WO2021175230A1 - Protéine de cas13 séparée - Google Patents

Protéine de cas13 séparée Download PDF

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WO2021175230A1
WO2021175230A1 PCT/CN2021/078776 CN2021078776W WO2021175230A1 WO 2021175230 A1 WO2021175230 A1 WO 2021175230A1 CN 2021078776 W CN2021078776 W CN 2021078776W WO 2021175230 A1 WO2021175230 A1 WO 2021175230A1
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isolated
cas13 protein
seq
rna
derived
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PCT/CN2021/078776
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Chinese (zh)
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陈玲玲
杨力
杨良中
高宝青
李斯琪
黄友葵
王滢
徐奕锋
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中国科学院分子细胞科学卓越创新中心
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Priority claimed from CN202010136188.8A external-priority patent/CN113337488B/zh
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Publication of WO2021175230A1 publication Critical patent/WO2021175230A1/fr

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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the field of biotechnology, in particular to an isolated Cas13 protein.
  • CRISPR-associated CRISPR-associated, CRISPR-Cas
  • CRISPR-Cas The clustered regularly interspaced shortpalindromic repeat (CRISPR-associated, CRISPR-Cas) is an important immune defense system for archaea and bacteria against foreign nucleic acid infection, and is used to resist the invasion of phage viruses, etc. .
  • the CRISPR/Cas system can specifically recognize and cut foreign DNA or RNA, thereby silencing the expression of foreign genes.
  • CRISPR-Cas systems There are many types of CRISPR-Cas systems currently known. Among them, the CRISPR-Cas9 system is widely used in gene knockout, knock-in, base editing, etc., which overcomes the cumbersome steps, time-consuming, and low efficiency of traditional gene editing techniques. Disadvantages, with its fewer ingredients, convenient operation and higher efficiency, it meets the gene editing needs of most fields, and has potential and huge clinical application value.
  • CRISPR/Cas13 is an RNA-targeted CRISPR system, with four subtypes VI-A (Cas13a/C2c2), VI-B (Cas13b), VI-C (Cas13c) and VI-D (Cas13d), starting in 2015 Since its discovery, it has been applied to many aspects such as RNA knockdown, RNA base editing and RNA splicing regulation. In addition to its RNA-specific cleavage activity, CRISPR/Cas13 protein can cleave other RNAs non-specifically after being activated (accessory cleavage). This new feature provides key theoretical support for the nucleic acid detection platform based on the CRISPR/Cas13 system. Using this new CRISPR technology: CRISPR-Cas13 can detect diseases including MERS and other RNA virus infections with high sensitivity, and the combination of different Cas13 proteins can achieve simultaneous detection of multiple nucleic acids.
  • the purpose of the present invention is to provide an isolated Cas13 protein to solve the problems in the prior art.
  • the amino acid sequence of the Cas13 protein includes:
  • the isolated Cas13 protein can specifically cleave target RNA.
  • the target RNA is derived from the novel coronavirus COVID-19.
  • the isolated Cas13 protein can cleave other RNAs non-specifically after being activated.
  • the isolated Cas13 protein is derived from human gut metagenome
  • the isolated Cas13 protein is derived from human metagenome
  • the isolated Cas13 protein is derived from Malacobacter halophilus
  • the isolated Cas13 protein is derived from Prolixibacteraceae bacterium;
  • the isolated Cas13 protein is derived from Planctomycetes bacterium;
  • the isolated Cas13 protein is derived from Lentisphaeria bacterium;
  • the isolated Cas13 protein is derived from Empedobacter sp.;
  • the isolated Cas13 protein is derived from Rhodovulum viride
  • the isolated Cas13 protein is derived from Bacteroidales bacterium;
  • the isolated Cas13 protein is derived from Flavobacteriales bacterium;
  • the isolated Cas13 protein is derived from Verrucomicrobia bacterium;
  • the isolated Cas13 protein is derived from microbial mat metagenome
  • the isolated Cas13 protein is derived from oral metagenome
  • the isolated Cas13 protein is derived from Prevotellaceae bacterium;
  • the isolated Cas13 protein is derived from Saprospiraceae bacterium;
  • the isolated Cas13 protein is derived from metagenome
  • the isolated Cas13 protein is derived from the bacterium A37T11;
  • the isolated Cas13 protein is derived from Micavibrio sp;
  • the isolated Cas13 protein is derived from Proteiniphilum sp.;
  • the isolated Cas13 protein is from Listeria sp.;
  • the isolated Cas13 protein is derived from Ruminococcus sp.;
  • the isolated Cas13 protein is derived from Thiothrix sp.
  • the isolated Cas13 protein is derived from Bacteroides graminisolvens
  • the isolated Cas13 protein is derived from Prevotella intermedia
  • the isolated Cas13 protein is derived from Prevotellacae bacterium.
  • the HEPN motif of the RNase activity domain of the isolated Cas13 protein loses RNA cleavage activity after mutation, but the isolated Cas13 protein still retains the ability to bind RNA.
  • Another aspect of the present invention provides an isolated polynucleotide encoding the isolated Cas13 protein.
  • constructs containing the isolated polynucleotide.
  • the construct is a vector, including expression vectors and integrative vectors.
  • Another aspect of the present invention provides an expression system that contains the construct or the exogenous polynucleotide integrated into the genome.
  • the host cell of the expression system is selected from eukaryotic cells or prokaryotic cells, preferably selected from human cells.
  • Another aspect of the present invention provides the isolated Cas13 protein, the isolated polynucleotide, the construct, the expression system, or the base editing system described below in any one of the embodiments in RNA Use in horizontal gene editing.
  • the use is specifically the use in gene editing at the RNA level of eukaryotes or in nucleic acid detection.
  • Another aspect of the present invention provides a base editing system, including the isolated Cas13 protein, and the base editing system further includes gRNA.
  • Another aspect of the present invention provides an RNA-level gene editing method, including: performing gene editing through the isolated Cas13 protein or the base editing system.
  • Another aspect of the present invention provides a nucleic acid detection method, including: performing nucleic acid detection through the isolated Cas13 protein or the base editing system.
  • the nucleic acid detection method includes:
  • step 2) Combining and cutting the amplified product provided in step 1) through the isolated Cas13 protein or the base editing system;
  • the nucleic acid detection method is a detection method of a novel coronavirus COVID-19.
  • the nucleic acid detection method further includes: judging the detection result through the probe.
  • the present invention also provides the isolated Cas13 protein, the isolated polynucleotide, the construct, the expression system, or the base editing system of any one of the embodiments herein.
  • the kit contains the isolated Cas13 protein described in any of the embodiments herein, and optionally one or more of the following reagents: and RNA to be tested or edited.
  • GRNA, RNA cleavage buffer, fluorescent RNA substrate, T7 polymerase, rNTP, yeast tRNA and RNase inhibitor that specifically bind to and can be recognized by the Cas13 protein contained in the detection kit.
  • the kit further includes a test paper that can capture the complete fluorescent marker-RNA-biotin marker complex at the quality control line C, and capture and cut the complex at the test line T Free fluorescein produced.
  • Figure 1 shows a schematic diagram of the genome data analysis process in Example 1 of the present invention.
  • Fig. 2 is a schematic diagram showing the prediction result of the secondary structure of the palindrome repeat sequence corresponding to the protein in Example 1 of the present invention.
  • Figure 3 shows a schematic diagram of the results of protein purification in Example 2 of the present invention.
  • Figure 4 shows a schematic diagram of the accessory cleavage activity of the novel protein in Example 2 of the present invention.
  • Figure 5 is a schematic diagram showing the sequence preference at both ends of the targeting site of HmeCas13a in Example 3 of the present invention.
  • Fig. 6 is a schematic diagram showing the sequence preference at both ends of the target site of MhdCas13c in Example 3 of the present invention.
  • Figure 7 is a schematic diagram showing the base preference results of the HmeCas13a cleavage site in Example 4 of the present invention.
  • Figure 8 is a schematic diagram showing the base preference results of the MhdCas13c cleavage site in Example 4 of the present invention.
  • Figure 9 is a schematic diagram showing the comparison of the activities of MhdCas13c and LwaCas13c at different target sites in Example 5 of the present invention.
  • Example 10 is a schematic diagram of the activity of MhdCas13c in Example 6 of the present invention when the length of the complementary sequence of the gRNA and the target position is different.
  • Fig. 11 is a schematic diagram of the activity of MhdCas13c in Example 7 of the present invention when a single-base mismatch occurs between the complementary sequence of the gRNA and the target position.
  • Fig. 12 is a schematic diagram of the activity of MhdCas13c in Example 7 of the present invention when there is a double-base mismatch between the complementary sequence of the gRNA and the target position.
  • Figure 13 is a schematic diagram showing the screening results of the HmeCas13a protein used to detect COV-Target gRNA in Example 8 of the present invention.
  • Fig. 14 is a schematic diagram showing the screening results of the MhdCas13c protein used to detect COV-Target gRNA in Example 8 of the present invention.
  • Figure 15 a shows the schematic diagram of the sensitivity of the HmeCas13a protein used in the direct detection of COV-Target in Example 8 of the present invention; b shows the schematic diagram of the sensitivity result of the direct detection of COV-Target using the MhdCas13c protein in Example 8 of the present invention.
  • Figure 16 shows a schematic diagram of the screening results of ERA primers in Example 8 of the present invention.
  • Fig. 17 is a schematic diagram showing the sensitivity result of detection of COV-Target standard product by SHELOCK based on HmeCas13a in Example 8 of the present invention.
  • Fig. 18 is a schematic diagram showing the sensitivity results of detection of COV-Target standard products by SHELOCK based on MhdCas13c in Example 8 of the present invention.
  • Fig. 19 is a schematic diagram showing the result of detecting COV-Target standard test paper by SHELOCK based on MhdCas13c in Example 9 of the present invention.
  • Figure 20 shows a schematic diagram of the genome data analysis process in Example 1 of the present invention.
  • Figure 21 is a schematic diagram showing the results of protein purification in Example 10 of the present invention.
  • Figure 22 is a schematic diagram of the northern blot identification result in Example 10 of the present invention.
  • Figure 23a shows a schematic diagram of the accessory cleavage activity of Pin4Cas13b in Example 11 of the present invention.
  • Figure 23b shows a schematic diagram of the accessory cleavage activity of BgrCas13a in Example 11 of the present invention.
  • Figure 24 is a schematic diagram showing the results of identifying knockdown RNA activity in Example 12 of the present invention.
  • Figure 25 is a schematic diagram of the identification result of labeled RNA ability in Example 13 of the present invention.
  • Figure 26a is a schematic diagram showing the result of using Cas13 protein to detect sno-lncRNA1 in Example 14 of the present invention.
  • Figure 26b is a schematic diagram showing the result of using Cas13 protein to detect sno-lncRNA2 in Example 14 of the present invention.
  • Figure 26c is a schematic diagram showing the result of using Cas13 protein to detect sno-lncRNA3 in Example 14 of the present invention.
  • Figure 26d is a schematic diagram showing the result of using Cas13 protein to detect sno-lncRNA4 in Example 14 of the present invention.
  • Figure 26e shows a schematic diagram of the result of using Cas13 protein to detect sno-lncRNA5 in Example 14 of the present invention.
  • Figure 27 is a schematic diagram showing the changes in the fluorescence of the substrate RNA in Example 15 of the present invention.
  • Figure 28 is a schematic diagram showing the test results of the test paper in Example 16 of the present invention.
  • Figure 29 is a schematic diagram showing the comparison of RNA detection in Example 16 of the present invention.
  • Figure 30 is a schematic diagram of the construction of a novel coronavirus-specific target in Example 17 of the present invention.
  • Figure 31 is a schematic diagram showing the results of gRNA screening of the novel coronavirus in Example 17 of the present invention.
  • Figure 32 is a schematic diagram showing the test results of the test paper in Example 17 of the present invention.
  • the inventors of the present invention have provided a new type of isolated Cas13 protein that has the ability to bind RNA and has RNA-targeted cleavage activity when matched with gRNA, so that it can be used for RNA level
  • the present invention has been completed on the basis of gene editing, nucleic acid detection, etc.
  • the first aspect of the present invention provides an isolated Cas13 protein, the amino acid sequence of the Cas13 protein includes:
  • amino acid sequence in b) specifically refers to: the amino acid sequence shown in one of SEQ ID NOs: 1 to 43 has been substituted, deleted, or added one or more (specifically, 1-50, 1- 30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids, or added at the N-terminus and/or C-terminus One or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids and The obtained amino acid sequence has the function of the polypeptide shown in one of SEQ ID NOs: 1 to 43 and 287 to 289.
  • SEQ ID NOs: 1 to 43 and 287 to 289 include cooperating with gRNA to perform specific or non-specific cleavage of RNA.
  • the amino acid sequence in b) can be 80%, 85%, 90%, 93%, 95%, 97% or 99% of the amino acid sequence shown in one of SEQ ID NOs: 1 to 43 and 287 to 289. The similarities above.
  • the isolated Cas13 protein is derived from human gut metagenome, human metagenome, Malacobacter halophilus, Prolixibacteraceae bacterium, Planctomycetes bacteriu, Lentisphaeria bacterium, Empedobacter sp., Rhodovulum viride, Bacteroidal bacteria, bacterium, bacteria, metabolic microe Prevotellaceae bacterium, Saprospiraceae bacterium, metagenome, bacterium A37T11, Micavibrio sp, Proteiniphilum sp., Listeria sp. 102, Ruminococcus sp. or Thiothrix sp..
  • the protein is derived from human gut metagenome, human metagenome, Malacobacter halophilus, Prolixibacteraceae bacterium, Planctomycetes bacteriu, Lentisphaeria bacterium, Empedobacter sp., Rhodovulum viride, Bacteroidales bacterium, Flavobacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria, bacteria
  • the Cas13 protein of the present invention with SEQ ID NO: 1, 3, 17, 18, 19, 20, 21 or 23 has 80%, 85%, 90%, 93%, 95%, 97% Or more than 99% similarity, and derived from human gut metagenome; or 80%, 85%, 90%, 93%, 95%, 97%, or more than 99% similarity with SEQ ID NO: 2, and source From Malacobacter halophilus (such as DSM 18805 strain); or with SEQ ID NO: 4 with 80%, 85%, 90%, 93%, 95%, 97% or 99% similarity, and derived from Prolixibacteraceae bacterium (such as UBA12447); or with SEQ ID NO: 5 with 80%, 85%, 90%, 93%, 95%, 97% or 99% similarity, and derived from Planctomycetes bacterium (such as RBG_16_55_9); or with SEQ ID NO:6 has 80%, 85%, 90%, 93%, 95%, 97% or 99% similarity with SEQ
  • SEQ ID NO: 41 (such as UBA6147) ; Or with SEQ ID NO: 41 with 80%, 85%, 90%, 93%, 95%, 97% or 99% similarity, and derived from Proteiniphilum sp. (such as UBA4907); or with SEQ ID NO :42 has 80%, 85%, 90%, 93%, 95%, 97% or 99% similarity, and is derived from Ruminococcus sp. (such as AM40-10AC); or has 80% with SEQ ID NO: 43 %, 85%, 90%, 93%, 95%, 97% or more than 99% similarity, and derived from Thiothrix sp. (such as UBA2332).
  • the Cas13 protein of the present invention has the amino acid sequence shown in SEQ ID NO: 2, or 80%, 85%, 90%, 93%, 95%, 97% or 80%, 85%, 90%, 93%, 95%, 97% of SEQ ID NO: 2 Similarity of more than 99%.
  • the Cas13 protein is derived from Malacobacter halophilus (such as DSM 18005 strain); or has the amino acid sequence shown in SEQ ID NO: 9, or has 80%, 85 %, 90%, 93%, 95%, 97%, or 99% or more similarity, preferably, the Cas13 protein is derived from human metagenome; or has the amino acid sequence shown in SEQ ID NO: 287, or is similar to SEQ ID NO: 287 has 80%, 85%, 90%, 93%, 95%, 97%, or 99% or more similarity.
  • the Cas13 protein is derived from Bacteroides graminisolvens; or has SEQ ID NO: 288
  • the amino acid sequence of SEQ ID NO: 288 is 80%, 85%, 90%, 93%, 95%, 97% or 99% similar, preferably, the Cas13 protein is derived from Prevotellaceae bacterium; or Having the amino acid sequence shown in SEQ ID NO: 289, or having 80%, 85%, 90%, 93%, 95%, 97%, or more than 99% similarity with SEQ ID NO: 289, preferably, the The Cas13 protein is derived from Prevotella Intermedia.
  • the isolated Cas13 protein provided by the present invention can be matched with a specific gRNA, so that the isolated Cas13 protein can specifically cleave the target RNA, and at the same time, the isolated Cas13 protein can be activated but not specifically cleave other RNAs.
  • the isolated Cas13 protein can be activated by binding gRNA and target RNA.
  • the target RNA may be derived from the novel coronavirus COVID-19.
  • the target RNA may be derived from an individual suffering from chubby Wiley syndrome.
  • the isolated Cas13 protein provided by the present invention may have the ability to bind RNA.
  • the isolated Cas13 protein may be Used for RNA labeling.
  • the HEPN motif of the RNase activity domain of the isolated Cas13 protein has a mutation, thereby losing the activity of cutting RNA but retaining the ability to bind RNA.
  • gRNA usually includes two parts: target binding region and Cas protein recognition region.
  • the target binding region and the Cas13 protein recognition region are usually connected in a 5'to 3'direction.
  • the length of the target binding region is usually 15 to 35 bases, more usually 26 to 30 bases, such as 30 bases.
  • the target binding region specifically binds to RNA, thereby recruiting Cas13 to a predetermined site.
  • the Cas protein recognition region of the gRNA is determined according to the Cas protein used, which is mastered by those skilled in the art.
  • the gRNA can be prepared by conventional methods in the art, for example, by conventional chemical synthesis methods.
  • the gRNA can also be transferred into the cell via an expression vector, and the gRNA can be expressed in the cell.
  • the gRNA expression vector can be constructed using methods well known in the art. Generally, gRNA contains 60-80 nucleotides.
  • the second aspect of the present invention provides an isolated polynucleotide encoding the isolated Cas13 protein provided in the first aspect of the present invention.
  • the present invention also includes the complementary sequence of the polynucleotide, and the 15-50 base-long fragment of the polynucleotide and its complementary sequence.
  • the third aspect of the present invention provides a construct comprising the isolated polynucleotide provided in the second aspect of the present invention and one or more regulatory sequences operably linked to the polynucleotide sequence.
  • the control sequence can be a suitable promoter sequence.
  • the promoter sequence is usually operably linked to the coding sequence of the protein to be expressed.
  • the promoter can be any nucleotide sequence that shows transcriptional activity in the host cell of choice, including mutant, truncated and hybrid promoters, and can be derived from extracellular sequences that are homologous or heterologous to the host cell. Or the intracellular polypeptide gene is obtained.
  • the control sequence may also be a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription.
  • the terminator sequence is operatively linked to the 3'end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the host cell of choice can be used in the present invention.
  • the control sequence can also be a suitable leader sequence, the untranslated region of the mRNA that is important for the host cell's translation.
  • the leader sequence is operably linked to the 5'end of the nucleotide sequence encoding the polypeptide. Any leader sequence that is functional in the host cell of choice can be used in the present invention. Therefore, the construct can be an expression cassette consisting of a promoter, the isolated polynucleotide described herein, and polyA. In some embodiments, the construct is a vector, including expression vectors and integrative vectors.
  • the expression vector is used to express the Cas13 protein of the present invention; the integrated vector is used to integrate the coding sequence of the Cas13 protein of the present invention or the expression cassette of the present invention into the genome of the host cell.
  • Vectors can refer to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art. As long as it can replicate and stabilize in the host, any plasmid, virus, and phage can be used.
  • the expression vector may be a pCMV vector or the like.
  • the fourth aspect of the present invention provides an expression system containing the isolated polynucleotide according to the second aspect of the present invention.
  • the isolated polynucleotide may be present in the expression system in the form of a vector, or may be present in the expression system in the form of being integrated into the genome.
  • the expression system may be a host cell, and the host cell may express the isolated Cas13 protein as described above, and the isolated Cas13 protein may cooperate with gRNA, so that the isolated Cas13 protein can be located to the target region, The combination of the target area is realized, and the cleavage of the target area of the nucleic acid can be further realized.
  • the host cell may be a eukaryotic cell and/or a prokaryotic cell, etc., for example, a human cell or the like.
  • the host cell may be a cell line such as HeLa and 293FT.
  • the fifth aspect of the present invention provides a gene/base editing system, including the isolated Cas13 protein provided in the first aspect of the present invention.
  • the system can also include gRNA.
  • gRNA Those skilled in the art can select a suitable gRNA that targets a specific site according to the target editing region of the gene.
  • the sequence of the gRNA can usually be at least partially complementary to the target region, so that it can cooperate with the isolated Cas13 protein to locate the Cas13 protein to the target region to achieve the binding of the Cas13 protein to the target region, and further Realize the cleavage of the target area of nucleic acid.
  • the gRNA can target the nucleic acid of the novel coronavirus COVID-19, and the polynucleotide sequence of the gRNA can specifically include the sequence shown in one of SEQ ID NOs: 250 to 274.
  • the gene/base editing system can also include various reagents required by other CRISPR-Cas13 systems, for example, can also include RNase inactivator, recombinase polymerase amplification reaction reagent, T7 RNA polymerase, rNTP, Cas13 One or more of protein reaction buffer, fluorescent RNA substrate and yeast tRNA.
  • the RNase inactivator can be used to inhibit RNase activity in the sample, thereby inhibiting its degradation of RNA, and effectively protecting the RNA to be detected.
  • the rNTP may be ribonucleoside triphosphate, which plays a role as a raw material in RNA synthesis.
  • Yeast tRNA is used to improve specificity.
  • the gene/base editing system of the present invention contains the isolated Cas13 protein described in the first aspect of the present invention, which specifically binds to the RNA to be tested or edited and can be recognized by the Cas13 protein used in the system GRNA, RNA cleavage buffer, RNase inactivator and fluorescent RNA substrate.
  • the gene/base editing system of the present invention contains the isolated Cas13 protein described in the first aspect of the present invention, and the Cas13 protein that specifically binds to the RNA to be tested or edited and can be used by the system Recognized gRNA, RNA cleavage buffer, RNase inactivator, fluorescent RNA substrate, T7 polymerase, rNTP and optional yeast tRNA; add amplification products (such as enzymatic recombination isothermal amplification products) to this system , Can detect the amplified product.
  • the 5'end of the sequence selected for the fluorescent RNA substrate is marked with a fluorescent group, and the 3'end is marked with a quenching group.
  • the fluorescent group can be selected from one of FAM, VIC, HEX, TRT, Cy3, Cy5, ROX, JOE and Texas Red, and the quenching group can be selected from TAMRA, DABCYL, MGB, Bio, BHQ- 1.
  • the fluorescent RNA substrate may be a fluorescent label-RNA-biotin label complex.
  • the nucleotide sequence of an exemplary fluorescent marker-RNA-biotin marker complex may be as shown in SEQ ID NO: 286.
  • An exemplary fluorescent RNA substrate can be 6-FAM/mArArUrGrGrCmAmArArUrGrGrCmA/3-Bio, the 5'end label of the fluorescent probe sequence is FAM group, and the 3'end label is Bio group.
  • the gene/base editing system of the present invention may be in the form of an aqueous solution.
  • the Cas13 protein of the present invention has the amino acid sequence shown in SEQ ID NO: 2, or 80%, 85%, 90%, 93%, 95%, 97%, or 80%, 85%, 90%, 93%, 95%, 97% of SEQ ID NO: 2 Similarity of more than 99%, preferably, the Cas13 protein is derived from Malacobacter halophilus (such as DSM 18005 strain); the gRNA specifically matches the reverse complement of the COVID-19 characteristic sequence shown in SEQ ID NO: 248. The sequence specificity Combine.
  • the sequence of the gRNA used in conjunction with the Cas13 protein is as described in any one of SEQ ID NO: 268-274, and more preferably as shown in SEQ ID NO: 273 or 274.
  • the Cas13 protein of the present invention has the amino acid sequence shown in SEQ ID NO: 9, or 80%, 85%, 90%, 93%, 95%, 97% or 80%, 85%, 90%, 93%, 95%, 97% of SEQ ID NO: 9 Similarity of more than 99%, preferably, the Cas13 protein is derived from human metagenome; gRNA specifically binds to the reverse complementary pair sequence of the COVID-19 characteristic sequence shown in SEQ ID NO: 248.
  • the sequence of the gRNA used in conjunction with the Cas13 protein is as described in any one of SEQ ID NO: 250-267, and more preferably as shown in SEQ ID NO: 253.
  • the Cas13 protein of the present invention has the amino acid sequence shown in SEQ ID NO: 289, or 80%, 85%, 90%, 93%, 95%, 97% or 80%, 85%, 90%, 93%, 95%, 97% of SEQ ID NO: 289. Similarity of more than 99%, preferably, the Cas13 protein is derived from Prevotella Intermedia; preferably, the sequence of the gRNA used in conjunction with the Cas13 protein is as described in any one of SEQ ID NO: 318-327.
  • the Cas13 protein of the present invention has the amino acid sequence shown in SEQ ID NO: 289, or 80%, 85%, 90%, 93%, 95%, 97% or 80%, 85%, 90%, 93%, 95%, 97% of SEQ ID NO: 289. Similarity of more than 99%, preferably, the Cas13 protein is derived from Prevotella intermedia; gRNA specifically binds to the reverse complementary pair sequence of the COVID-19 characteristic sequence shown in SEQ ID NO: 332.
  • the sequence of the gRNA used in conjunction with the Cas13 protein is as described in any of SEQ ID NO: 335-344, and more preferably SEQ ID NO: 344.
  • the Cas13 protein described in the gene/base editing system of the present invention has the amino acid sequence shown in SEQ ID NO: 2, or 80%, 85%, 90% of SEQ ID NO: 2 %, 93%, 95%, 97% or more than 99% similarity, preferably, it is the Cas13 protein derived from Malacobacter halophilus (such as strain DSM18005), and the sequence of the gRNA is shown in SEQ ID NO: 273 or 274 ; Or the Cas13 protein has the amino acid sequence shown in SEQ ID NO: 9, or is 80%, 85%, 90%, 93%, 95%, 97% or more similar to SEQ ID NO: 2 Preferably, it is a Cas13 protein derived from human metagenome (such as DSM 18005 strain), and the sequence of the gRNA is shown in SEQ ID NO: 253; or the Cas13 protein has the amino acid sequence shown in SEQ ID NO: 289 , Or have 80%, 85%, 90%, 93%, 95%
  • the sixth aspect of the present invention provides any one or more of the isolated Cas13 protein, isolated polynucleotide, construct, expression system, and gene/base editing system of the present invention in RNA editing or nucleic acid detection Uses are preferably used in RNA editing or nucleic acid detection of eukaryotes; or used in the preparation of reagents or kits for these purposes, especially in preparation for the detection/diagnosis of chubby Wiley syndrome or novel Coronavirus COVID-19 detection/diagnostic reagents or use in detection/diagnostic kits.
  • the eukaryotic organism may specifically be a metazoan, specifically including but not limited to humans, mice, nematodes, planarians and the like.
  • the nucleic acid may be DNA and/or RNA.
  • the purpose can specifically be through the cooperation of the isolated Cas13 protein with a specific gRNA, so that the isolated Cas13 protein can specifically cleave the target RNA, thereby achieving RNA editing of target fragments (for example, RNA knockdown) , Knockout, etc.), or to achieve nucleic acid detection of target fragments.
  • the use can also specifically be to realize the RNA labeling of the target fragment by the isolated Cas13 protein that loses the activity of cutting RNA but retains the ability to bind the RNA, so as to realize the detection of the nucleic acid of the target fragment.
  • the use can also be to combine the isolated Cas13 protein, isolated polynucleotide, construct, expression system, gene/base editing system provided by the present invention with other technologies (for example, RPA technology, etc.),
  • the DNA is amplified and the T7 promoter is introduced upstream so that it can be transcribed into RNA by T7 RNA polymerase, so that it can be detected by Cas13 for DNA detection.
  • the seventh aspect of the present invention provides an RNA editing method, including: editing by the isolated Cas13 protein provided in the first aspect of the present invention or the base editing system provided in the sixth aspect of the present invention.
  • the RNA editing method may include: culturing the expression system provided in the fourth aspect of the present invention under appropriate conditions, thereby expressing the isolated Cas13 protein, and the isolated Cas13 protein may be located in the target region that cooperates with it. In the presence of the gRNA, base editing is performed on the target region.
  • the method of providing the conditions under which the gRNA exists should be known to those skilled in the art.
  • the expression system may be an expression system capable of expressing the gRNA under appropriate conditions, and the expression system may include The host cell of the expression vector of the gRNA polynucleotide, or the host cell in which the polynucleotide encoding the gRNA is integrated into the chromosome.
  • the gRNA and the isolated Cas13 protein may be expressed in the same host cell, and the host cell may be a target cell.
  • the gene editing is in vitro gene editing.
  • the eighth aspect of the present invention provides a nucleic acid detection method, including: performing nucleic acid detection through the isolated Cas13 protein provided in the first aspect of the present invention or the base editing system provided in the sixth aspect of the present invention.
  • the nucleic acid detection method may include: culturing the expression system provided in the fourth aspect of the present invention under appropriate conditions, thereby expressing the isolated Cas13 protein, and the isolated Cas13 protein may be located in the target target region coordinated with it. In the presence of the gRNA, the target region is bound or cleaved.
  • the gRNA and the isolated Cas13 protein may be expressed in the same host cell, and the host cell may be a target cell.
  • the nucleic acid detection is in vitro nucleic acid detection.
  • the nucleic acid detection method may include: 1) providing amplified target fragments; 2) using the isolated Cas13 protein provided in the first aspect of the present invention, or the base editing system provided in the sixth aspect of the present invention Step 1) The amplified product provided is bound or cleaved.
  • Appropriate methods for providing amplified target fragments should be known to those skilled in the art.
  • polynucleotides such as SEQ ID NO: 275 to SEQ ID NO: 285 can be used to perform primers on the target fragment.
  • the target fragment may be the nucleic acid sequence of the novel coronavirus COVID-19.
  • the nucleic acid detection method is the detection method of chubby Wiley syndrome or the novel coronavirus COVID-19. In another specific embodiment of the present invention, the nucleic acid detection is in vitro nucleic acid detection.
  • the nucleic acid detection method provided by the present invention may further include: judging the detection result by the probe.
  • Suitable probes should be known to those skilled in the art.
  • the probe may be a reporter gene, and the reporter gene may be combined with the isolated Cas13 protein, so as to be used to judge the detection result.
  • the probe may be a nucleic acid capable of reacting with the activated Cas13 protein and may be labeled with a reporter system.
  • the reporter system may be a fluorescent reporter reagent, biotin, etc., and the activated Cas13 protein may be combined with the probe.
  • the reaction can be used to determine the detection result.
  • the probe may be a nucleic acid sequence connected with biotin and a fluorescent reporter reagent, and the nucleic acid sequence usually includes the base U.
  • the present invention also provides a detection kit, which includes the isolated Cas13 protein according to any embodiment of the present invention, and optionally one or more of the following reagents: GRNA, RNA cleavage buffer, fluorescent RNA substrate, T7 polymerase, rNTP, yeast tRNA, and RNase inhibitor that specifically bind to the RNA to be detected or edited and can be recognized by the Cas13 protein contained in the detection kit;
  • the kit further includes a test paper that can capture the complete fluorescent marker-RNA-biotin marker complex at the quality control line C, and capture the free fluorescence generated after cutting at the test line T White.
  • the detection kit of the present invention includes the gene/base editing system according to any embodiment of the present invention.
  • the detection kit of the present invention may also include reagents required for DNA amplification, including but not limited to one or more of RNA polymerase, RNase inhibitor, rNTP, and T7 enzyme mixture; or recombination Enzyme polymerase reagents required for amplification reactions.
  • reagents required for DNA amplification including but not limited to one or more of RNA polymerase, RNase inhibitor, rNTP, and T7 enzyme mixture; or recombination Enzyme polymerase reagents required for amplification reactions.
  • the novel Cas13 protein provided by the present invention has the ability to bind RNA and has RNA targeted cleavage activity when matched with gRNA, so that it can be used for RNA editing, nucleic acid detection, etc., based on the base editing system of the novel Cas13 protein It can achieve high-sensitivity and high-precision molecular detection at 37°C, with good specificity and compatibility, high detection sensitivity, low detection cost, convenient and fast operation, and wide application range. It has good nucleic acid detection. Application prospects.
  • MOLECULAR CLONING A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel, etc., Current PROTOCOLS IN MOLECULAR BI, John Wi , New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolfe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998;METHODS Chromatin, Vol ENZYMOLOGY, Vol. and AP Wolfe, eds.), Academic Press, San Diego, 1999; and Methods IN MOLECULAR BIOLOGY, Vol. 119, Chromatin Protocols (PBBecker, ed.) Humana Press, Totowa, 1999, etc.
  • the genomic data was analyzed according to the flowchart shown in Figures 1 and 20.
  • pairwise comparisons were made to find out the conservative features (seed), and then downloaded from the NCBI to all public information provided on September 16, 2018.
  • Prokaryotic genome data extract all open reading frames with potential protein coding ability, and screen out the open reading frame containing seed as potential Cas13 protein.
  • Further comparison and cluster analysis revealed a new type of Cas13 protein (SEQ ID NO: 1-43, 287-289) (as shown in Table 1). These new cas13 proteins do not have the same amino acid sequence as the cas13 published in NCBI before 2020.8.31.
  • the sequence of the clustered regularly spaced short palindrome repeat sequence CRISPR-DR corresponding to the protein shown in SEQ ID NO: 1-43 is shown in Table 2.
  • the DR sequence of these proteins analyzed by RNAfold online software is shown in Figure 2.
  • RNA in vitro cleavage reaction system is shown in Table 3 below:
  • the ssRNA1 sequence used is shown in SEQ ID NO:87.
  • the multifunctional microplate detector BioTek SynergyNEO was used to detect the changes in the fluorescence of the substrate RNA, and the specific results are shown in Figure 4.
  • the gRNA sequence corresponding to each protein used is shown in Table 4:
  • HmeCas13a has no obvious base preference, and MhdCas13c has a significant decrease in activity when any one of the two bases at the 5'end of the target site is C.
  • HmeCas13a tends to recognize the C base and cut at the 3'end.
  • MhdCas13c tends to recognize the A base and cut at the 5'end.
  • the gRNA sequences are shown in Table 7 below:
  • the experiment designed 9 gRNAs with a target length ranging from 14-30 nt for ssRNA1 (sequence SEQ ID NO.87).
  • the gRNA sequences are shown in Table 8 below:
  • the experimental procedure is the same as in Example 2. The result is shown in Figure 10.
  • the active length of gRNA is shorter than the previous shortest length (22nt).
  • MhdCas13c can still maintain strong RNA cleavage activity, and when the target sequence length is low The effect of cutting RNA will be completely lost until 14 nt.
  • the experiment performed single-base and double-base mismatches on the complementary pairing regions of the gRNA and the target RNA, respectively.
  • the gRNA sequence used is shown in the following 9:
  • the experimental procedure is the same as in Example 2.
  • the results are shown in Figures 11 and 12, the first four bases of the 3'end of the complementary pairing region between gRNA and the target RNA have a single base mismatch, MhdCas13c has a relatively small effect on the enzyme activity, and the first two bases have a double base mismatch MhdCas13c.
  • the activity effect is relatively small, and single-base or double-base mismatches in other positions will greatly reduce the activity of MhdCas13c (the 16th and 17th bases are affected by single-base or double-base mismatches It is very small, which may be caused by the extremely high GC content at this location).
  • Cas13 protein is used to detect the new coronavirus COVID-19
  • the new type of coronavirus COVID-19 has directly caused more than 20 million people to suffer directly.
  • the experiment used the verified HmeCas13a protein and MhdCas13c to detect the COVID-19 standard.
  • SEQ ID NO: 248 the specific target sequence COV-Target of the new coronavirus is shown in SEQ ID NO: 248.
  • the target sequence is synthesized, and DNA amplification is performed.
  • the amplified products are transcribed forward and reverse nucleic acid strands in vitro, and the system is shown in Table 10:
  • COV-Target's reverse complementary paired sequence nucleic acid strand COV-Target-reverse sequence SEQ ID NO: 249
  • the gRNA sequence is shown in Table 11 below:
  • Cas13 Under the “guide” of gRNA, Cas13 binds to the target RNA to exert its cleavage activity. In addition to cutting the target RNA, the "activated" Cas13 also cleaves other RNAs non-specifically. Therefore, the ability of Cas13 to cleave fluorescent RNA substrates is used to screen high-efficiency gRNAs.
  • the fluorescent RNA substrate RNAse Alert v2 was purchased from the company (Integrated DNA Technologies).
  • the Cas13 cleavage RNA reaction system is shown in Table 12 below:
  • HmeCas13a Hme-gT3-4 can achieve the best results.
  • MhdCas13c has higher detection activity under the action of Mhd-gT3-6 and Mhd-gT3-7.
  • HmeCas13a can directly detect 10 10 COV-Target-reverse molecules under the action of Hme-gT3-4
  • MhdCas13c can directly detect as low as 10 10 COV-Target-reverse molecules under the action of Mhd-gT3-6.
  • the present invention utilizes the recombinase polymerase to amplify the COV-Target molecular target.
  • the primers were synthesized by Shenggong Bioengineering (Shanghai) Co., Ltd.
  • the nucleotide sequence of the specific recombinase polymerase amplification primer is shown in Table 13 below:
  • RT-ERA nucleic acid amplification reagent (basic) (Suzhou Xianda Gene Technology Co., Ltd.) to amplify the target RNA by recombinase polymerase.
  • RT-ERA nucleic acid amplification reagent (basic) (Suzhou Xianda Gene Technology Co., Ltd.) to amplify the target RNA by recombinase polymerase.
  • the recombinase polymerase amplification reaction system and process are shown in Table 14 below:
  • the experiment is equipped with an aqueous solution containing COV-Target molecules with different concentration gradients such as 10 3 , 10 2 , 10 1 copy/ ⁇ l, and HmeCas13a and MhdCas13c detection are carried out after the amplification reaction of recombinase polymerase.
  • the experimental process is the same as the amplification primer screening process in step 3).
  • This embodiment provides a test paper detection method for the novel coronavirus based on the novel Cas13 protein, and the detection method includes the following steps:
  • this detection method uses constant temperature amplification reaction RPA to amplify the target RNA, thereby amplifying the signal.
  • Cas13 When a new type of coronavirus is present in the test sample, Cas13 will be activated after recognizing viral RNA, and the activated Cas13 can non-specifically cleave the fluorescent marker-RNA-biotin marker complex.
  • the fluorescent marker-RNA-biotin marker complex can be cleaved to produce free fluorescent markers.
  • the complete fluorescent marker-RNA-biotin marker complex will be on the test paper. It is limited to the quality control line C, so that the quality control line has bands, and the free fluorescein produced after cutting will continue to be chromatographed to the detection line T band to be detected.
  • the fluorescent marker-RNA-biotin marker complex was synthesized by Shanghai Sanggong Bioengineering (Shanghai) Co., Ltd., and the RNA sequence information therein is shown in SEQ ID NO: 286:
  • the structure of the fluorescent marker-RNA-biotin marker complex is shown in Table 16 below:
  • HybriDetect detection buffer 80 ⁇ l HybriDetect detection buffer to the above reaction tube, mix well, add HybriDetect Dipsticks into the reaction tube, and react for 3 minutes.
  • RNA in vitro cleavage reaction system is shown in Table 17 below:
  • the gRNA sequence BgrCas13a-gssRNA1 of BgrCas13a used in the experiment is shown in SEQ ID NO: 290
  • the gRNA sequence of Pin4Cas13b used in the experiment Pin4Cas13b-gssRNA1 is shown in SEQ ID NO: 291
  • the sequence of the substrate ssRNA1 used in the experiment is shown in SEQ ID NO: 292 .
  • RNA accessory cleavage reaction system is shown in Table 18 below:
  • Cas13 RNA-specific cleavage is applied to mammalian cells and can be used to degrade and knock down specific RNA, providing a simple and convenient tool for gene functional research.
  • an experimentally constructed Cas13 protein and a 293FT cell expression system for ACTB gRNA is a pHAGE-IRES-puro vector, which encodes the sequence of the Cas13 protein. Inserted into the vector, the insertion sites are NheI and XbaI, and the inserted protein sequence is shown in SEQ ID NO: 287-289.
  • the ACTB gRNA expression vector is pUC19.
  • the U6 promoter and gRNA sequence are inserted at HindIII and KpnI sites.
  • the gRNA See (SEQ ID NO: 293 ⁇ 298) for the sequence. It was transfected into 293FT cells, and after 48 hours, the cells were harvested and RNA was extracted and subjected to RT-qPCR test.
  • BgrCas13a and PbaCas13b have a certain knockdown effect on ACTB under the mediation of gRNA2, while the knockdown of Pin4Cas13b The low effect is not very obvious.
  • Cas13 protein By mutating the RNase activity domain HEPN motif of Cas13 protein, Cas13 protein can lose its ability to cleave RNA, but still retain its ability to bind RNA.
  • the formed dCas13 protein is often used for RNA labeling, RNA-directed editing, RNA splicing regulation, and RNA Purification etc.
  • BgrCas13a, PbaCas13b, and Pin4Cas13b were mutated with HEPN motif and fused with the green fluorescent protein EGFP.
  • the resulting dBgrCas13a-EGFP sequence is shown in SEQ ID NO:302.
  • the sequence of dPbaCas13b-EGFP is shown in SEQ ID NO: 303, and the sequence of dPin4Cas13b-EGFP is shown in SEQ ID NO: 304.
  • the expression vector of protein and gRNAs is the same as that in Example 12, and the gRNA for MUC4RNA with highly repetitive sequence
  • the sequence of BgrCas13a-gMUC4 is shown in SEQ ID NO: 305
  • the sequence of PbaCas13b-gMUC4 is shown in SEQ ID NO: 306, and the sequence of Pin4Cas13b-gMUC4 is shown in SEQ ID NO: 307.
  • Cells are transferred to a 35mm glass-bottom dish (Cellvis).
  • Cas13 protein is used to detect the molecular marker nucleic acid sno-lncRNAs of PWS patients
  • the combination of sno-lncRNAs molecular markers for chubby Wiley’s syndrome includes 5 long non-coding RNAs, namely sno-lncRNA1 (SEQ ID NO: 308), sno-lncRNA2 (SEQ ID NO: 309), and sno-lncRNA3 (SEQ ID NO: 308).
  • ID NO: 310 sno-lncRNA4 (SEQ ID NO: 311), sno-lncRNA5 (SEQ ID NO: 312).
  • the sequence is obtained from NCBI Gene Expression Omnibus database (https://www.ncbi.nlm.nih.gov/geo/; accession number GSE38541).
  • the present invention utilizes recombinase polymerase to amplify sno-lncRNAs molecular markers.
  • the nucleotide sequence of gRNA is shown in Table 19 below:
  • RNA fluorescence report detection experiment is the same as the RNA accessory cleavage experiment in Example 10.
  • the results are shown in Figures 26a-26e, where Figures 26a-26e are the schematic diagrams of the results of Cas13 protein used to detect sno-lncRNA1, sno-lncRNA2, sno-lncRNA3, sno-lncRNA4, and sno-lncRNA5, respectively, as shown in Figures 26a-26e. It can be seen that for the selected gRNA, Pin4Cas13b can effectively detect sno-lncRNA3, sno-lncRNA4, and sno-lncRNA5.
  • the whole blood sample was collected from the Children's Hospital of Zhejiang University School of Medicine, and the collection time was July 2018. There were 4 whole blood samples from patients with chubby Wiley syndrome and 4 samples from normal people, corresponding to the chubby Wiley syndrome group and the normal control group. All the above-mentioned specimens were obtained with the consent of the test subjects and approved by the organization ethics committee.
  • RNA stabilizer Take fresh anticoagulant blood and add blood RNA stabilizer (TIANGEN) in a ratio of 1:3 (for example, take 300 ⁇ l of healthy human fresh whole blood and add 900 ⁇ l of blood RNA stabilizer), immediately cover the tube cap, and mix upside down for 8-10 times. Place at room temperature for 2h.
  • TIANGEN blood RNA stabilizer
  • TIANGEN RNAprep Pure Blood Total RNA Extraction Kit
  • the sample supernatant after RNase inactivation is added to a tube-type recombinase polymerase amplification reaction system, the reaction system includes sno-lncRNA1 recombinase polymerase amplification specific primer sno-lncRNA4-RPA-F sequence It is shown in SEQ ID NO: 328 and the sequence of sno-lncRNA4-RPA-R is shown in SEQ ID NO: 329. React at 37° for 30 minutes, and the reaction system and conditions are shown in Table 20 below:
  • a multifunctional microplate detector (BioTek SynergyNEO) was used to monitor the changes in the fluorescence of the substrate RNA (as shown in Figure 27).
  • the normal group can detect the obvious fluorescent signal, while the patient group's signal is at a very low level, indicating that the Pin4Cas13b attached fluorescent reporter substrate system can effectively detect the expression of sno-lncRNA4 molecular markers in clinical samples (blood).
  • This embodiment provides a test strip detection method based on the novel Cas13 protein for chubby Wiley syndrome, and the detection method includes the following steps:
  • this detection method uses the constant temperature amplification reaction RPA to amplify the target RNA to amplify the signal;
  • Pin4Cas13b When sno-lncRNA4 is present in the test sample, Pin4Cas13b will be activated after recognizing sno-lncRNA4. The activated Pin4Cas13b can non-specifically cleave the fluorescent marker-RNA-biotin marker complex.
  • the fluorescent marker-RNA-biotin marker complex can be cleaved to produce free fluorescent markers.
  • the complete fluorescent marker-RNA-biotin marker complex will be on the test paper. It is limited to the quality control line C, so that the quality control line has bands, and the free fluorescein produced after cutting will continue to be chromatographed to the detection line T band to be detected.
  • the fluorescent marker-RNA-biotin marker complex was synthesized by Shanghai Shenggong Bioengineering (Shanghai) Co., Ltd., and the RNA sequence information therein is shown in SEQ ID NO: 330:
  • the structure of the fluorescent marker-RNA-biotin marker complex is shown in Table 23 below:
  • the genome comparison of the new coronavirus, SARS coronavirus and bat SARS-like coronavirus, and find out the differences in sequence as specific targets for the new coronavirus are shown in Figure 30.
  • the new type of coronavirus used (EPI_ISL_402119), SARS coronavirus (SARS-CoV NC_004718.3 SARS coronavirus), MERS coronavirus (MERS-CoV NC_019843.3 Middle East respiratory), bat-like coronavirus (bat-SL-CoVZC45MG772933).
  • the specific target sequence nCoV-ssRNA1 of the new coronavirus is shown in SEQ ID NO: 332.
  • the target sequence is synthesized, and DNA amplification is performed.
  • the amplified products are transcribed in vitro, and the system is shown in Table 24 below:
  • the present invention utilizes the recombinase polymerase to amplify the nCoV-ssRNA1 molecular target.
  • the primers were synthesized by Beijing Kinco Xinye Biotechnology Co., Ltd.
  • the nucleotide sequence of the specific recombinase polymerase amplification primer is shown in Table 25 below:
  • RT RAA nucleic acid amplification reagent (basic) (Hangzhou Zhongce Biotechnology Co., Ltd.) to carry out recombinase polymerase amplification primers on the target RNA.
  • the recombinase polymerase amplification reaction system and process are shown in Table 26 below:
  • the amplified product of recombinase polymerase was used as the substrate for RNA screening.
  • the nucleotide sequence of gRNA is shown in Table 27 below:
  • Cas13a Under the “guidance” of gRNA, Cas13a binds to the target RNA to exert its cleavage activity. In addition to cutting the target RNA, the "activated" Cas13a also cleaves other RNAs non-specifically. Therefore, the ability of Cas13 to cleave fluorescent RNA substrates is used to screen high-efficiency gRNAs.
  • the fluorescent RNA substrate RNAse Alert v2 was purchased from the company (Integrated DNA Technologies).
  • the Cas13 cleavage RNA reaction system is shown in Table 28 below:
  • the experiment tested the 2019-nCoV virus standard nCoV-ssRNA1 with test strips.
  • the detection steps are the same as in Example 16.
  • the gRNA used in the experiment is nCoV-ssRNA1-gRNA10, and the results are as follows Shown in Figure 32. It can be seen from Figure 32 that nCoV-ssRNA1-gRNA10 can accurately detect virus standards.
  • the present invention effectively overcomes various shortcomings in the prior art and has a high industrial value.

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Abstract

L'invention porte sur une protéine de CAS13 séparée. Une séquence d'acides aminés de la protéine Cas13 comprend une séquence d'acides aminés représentée par l'une quelconque des SEQ ID NO : 1 à 43 et 287 à 289 ; ou une séquence d'acides aminés ayant une similarité de 80 % ou plus à celle représentée par l'une quelconque des SEQ ID NO : 1 à 43 et 287 à 289. La nouvelle protéine Cas13 fournie a la capacité de lier l'ARN et possède une activité de clivage de l'ARN cible dans le cas d'un ARNg correspondant, de sorte que la protéine peut être utilisée pour l'édition de l'ARN et la détection des acides nucléiques, etc. Un système d'édition de base basé sur la nouvelle protéine Cas13 peut mettre en oeuvre une détection moléculaire de haute sensibilité et de haute précision à 37°C, a une bonne spécificité et compatibilité, a une sensibilité de détection élevée, des coûts de détection faibles, un fonctionnement pratique et rapide, et une large gamme d'applications, et a de bonnes perspectives d'application dans le domaine de la détection des acides nucléiques.
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