the amino acid sequence shown in any one of SEQ ID No: 1-11. , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical Cas12a protein, or any one of SEQ ID No: 1-11
  • the amino acid sequence shown has a variant of Cas12a with one or several amino acid deletions, substitutions or additions, and the variant has the functional conservation of Cas12a.
  • the deletion, substitution or addition of one or several amino acids can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid deletions, substitutions Or add.
  • the Cas12a protein is derived from Lachnospiraceae, Acidaminococcus, Porphyromonas macacae, Moraxella bovoculi and/or Thiomicrospira sp.).
  • the Cas12 protein can also form a fusion protein with a heterologous polypeptide (fusion ligand).
  • fusion ligand a heterologous polypeptide
  • the heterologous polypeptide can provide subcellular localization, that is, the heterologous polypeptide contains a subcellular localization sequence (for example, a nuclear localization signal (NLS) for targeting the nucleus, for keeping the sequence of the fusion protein outside the nucleus.
  • a subcellular localization sequence for example, a nuclear localization signal (NLS) for targeting the nucleus, for keeping the sequence of the fusion protein outside the nucleus.
  • NLS nuclear localization signal
  • NES Output sequence
  • a sequence that keeps the fusion protein retained in the cytoplasm is used to target the mitochondrial mitochondrial localization signal, is used to target the chloroplast-targeted chloroplast localization signal, the Golgi localization signal, etc.
  • the NLS may include the NLS sequence derived from the following: the NLS of the SV40 virus large T antigen, with the amino acid sequence PKKKRKV; the NLS from the nucleoplasmic protein (for example, the nucleoplasmic protein dyad NLS with the sequence KRPAATKKAGQAKKKK); c -myc NLS, with the amino acid sequence PAAKRVKLD or RQRRNELKRSP; hRNPA1M9NLS, with the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY.
  • the NLS sequence derived from the following: the NLS of the SV40 virus large T antigen, with the amino acid sequence PKKKRKV; the NLS from the nucleoplasmic protein (for example, the nucleoplasmic protein dyad NLS with the sequence KRPAATKKAGQAKKKK); c -myc NLS, with the amino acid sequence PAAKRVKLD or RQRR
  • the heterologous polypeptide may be provided with a tag (a detectable label) to facilitate tracking and/or purification.
  • the tag may include fluorescent protein (eg, green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), blue fluorescent protein (CFP), etc.), mCherry, tdTomato, histidine Labels (for example, 6 ⁇ His label), hemagglutinin (HA) label, FLAG label, Myc label, biotin label, nemycin label, etc.
  • the positioning sequence or tag contained in the fusion protein may be any one or more of the above, and each tag or positioning sequence may be one or more repeats.
  • the indicator nucleic acid contains a detectable label.
  • the detectable label includes a fluorescent label.
  • the fluorescent label is a fluorescent signal molecule pair, such as a fluorescence resonance energy transfer (FRET) pair or a quencher/fluorescent agent pair.
  • FRET fluorescence resonance energy transfer
  • the fluorescent signal molecule pair is respectively labeled on the 5'end and 3'end of the single-stranded DNA molecule, which is the indicator nucleic acid.
  • the strand of the indicator nucleic acid molecule is broken so that the relative position of the fluorescent signal molecule pair changes, thereby generating detectable signal changes before and after cleavage.
  • the indicator nucleic acid may be cleaved by the V-type CRISPR/Cas effector protein; the cleavage of the indicator nucleic acid by the V-type CRISPR/Cas effector protein may be the same as that of the V-type CRISPR/Cas effector protein on the target nucleic acid.
  • the cleavage of the single-stranded amplification product is associated.
  • the signal change can be used as an indicator for detecting the target nucleic acid.
  • the signal change may include: the indicator nucleic acid generates a certain amount of detectable signal before being cleaved, and when the indicator nucleic acid is cleaved, the amount of the detectable signal is reduced or quenched.
  • the signal change may include: the indicator nucleic acid generates a first detectable signal before being cleaved, and when the indicator nucleic acid is cleaved, a second detectable signal is generated.
  • the signal change may include: indicating that the nucleic acid does not generate a detectable signal before being cleaved, and when the indicating nucleic acid is cleaved, generating the detectable signal.
  • the signal change may include: the indicator nucleic acid generates a certain amount of detectable signal before being cleaved, and when the indicator nucleic acid is cleaved, the amount of the detectable signal increases.
  • the fluorescence resonance energy transfer (FRET) pair may include a donor and an acceptor.
  • the donor and acceptor of the fluorescence resonance energy transfer (FRET) pair are known to those skilled in the art, and the donor and acceptor applicable to this application can be selected adaptively.
  • FRET fluorescence resonance energy transfer
  • the fluorescence resonance energy transfer (FRET) pair may include:
  • IAEDANS ((5-(2-iodoacetylaminoethyl)aminonaphthalene-1-sulfonic acid)/DDPM(N-(4-dimethylamino-3, 5-Dinitrophenyl)maleimide), BFP/DsRFP, dansyl/FITC, dansyl/Octadecylrhodamine, Cyan fluorescent protein )/Green Fluorescent Protein (GFP), CF (Carboxyfluorescein Succinimidyl Ester)/TexasRed, Fluorescein/Tetramethylrhodamine, Cy3/Cy5, Green Fluorescent Protein (GFP)/Yellow Fluorescent protein (YFP), Rhodamine 110/Cy3, Rhodamine 6G/Malachite Green, FITC/Eosin Thiosemicarbazide, B-Phycoerythrin/Cy5, Cy5 /Cy5.5
  • the quencher/fluorescer pair may include a quencher group and a fluorescent group.
  • the fluorescent group can emit a detectable signal.
  • the detectable signal is completely or partially quenched by the quenching group.
  • the quenching groups and fluorescent groups are known to those skilled in the art, and the quenching groups and fluorescent groups applicable to this application can be selected adaptively.
  • the fluorescent group may include: carboxyfluorescein (FAM, Carboxy fluorescein), fluorescein isothiocyanate (FITC, Fluorescein isothiocyanate), tetrachloro-6-carboxyfluorescein (TET, Tetrachloro fluorescein), hexachlorofluorescein -6-Methyl fluorescein (HEX, Hexachloro fluorescein), 2,7-dimethyl-4,5-dichloro-6-carboxy fluorescein (JOE), rhodamine dyes, such as R110, TAMRA, Texas Red, etc.), ROX, AlexaFluor dyes (e.g.
  • Alexa 350 Alexa 405,Alexa 430,Alexa 488,Alexa 500, Alexa 514,Alexa 532,Alexa 546,Alexa 555,Alexa 568,Alexa 594,Alexa 610,Alexa 633,Alexa 635,Alexa 647,Alexa 660,Alexa 680, Alexa 700, Alexa 750, Alexa 790), ATTO dyes (e.g.
  • the quenching group may include DABCYL, TAMRA, MGB, BHQ-0, BHQ-1, BHQ-2, and/or BHQ-3.
  • the single-stranded DNA molecule may have any length capable of realizing the signal change before and after cutting.
  • the length of the single-stranded DNA molecule can be 3-180 nucleotides, for example, 5-100, 5-80, 10-50, 5-30, 10-60, 10- 70, 10-30, 15-50, 12-40, 8-80, 12-28, 18-40, 100-180, 80-180, 70-100, 30- 80 nucleotides.
  • the single-stranded DNA molecule does not hybridize to the guide sequence of the gRNA.
  • the non-hybridization with the guide sequence of the gRNA usually means that the identity of the single-stranded DNA molecule and the guide sequence is not sufficient to form a double-stranded structure through complementary base pairing between the two.
  • the single-stranded DNA molecule has 40% or less identity with the guide sequence, such as 30% or less, such as 20% or less, such as 15% or less, such as 10% or less, such as 5 % Or less, for example, no identity.
  • the single-stranded DNA probe is 5-FAM/TTATTAATTATA/BHQ1-3.
  • the sample contains target nucleic acids derived from one or more of the following: cells of an organism, body fluids of an organism, and/or nucleic acid molecules of an organism.
  • the sample may contain nucleic acid sequences other than the target nucleic acid.
  • the sample contains target nucleic acid derived from cells of the organism.
  • the cells of the organism may be in vitro cells (for example, an established cultured cell line), or may be isolated cells (cultured cells from an individual, primary cells).
  • the cell may be a cell in the body (a cell in a biological individual).
  • the sample may be a biological cell, a lysate of biological cells, or a homogenate of biological tissues, and the lysate or homogenate may be prepared by a method known to those skilled in the art (e.g., RIPA lysis method). , Ultrasonic crushing, grinding homogenate, etc.).
  • the sample may also be a product of further purification of the cell lysate, for example, some ions or organics are removed to reduce its influence on subsequent operations.
  • the biological cells may include animal cells, plant cells, and microbial cells.
  • the plant cells may include Arabidopsis thaliana cells, and may also include cells of agricultural crops, such as plant somatic cells such as wheat, corn, rice, sorghum, millet, soybeans, etc.; the plant cells may also include cells of fruit and nut plants , For example, produce apricots, oranges, lemons, apples, plums, pears, almonds, walnuts and other plants.
  • the plant cell may be a cell derived from any part of the plant body, for example, root cells, leaf cells, xylem cells, phloem cells, cambium cells, apical meristem cells, parenchyma cells.
  • the microbial cells may include bacteria (e.g. Escherichia coli, archaea), fungi (e.g. yeast), actinomycetes (e.g.), rickettsiae, mycoplasma, chlamydia, spirochetes, and the like.
  • bacteria e.g. Escherichia coli, archaea
  • fungi e.g. yeast
  • actinomycetes e.g.
  • rickettsiae mycoplasma
  • chlamydia chlamydia
  • spirochetes and the like.
  • the animal cell may include invertebrate (e.g., Drosophila, nematode, planarian, etc.) cells, and vertebrate (e.g., zebrafish, chicken, mammalian) cells.
  • invertebrate e.g., Drosophila, nematode, planarian, etc.
  • vertebrate e.g., zebrafish, chicken, mammalian cells.
  • the mammalian cells may include mice, rats, rabbits, pigs, dogs, cats, monkeys, humans, and the like.
  • the animal cells may include cells from any tissue in the organism, such as stem cells, induced pluripotent stem (iPS) cells, germ cells (eg oocytes, egg cells, sperm cells, etc.), adult stem cells, somatic cells ( For example, fibroblasts, hematopoietic cells, cardiomyocytes, neurons, muscle cells, bone cells, liver cells, pancreatic cells, epithelial cells, immune cells and those derived from lung, spleen, kidney, stomach, large intestine, small intestine and other organs or tissues Any cell) and embryos at any stage in vitro or in vivo.
  • stem cells eg oocytes, egg cells, sperm cells, etc.
  • germ cells eg oocytes, egg cells, sperm cells, etc.
  • adult stem cells e.g., etc.
  • somatic cells fibroblasts, hematopoietic cells, cardiomyocytes, neurons, muscle cells, bone cells, liver cells, pancreatic cells, epit
  • the sample contains the target nucleic acid derived from the body fluid of the organism.
  • the body fluid of the organism may include cerebrospinal fluid, aqueous humor, lymph, digestive juice (e.g., saliva, gastric juice, small intestinal fluid, bile, etc.), breast milk, blood, urine, sweat, tears, feces, respiratory secretions, reproduction Organ secretions (such as semen, cervical mucus), etc.
  • the sample may include nucleic acid molecules of the organism.
  • the nucleic acid molecule can be isolated and extracted from any organism by the technical means known to those skilled in the art to separate nucleic acid molecules, including DNA and RNA.
  • the nucleic acid molecule is extracted from the above-mentioned biological cells or body fluids of the biological body.
  • the target nucleic acid may be a polynucleotide from any source, for example, it may include double-stranded DNA and/or single-stranded DNA.
  • the polynucleotide may include an exogenous polynucleotide that resides in the nucleus of a eukaryotic cell (for example, the target polynucleotide may be a viral genome sequence); for example, the target polynucleotide may include a coding gene The sequence of the product (e.g., protein) or a non-coding sequence (e.g., regulatory polynucleotide or useless DNA).
  • the product e.g., protein
  • a non-coding sequence e.g., regulatory polynucleotide or useless DNA
  • the genome sequence of the virus may include papillomavirus (for example, human papilloma virus (HPV), polyoma virus, etc.), and hepatitis virus (for example, hepatitis B virus (HBV), hepatitis C virus (HCV) Etc.), herpes virus (such as herpes simplex virus (HSV), varicella-zoster virus (VZV), cytomegalovirus (CMV), herpes lymphovirus, pityriasis erythema, Kaposi’s sarcoma-associated herpes virus, etc.), Adenovirus, adeno-associated virus, poxvirus (such as smallpox, vaccinia virus, monkeypox virus), African swine fever virus and/or human immunodeficiency virus (HIV).
  • papillomavirus for example, human papilloma virus (HPV), polyoma virus, etc.
  • hepatitis virus
  • the sequence encoding the gene product may include a sequence encoding a tumor antigen
  • the tumor antigen may include a TNF receptor family member B cell maturation antigen (BCMA), Tn antigen (such as Tn'Ag, GalNAc ⁇ -Ser/Thr) , Prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1), Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72), CD38, CD44v6, cancer Embryonic antigen (CEA), epithelial cell adhesion molecule (EPCAM), B7H3 (CD276), KIT (CD117), interleukin-13 receptor subunit ⁇ -2 (IL-13Ra2 or CD213A2), mesothelin, interleukin 11 receptor ⁇ (IL-11Ra), prostate stem cell antigen (PSCA), p53, p53 mutant, prostate specific protein (
  • BCMA
  • the target nucleic acid may also include a sequence containing a SNP site in the genome of an organism.
  • the target nucleic acid may also include the gene sequence of pathogenic bacteria, parasites and the like.
  • the concentration of the target nucleic acid is at least 1*1E2 copies or more, 1*1E3 copies or more, 1*1E4 copies or more, for example, 0.2*1E5 copies or more, 0.3*1E5 copies or Above, 0.4*1E5 copies or more, 0.5*1E5 copies or more, 0.6*1E5 copies or more, 0.7*1E5 copies or more, 0.8*1E5 copies or more, 0.9*1E5 copies or more, 1*1E5 copies or more, 3*1E5 copies or more, 6*1E5 copies or more.
  • the concentration of the target nucleic acid is at least 1 amol or more, 10 amol or more, 30 amol or more, 80 amol or more, 150 amol or more ( amol) or more, 200 amol or more, 300 amol or more, 500 amol or more, 700 amol or more, and 900 amol or more.
  • the concentration of the target nucleic acid is at least 1 femtomole (fmol) or more, 10 femtomole (fmol) or more, 30 femtomole (fmol) or more, 80 femtomole (fmol) or more, 150 femtomole (fmol) or more ( fmol) or more, 200 femtomole (fmol) or more, 300 femtomole (fmol) or more, 500 femtomole (fmol) or more, 700 femtomole (fmol) or more, and 900 femtomole (fmol) or more.
  • the concentration of the target nucleic acid is at least 1 picomoles (pmol) or more, 10 picomoles (pmol) or more, 30 picomoles (pmol) or more, 80 picomoles (pmol) or more, 150 picomoles (pmol) or more ( pmol) or more, 200 picomoles (pmol) or more, 300 picomoles (pmol) or more, 500 picomoles (pmol) or more, 700 picomoles (pmol) or more, 900 picomoles (pmol) or more.
  • the concentration of the target nucleic acid is at least 1 nanomole (nmol) or more, 10 nanomole (nmol) or more, 30 nanomole (nmol) or more, 80 nanomole (nmol) or more, 150 nanomole ( nmol) or more, 200 nanomole (nmol) or more, 300 nanomole (nmol) or more, 500 nanomole (nmol) or more, 700 nanomole (nmol) or more, 900 nanomole (nmol) or more.
  • the concentration of the target nucleic acid is at least 1 micromole ( ⁇ mol) or more, for example, 30 micromole ( ⁇ mol) or more, 50 micromole ( ⁇ mol) or more, and 80 micromole ( ⁇ mol) or more.
  • the method for detecting target nucleic acid in a sample includes:
  • the single-stranded amplification product with i) a type V CRISPR/Cas effector protein, ii) gRNA, and iii) an indicator nucleic acid, wherein the gRNA includes a region that binds to the type V CRISPR/Cas effector protein and A guide sequence that hybridizes with the target sequence in the target nucleic acid, the sequence adjacent to the 5'end or 3'end of the target sequence in the target nucleic acid does not contain a PAM sequence, and the indicator nucleic acid is a single-stranded nucleic acid molecule and does not interact with The guide sequence of the gRNA hybridizes; and
  • the contact may occur under in vivo, in vitro or ex vivo conditions, for example, the V-type CRISPR/Cas effector protein can bind to the gRNA, the gRNA may include a guide sequence, and the single-stranded amplification product A target sequence may be included, and the guide sequence can hybridize to the target sequence so that the V-type CRISPR/Cas effector protein targets the single-stranded amplification product.
  • the interaction of the V-type CRISPR/Cas effector protein and the single-stranded amplification product can correlate with the non-specific cleavage of the indicator nucleic acid by the V-type CRISPR/Cas effector protein.
  • V-type CRISPR/Cas effector protein can cleave the single-stranded amplification product.
  • the cleavage of the single-stranded amplification product enables the V-type CRISPR/Cas effector protein to perform non-specific cleavage of the indicator nucleic acid.
  • the indicator nucleic acid molecule strand breaks can cause changes in the relative positions of the fluorescent signal molecule pairs respectively connected to the 5'and 3'ends of the indicator nucleic acid DNA strand, thereby generating detectable signal changes before and after cleavage. .
  • the detection of the target nucleic acid can be achieved by detecting the change in the signal.
  • the double-stranded target nucleic acid and/or its double-stranded amplification product can avoid being cut or degraded by the V-type CRISPR/Cas effector protein, so that the amplification and detection of the target nucleic acid can be achieved in the same system conduct.
  • the double-stranded target nucleic acid and/or its double-stranded amplification product can avoid being cleaved or degraded by the V-type CRISPR/Cas effector protein by being adjacent to the 5'end or 3'end of the target sequence in the target nucleic acid This is achieved without PAM sequence in the sequence.
  • the above a) and the above b) are carried out in the same reaction system.
  • the amplification may include any amplification method that can produce a single-stranded amplification product, and the V-type CRISPR/Cas effector protein can still be maintained under the amplification conditions (such as temperature, ion concentration, etc.)
  • Its enzymatic properties, such as cleaving the single-stranded amplification product and the activity of the indicator nucleic acid, for example can maintain at least 30%, for example, at least 40% of its activity under conventional conditions (such as the best known activity conditions) , At least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100%, the conventional conditions are known to those skilled in the art, such as those reported in the known technical literature Use conditions of different CRISPR/Cas effector proteins.
  • the CRISPR/Cas effector protein is Cas12, and the conventional use conditions of Cas12 can be found in Doud
  • the CRISPR/Cas effector protein can realize the function of detecting the target nucleic acid described in the present application under the amplification conditions (such as temperature, ion concentration, etc.).
  • V-type CRISPR/Cas effector proteins that is, V-type CRISPR/Cas effectors from various species
  • V-type CRISPR/Cas effectors from various species can be selected according to the amplification conditions to facilitate the use in various provided amplification methods to take advantage of all Desired characteristics (for example, specific enzymatic properties).
  • different amplification methods can be selected according to the enzymatic characteristics of different V-type CRISPR/Cas effector proteins (that is, V-type CRISPR/Cas effector proteins from various species), so as to facilitate the selection of various amplification methods provided. Use to take advantage of the specific enzymatic properties required.
  • the amplification may include asymmetric amplification.
  • the asymmetric amplification may include the following types: 1) Upstream and downstream primers of different concentrations are used for asymmetric amplification. As the circulation increases, the primers with a small amount are gradually depleted, and the primers with an excessive amount can continue to amplify to generate single-stranded DNA (Gyllensten and Erlich, Proc, Natl. Acad Sci. USA, 1988, 85: 7652-7656) ; 2) Upstream and downstream primers of different lengths are used for asymmetric amplification.
  • a forward amplification primer and a reverse amplification primer are used in the amplification, and the concentration ratio of the forward amplification primer and the reverse amplification primer is 1:10-1:80, for example, The concentration ratio is 1:10-1:70, 1:10-1:60, 1:10-1:50, 1:10-1:40, 1:15-1:40, 1:20-1 :40, 1:10-1:30, 1:10-1:20, 1:15-1:30. For example, the concentration ratio is 1:10-1:40.
  • a forward amplification primer and a reverse amplification primer are used in the amplification, and the ratio of the reverse amplification primer and the forward amplification primer is 1:10-1:80, for example, the The concentration ratio is 1:10-1:70, 1:10-1:60, 1:10-1:50, 1:10-1:40, 1:15-1:40, 1:20-1:40 , 1:10-1:30, 1:10-1:20, 1:15-1:30.
  • the concentration ratio is 1:10-1:40.
  • the amplification includes isothermal amplification.
  • the isothermal amplification refers to a method of amplifying nucleic acid molecules under constant temperature conditions.
  • it may include loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), and recombinase polymerase.
  • LAMP loop-mediated isothermal amplification
  • HDA helicase-dependent amplification
  • recombinase polymerase recombinase polymerase
  • RPA strand displacement amplification
  • SDA nucleic acid sequence-based amplification
  • NASBA nucleic acid sequence-based amplification
  • TMA transcription-mediated amplification
  • NEAR nickase amplification reaction
  • RCA rolling circle amplification
  • MDA Multiple displacement amplification
  • cHDA single primer isothermal amplification
  • SPIA single primer isothermal amplification
  • SMART RNA technology signal-mediated amplification
  • GEAR genomic exponential amplification reaction
  • IMDA isothermal multiple displacement amplification
  • the amplification includes recombinase polymerase amplification (RPA).
  • the amplification system amplified by the recombinase polymerase may include recombinase, single-stranded DNA-binding protein (SSB), and single-stranded displacement polymerase.
  • the amplification temperature of the recombinase polymerase may be 10-50°C, such as 15-50°C, 18-50°C, 20-50°C, 25-50°C, 30-50°C, 35-50°C , 10-45°C, 20-45°C, 25-45°C, 28-45°C, 30-45°C, 35-45°C, 37-42°C.
  • the amplification is an amplification method in which the asymmetric amplification and the isothermal amplification are combined.
  • the combined amplification method is to obtain the amplification product under isothermal conditions.
  • the detection of the signal change is a fluorescence detection system
  • the fluorescence detection system may include the following modules: 1) A temperature control module, the temperature control range is 0-100°C, and a specific temperature can be selected according to the temperature conditions of the amplification reaction.
  • Temperature control conditions for example, 25-50 °C, for example, 37-42 °C; 2) fluorescence detection module, according to the different pairs of fluorescent signal molecules set different excitation wavelength and/or emission wavelength, for example, excitation light wavelength 490nm, emission wavelength is 520nm, for example, excitation light wavelength is 535nm, emission wavelength is 560nm; 3) Timing detection function module, can detect the set fluorescence every 0.5-120 minutes, for example, every 2 Perform a test every 15 minutes, for example, perform a test every 2-5 minutes, for example, the duration of the test is 10 minutes to 3 hours, for example, 15 minutes to 2 hours.
  • the fluorescence detection system may be BioTekCytation 3; for example, the fluorescence detection system may be ThermoVarioskan TM LUX; for example, the fluorescence detection system may be fluorescence quantitative PCR, for example, the fluorescence detection system may be Applied Biosystems TM 7500 Real- Time PCR System.
  • the application also provides a kit for detecting target nucleic acid in a sample, which comprises i) a type V CRISPR/Cas effector protein, ii) a gRNA, and iii) an indicator nucleic acid; wherein the gRNA comprises the same type as the type V CRISPR/Cas The region where the effector protein binds and the guide sequence that hybridizes to the target sequence in the target nucleic acid; and the guide sequence is designed such that the sequence at the 5'end or 3'end of the target sequence to which it hybridizes does not contain a PAM sequence ;
  • the indicator nucleic acid is a single-stranded nucleic acid molecule and does not hybridize with the guide sequence of the gRNA.
  • the kit further includes reagents for obtaining a single-stranded amplification product containing the target nucleic acid.
  • the reagents for obtaining single-stranded amplification products include reagents required for asymmetric amplification.
  • the reagent may include a single primer or primer pair designed for the target nucleic acid to be detected, and the single primer or primer pair can hybridize with the target nucleic acid and initiate the amplification of the target nucleic acid.
  • the primer pair includes amplification
  • the upstream primer of the target nucleic acid and/or the downstream primer that amplifies the target nucleic acid may be located in different containers, or may be located in the same container, and set to be different concentration.
  • the upstream primer and the downstream primer may have different lengths, and the length difference between the upstream primer and the downstream primer is sufficient to enable the longer primer to anneal and hybridize to the target nucleic acid, while the shorter primer cannot Annealing and hybridizing with the target nucleic acid.
  • the reagents for obtaining single-stranded amplification products include reagents required for isothermal amplification.
  • the reagent may include any substance capable of achieving the isothermal amplification, and may be selected according to the enzymatic characteristics of the V-type CRISPR/Cas effector protein used in combination therewith to achieve the purpose of detecting the target nucleic acid.
  • the reagents required for the isothermal amplification may include RPA recombinase, RPA polymerase and/or RPA buffer.
  • the RPA polymerase may include a single-strand displacement polymerase
  • the RPA buffer may include a single-stranded binding protein and metal ions, for example, magnesium ions.
  • the reagents for the single-stranded amplification product and/or the reagents required for isothermal amplification may also include dATP, dGTP, dTTP, and dCTP.
  • the reagents for the single-stranded amplification product of the target nucleic acid and/or the reagents required for isothermal amplification and the V-type CRISPR/Cas effector protein and the gRNA are located in the same container.
  • the type V CRISPR/Cas effector protein includes Cas12 protease.
  • the Cas12 protease is selected from the group consisting of Cas12a protease and Cas12b protease.
  • the V-type CRISPR/Cas effector protein comprises the amino acid sequence described in any one of SEQ ID NOs. 1-11.
  • the indicator nucleic acid contains a detectable label.
  • the detectable label includes a fluorescent label.
  • the fluorescent label can be adapted to select the fluorescence resonance energy transfer (FRET) pair and/or the quencher/fluorescent agent pair.
  • the kit may also include other compounds (such as enzymes or indicators, etc.) to provide additional functions.
  • the detection of RNA and/or DNA can be achieved by adding reverse transcriptase.
  • the following examples are only to illustrate the chimeric antigen receptor, preparation method, and use of the present application, and are not used to limit the scope of the present application.
  • the examples do not include detailed descriptions of traditional methods, such as those used to construct vectors and plasmids, methods of inserting genes encoding proteins into such vectors and plasmids, or methods of introducing plasmids into host cells.
  • Such methods are well known to those of ordinary skill in the art, and are described in many publications, including Sambrook, J., Fritsch, EF and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd edition, Cold spring Harbor Laboratory Press.
  • the gRNA that only cuts ssDNA is designed, and the asymmetric RPA amplification method is used to generate a large number of ssDNA targets.
  • African swine fever protein K205R sequence shown in SEQ ID NO.36
  • design two gRNA gRNA5 and gRNA6 sequences (shown in SEQ ID NO.23-24, respectively) on the sense strand
  • design on the antisense strand Two gRNA (gRNA7 and gRNA8) sequences (shown in SEQ ID NO.25-26), and the corresponding amplification primer pair: primer-F1/primer-R1 (shown in SEQ ID NO.29-30, respectively) .
  • the 2xNA buffer formula is: 80mM Tris-HCl, 120mM NaCl, 12mM MgCl, PH 7.3;
  • For the preparation method of cas12a refer to JSChen, E.Ma, LB Harrington, M. Da Costa, X. Tian, JMPalefsky, et al.
  • the above 50 ⁇ l reaction system is reacted at 37°C for 60 minutes, and the FAM fluorescence value is read every 2 minutes (Fluorescence Quantitative PCR Instrument, ABI 7500).
  • Each gRNA is set with two replicates and an NTC negative control group.
  • the concentration of cas12a in the system is 250nM
  • the concentration of gRNA is 250nM
  • the concentration of the signal reporter probe (5'-FAM-TTATT-BHQ1-3') is 1uM
  • the concentration of ssDNA is 1E8 copies/ul.
  • the gRNA detection result of VP72 sequence is shown in Figure 1.
  • the gRNA test results are shown in Figure 2.
  • Figure 1-Figure 2 show that all 8 gRNAs can achieve the cutting effect, and gRNA4 and gRNA6 are selected for downstream verification.
  • primer-F1: primer-R1 1:1 Or 1:10 or 1:20 or 1:4. Screen out the primer concentration combination with the best detection effect.
  • RPA upstream primers, RPA downstream primers and DNA (plasmid) to be tested (shown in SEQ ID NO.31 and 36) were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.; cas12a was produced by Suzhou Cree Gene Biotechnology Co., Ltd. (Sequence (As shown in SEQ ID NO.1); gRNA was synthesized by GenScrip; RPA enzyme premix and magnesium acetate were purchased from Jiangsu Qitian Gene Company (Cat. No.: B190122AA) and used according to the product instructions; NTC is nuclease-free water, purchased from Invitrogen Corporation.
  • the concentration in Table 1 refers to the concentration of the original solution before each reagent is added to the reaction system.
  • the above 50 ⁇ l reaction system was reacted at 37°C for 60 minutes, and the FAM fluorescence value was read every 2 minutes (Fluorescence Quantitative PCR Instrument, ABI 7500). Two repetitions are set for each concentration ratio.
  • concentration of cas12a in the system is 250nM
  • concentration of gRNA is 250nM
  • concentration of DNA to be tested is 1E6 copies/ ⁇ l
  • the RPA enzyme premix and magnesium acetate are used in accordance with the product instructions.
  • the experimental results are shown in Figure 3 and Figure 4.
  • F:R-1:1 indicates that the concentration of primer-F is 400nM, and the concentration of primer-R is 400nM
  • F:R-10:1 indicates that the concentration of primer-F is 400nM, and the concentration of primer-R is 400nM
  • the concentration of F:R-20:1 indicates that the concentration of primer-F is 400nM and the concentration of primer-R is 20nM
  • F:R-40:1 indicates that the concentration of primer-F is 400nM and the concentration of primer-R It is 10nM.
  • F1R1-1:1 indicates that the concentration of primer-F1 is 400nM, the concentration of primer-R1 is 400nM; F1R1-1:10 indicates that the concentration of primer-F1 is 40nM, and the concentration of primer-R1 is 400nM; F1R1-1:20 indicates The concentration of primer-F1 is 20nM, the concentration of primer-R1 is 400nM; F1R1-1:40 means that the concentration of primer-F1 is 10nM, and the concentration of primer-R1 is 400nM. According to the results, the primer ratios of F:R-20:1 and F1:R1-1:20 were selected for downstream verification.
  • the single-stranded product obtained by primer-F is shown in SEQ ID NO.41; the single-stranded product obtained by primer-R is shown in SEQ ID NO.42; the single-stranded product obtained by primer-F1 is shown in SEQ ID NO.43 Show; the single-stranded product obtained by primer-R1 is shown in SEQ ID NO.44.
  • Example 4 Asymmetric RPA and symmetric RPA combined with Cas12a one-step method for lower detection limit verification
  • Select gRNA4 and gRNA6, select the primer concentration ratio F:R-1:1, F:R-20:1, F1:R1-1:1, F1:R1-1:20 to verify the detectable target amount and target amount Set to 1E6, 1E5, 1E4, 1E3, 1E2.
  • the detection effect of asymmetric RPA is better than that of symmetric RPA.
  • the primer F:R ratio is 20:1, and the target is as low as 1E2 copies.
  • the fluorescent signal can be detected at 40 minutes, and it is significantly different from the negative control. Test results.
  • Example 5 Asymmetric RPA and symmetric RPA combined with Cas12a one-step detection and DETECTR system comparison
  • Select gRNA6, prepare a 50 ⁇ l reaction system according to the method of Example 3, the primer concentration ratios are F1:R1-1:1, F1:R1-1:20, and the target amount is set to 1E6, 1E5, 1E4, 1E3, 1E2.
  • the DETECTR system as follows, where the gRNA9 sequence: UAAUUUCUACUAAGUGUAGAUAAGACCUGCUUUCAGCAGUA (SEQ ID NO.45), the primer concentration ratios are F1:R1-1:1, F1:R1-1:20, and the target scalar is set to 1E6, 1E5 , 1E4, 1E3, 1E2.
  • Example 6 Other asymmetric isothermal amplification combined with one-step detection of Cas12a
  • Select gRNA4 and gRNA6, select the primer concentration ratio F:R-20:1, F1:R1-1:20 to verify the detectable target amount, and the target amount is set to 1E6, 1E5, 1E4, 1E3, 1E2.
  • EMA Normal Temperature Nucleic Acid Amplification

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Abstract

L'invention concerne un procédé de détection d'un acide nucléique cible dans un échantillon, comprenant : l'amplification d'un acide nucléique cible dans un échantillon pour obtenir un produit d'amplification monocaténaire ; la mise en contact du produit d'amplification monocaténaire avec une protéine effectrice CRISPR/Cas de type V, un ARN guide et un acide nucléique indicateur, l'ARN guide comprenant une région liée à la protéine effectrice CRISPR/Cas de type V et une séquence de guidage hybridée à une séquence cible dans l'acide nucléique cible, la séquence adjacente à l'extrémité 5' ou à l'extrémité 3' de la séquence cible dans l'acide nucléique cible ne comprenant pas de séquence PAM, l'acide nucléique indicateur étant une molécule d'acide nucléique monocaténaire et n'étant pas hybridé à la séquence d'ARN guide ; et la détection d'un signal détectable généré par l'acide nucléique indicateur clivé par la protéine effectrice CRISPR/Cas de type V, permettant ainsi de détecter l'acide nucléique cible.
PCT/CN2021/074772 2020-02-03 2021-02-02 Procédé et kit de détection d'acide nucléique cible WO2021155775A1 (fr)

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