CN116622810A - Novel engineering CRISPR-Cas14a1 detection system, method and application - Google Patents

Novel engineering CRISPR-Cas14a1 detection system, method and application Download PDF

Info

Publication number
CN116622810A
CN116622810A CN202310311799.5A CN202310311799A CN116622810A CN 116622810 A CN116622810 A CN 116622810A CN 202310311799 A CN202310311799 A CN 202310311799A CN 116622810 A CN116622810 A CN 116622810A
Authority
CN
China
Prior art keywords
cas14a1
detection
target sequence
sgrna
detection system
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202310311799.5A
Other languages
Chinese (zh)
Inventor
邓仲良
何军
胡茜盼
翁兴墉
蒋婷清
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of South China
Original Assignee
University of South China
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of South China filed Critical University of South China
Publication of CN116622810A publication Critical patent/CN116622810A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/706—Specific hybridization probes for hepatitis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/35—Mycoplasma
    • 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

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Communicable Diseases (AREA)
  • Virology (AREA)
  • Hospice & Palliative Care (AREA)
  • Oncology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention relates to a novel engineered CRISPR-Cas14a1 detection system, a method and application, and relates to the field of gene detection, wherein a Cas14a1-sgRNA complex system comprises Cas14a1 protein, sgRNA and a fluorescent probe, and an isothermal amplification system comprises a target sequence to be detected; the target sequence to be detected comprises a target sequence capable of base complementary pairing with a target sequence in the sgRNA, and a PAM sequence is arranged at the upstream of the 5' end of the target sequence. The detection system provided by the invention combines an isothermal amplification technology, and the detection sensitivity is greatly improved by about 100 times and single base resolution capability through detecting a fluorescent or colloidal gold lateral flow chromatography test strip visual detection result, so that double-stranded DNA can be directly detected in a PAM (pulse-width modulation) mode, and the detection system is simple, quick, sensitive and specific to be applied to detection of mycoplasma pneumoniae, chlamydia psittaci, hepatitis B virus and lung tumor SNP, and provides an important technical support for rapid detection of infectious diseases and tumor single base mutations.

Description

Novel engineering CRISPR-Cas14a1 detection system, method and application
Technical Field
The invention relates to the field of gene detection, in particular to a novel engineered CRISPR-Cas14a1 detection system, a novel engineered CRISPR-Cas14a1 detection method and application.
Background
Gene detection, also known as nucleic acid detection, is an important component in the biotechnology field. In the face of sudden infectious diseases, rapid and accurate detection and identification of pathogens is critical for effective management and treatment of infectious diseases. The diagnosis method of infectious diseases mainly comprises methods of separation culture, immunological technology, molecular biology and the like. The molecular biological technology directly detects the nucleic acid of the pathogen, has higher sensitivity and specificity compared with antigen-antibody detection, and is particularly reliable for early diagnosis of infectious diseases. The current detection method comprises PCR amplification-based technology (common PCR, real-time fluorescent quantitative PCR method and the like), isothermal amplification technology (such as RPA, LAMP and the like), gene sequencing, gene chips and the like. The real-time quantitative PCR technology (Quantitative real-time PCR, qRT-PCR) is a conventional rapid identification method for detecting infectious diseases, has good sensitivity and specificity, and is widely applied to detection of various clinical samples because of being capable of accurately and reliably detecting pathogen nucleic acid. However, the existing devices are expensive, have high requirements on detection environment and operators, have high detection cost and long time consumption, and the defects greatly limit the application of the device in the diagnosis of infectious diseases, especially the diagnosis of new infectious diseases in underdeveloped medical areas.
Clustered regularly interspaced short palindromic repeats (Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR)/CRISPR-associated nuclease (CRISPR associated proteins, cas) technology is an immune defense system for prokaryotes (e.g., bacteria or archaebacteria) to resist the invasion of foreign genetic material (e.g., plasmids and phages). Cas12 of V type and Cas13 of VI type, which are commonly used for gene editing at present, particularly in the second type of system, are crRNA (CRISPR RNA) -dependent endonuclease with target gene-dependent collateral cleavage activity (collateral cleavage) and can be used for rapid detection of pathogen nucleic acid. Cas12 and Cas13 proteins need to rely on protospacer adjacent motifs (Protospacer adjacent motif, PAM) for target gene recognition, such as Cas12 needs to be rich in T nucleotide PAM (TTTN), whereas the PFS (PAM-like) 3' end of Cas13 is not a G base. After the Cas protein forms a ribonucleoprotein complex with the crRNA, the Cas protein recognizes the target gene PAM, while the crRNA complementarily binds to the target gene template strand, activating the Cas protein endonuclease activity to specifically recognize and cis-cleave the target strand and preserving the trans-nonspecific cleavage of adjacent other single-stranded nucleic acid strands or probe activities. Researchers establish a rapid, simple, sensitive and specific pathogen detection method by utilizing CRISPR-Cas protein accessory cleavage effect and combining isothermal amplification or other technologies such as PCR, and the method is known as a next-generation nucleic acid molecule diagnosis technology by journal of Science.
In recent years, two most common nucleic acid detection systems have been developed based on CRISPR Cas systems with high specificity for target gene recognition and with cleavage amplification effects, including SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing) [ Gootenberg JS, abudayyeh OO, lee JW, essletzbichler P, dy AJ, joung J, verdine V, donghia N, darringer NM, freij CA, myhrvold C, bhattacharyya RP, livny J, regv a, konin EV, hung DT, sabeti PC, collins JJ, zhang f.nucleic acid detection with CRISPR-Cas13 a/C2. Science.2017;356 438-442 ] and DETECTR (DNA Endonuclease Targeted CRISPR Trans Reporter, DETECTR) [ Chen JS, ma E, harrington LB, da Costa M, tian X, palefsky JM, doudna JA. CRISPR-Cas12a target binding unleashes indiscriminate single-structured DNase activity. Science.2018;360 (6387) 436-439 ], has the advantages of no dependence on a thermal cycler, simplicity, rapidness (1 hour), sensitivity, specificity, low cost and the like, and has been used for infectious pathogenic microorganism identification, virus typing, genetic disease SNP (single nucleotide polymorphism), tumor mutation, drug resistance gene and the like identification. However, cas13a relies on PFS to mainly recognize single-stranded RNA, requires in vitro transcription to single-stranded DNA after isothermal amplification, is time-consuming and has high environmental and operational requirements, and is widely applied in clinical applications or is a detection system and home [ Li SY, cheng QX, wang JM, li XY, zhang ZL, gao S, cao RB, zhao GP, wang j.crispr-Cas12a-assisted nucleic acid detection.cell discovery.2018; 4:20.]. Given the sensitivity, specificity, rapidness, simplicity and low cost of the CRISPR-Cas12 system detection method, an attractive alternative is provided for conventional methods such as gene sequencing and fluorescent quantitative PCR. It is worth noting that Cas12 and Cas13 have larger molecular weight (about 130 KD) and exceed adenovirus vector (AAV) transport capacity (< 4.7 kb) and are difficult to introduce during gene targeting delivery and gene therapy, so that a CRISPR-Cas system with small molecular weight and higher resolution is more needed to be developed during gene editing and molecular diagnosis.
11 months 2018, university of california Jennifer Doudna [ Harrington on LB, burstein D, chen JS, paez-Espino D, ma E, witte IP, cofsky JC, kyrpides NC, banfield JF, doudna ja.programmed DNA destruction by miniature CRISPR-Cas14 enzymes.science.2018;362 (6416):839-842.]The first time et al describe a novel second class of V-type system CRISPR/Cas14a1 featuring a small protein molecular weight (about 400-700 aa) doubled less than Cas9 and Cas12 (950-1400 aa), and found that Cas14a1 also has an attendant cleavage effect, which has the advantage that target single-stranded DNA recognition does not require dependenceDepending on PAM, the guide RNA formed by crRNA-tracrRNA is required to specifically recognize and cleave single-stranded DNA, thus establishing a DETECTR-Cas14, which can detect SNP of HERC2 gene responsible for expressing human eye color and has stronger resolving power than Cas12a for target gene SNP recognition. Although the Cas14 system can detect single-stranded DNA without depending on PAM, double-stranded DNA cannot be directly detected, if double-stranded DNA is to be detected, single-stranded DNA needs to be prepared, the operation is complex, the sensitivity is low, the application of the method in clinical detection is limited, and the method is only applied to SNP, non-nuclear targets (such as algae toxin determination and antibiotics) and other few applications at present, and is rarely used for reports of rapid diagnosis of infectious diseases and the like. Month 4 2020, tautvydas Karvelis [ Karvelis T, bigelyte G, young JK, hou Z, zedaveinyte R, budre K, paulraj S, djukanovic V, gasier S, silanska A, venclovas ]Siksnys V.PAM recognition by miniature CRISPR-Cas12f nucleases triggers programmable double-stranded DNA target cleavage.Nucleic Acids Res.2020；48(9):5016-5023.]The Cas14a1 (also called Un1Cas12f 1) can specifically identify and cleave double-stranded DNA when depending on PAM (TTTA/G), has the additional cleavage activity similar to Cas12, and has the capacity of non-specifically cleaving M13 phage single-stranded DNA; month 2 of 2021, takeda SN [ Takeda SN, nakagawa R, okazaki S, hirano H, kobayashi K, kusakizako T, nishizawa T, yamashita K, nishimasu H, nureki O.Structure of the miniature type V-F CRISPR-Cas effector enzyme.mol cell.2021;81 (3):558-570.]The formation of ribonucleoprotein complex between two Cas14a1 molecules and 1 molecule sgRNA (crRNA-tracrRNA) is observed by a cryoelectron microscope, which can specifically recognize and cleave target double-stranded DNA, and further confirm that Cas14a1 has the ability to specifically recognize and bind double-stranded DNA from the molecular structure. University of Stanford in the United states at 9 months of 2021 at 2-3 days [ Xu X, chemparthy A, zeng L, kempton HR, shang S, nakamura M, qi LS. Engineered minimum CRISPR-Cas system for mammalian genome regulation and edition. Mol cell.2021; s1097-2765 (21) 00648-1.]Korean Yong-Sam Kim laboratory [ Kim DY, lee JM, moon SB, chip HJ, park S, lim Y, kim D, koo T, ko JH, kim YS.efficiency CRISPR editing with a hy ]percompact Cas12f1 and engineered guide RNAs delivered by adeno-associated virus.Nat Biotechnol.2022；40(1):94-102.]And university of Shanghai science and technology team Ji Quanjiang [ Wu Z, zhang Y, yu H, pan D, wang Y, li F, liu C, nan H, chen W, ji q.programmed genome editing by a miniature CRISPR-Cas12F nucleic. 17 (11):1132-1138.]By engineering or identifying new CRISPR-Cas14a1 micronucleases, efficient gene editing in mammalian cells has been successfully achieved.
The above study shows that Cas14a1 has the additional cleavage activity similar to Cas12, can specifically recognize and cleave double-stranded DNA nucleic acid in a PAM-dependent manner, and additionally cleaves any single-stranded DNA activity, which provides a new idea for developing mini-precise gene editing and nucleic acid diagnosis, but it is not clear whether the Cas14a1 can be directly applied to rapid diagnosis of double-stranded DNA nucleic acid. Cas14a1 and Cas12 belong to the V-type system in the second class of systems, have small molecular weight, and similar to Cas12, need to rely on PAM to cut the target gene in cis and cut single-stranded DNA in trans, but the biggest difference is that Cas14a1 needs to rely on guide RNA formed by crRNA-tracrRNA to activate the collateral cutting effect, while Cas12 only needs single crRNA, cas14a1 forms sgRNA with molecular weight of about 220bp, the complex spatial conformation and redundant structure are the most important factors influencing interaction of CRISPR-Cas14a1 protein and the target gene, and engineering sgRNAs with different lengths and structures greatly influence gene editing efficiency and collateral cutting activity of Cas14a1 protein in vivo.
At present, a CRISPR/Cas14a1 detection system disclosed in a patent publication mainly relies on PAM to detect single-stranded DNA, and double-stranded DNA cannot be directly detected; for the detection of double stranded DNA, it is necessary to prepare single stranded DNA, for example, by using phosphorothioate modified primers followed by T7 exonuclease treatment or by asymmetric PCR, for example, patent Nos. CN112176035A, CN111808947A and CN113186258A. The existing CRISPR/Cas14a1 detection system is complex in operation, high in cost and low in sensitivity, and the application of the system in the aspects of infectious disease diagnosis, tumor mutation genes and the like is limited. In view of this, the present invention provides a novel engineered CRISPR-Cas14a1 detection system, method and application.
Disclosure of Invention
The invention aims to provide a novel engineering CRISPR-Cas14a1 detection system, a novel engineering CRISPR-Cas14a1 detection method and application. The method aims to construct a novel engineered CRISPR-Cas14a1 (Cas12f_g4.1) detection system by using engineering truncated sgRNA, and can directly detect double-stranded DNA in a PAM-dependent mode by combining an isothermal amplification technology, so that the detection sensitivity is greatly improved by about 100 times (the detection limit is about 5-10 copies/. Mu.l) and the single base resolution (the core seed sequence is enlarged), and the method is simple, quick, sensitive and specific to be applied to detection of procaryotes (mycoplasma pneumoniae and Chlamydia psittaci), hepatitis B viruses and non-small cell lung cancer SNP (EGFR), and greatly enriches the CRISPR/Cas detection system toolbox and provides important technical support for quick detection of infectious diseases and tumor single base mutations.
The present invention solves the above technical problems, a first object of the present invention is to screen to an optimal one of novel engineered CRISPR-Cas14a1 (Cas 12 f_g4.1) detection systems by using Cas14a1 in combination with different engineered sgrnas, comprising a Cas14a1-sgRNA complex system comprising Cas14a1 protein, sgrnas and fluorescent probes, and an isothermal amplification system comprising a target sequence to be detected; the Cas14a1 protein contains a sequence shown as SEQ ID NO. 49, the sgRNA contains a sequence shown as SEQ ID NO. 55 and a target sequence, the target sequence to be detected contains a target sequence capable of complementary pairing with the target sequence in the sgRNA, and the 5' -end upstream of the target sequence is provided with a PAM sequence.
The CRISPR-Cas14a1 belongs to Class II V-F type, also called Un1Cas12F1, cas14a1 protein contains 529 amino acids, is an endonuclease which depends on guide RNA (sgRNA for short), generally has nonspecific DNA cutting activity, and the engineered sgRNA is modified in original wild-type sgRNA (SEQ ID NO: 50) to remove some disordered neck ring structures and internal complementary structures to obtain an optimal sgRNA sequence (SEQ ID NO: 51), so as to form a novel engineered Cas12f_g4.1 system. When the Cas14a1 effector protein and engineered sgRNA complex is properly targeted to the target sequence to be detected of interest, the non-specific cleavage activity is activated. And by adding a fluorescent probe, the probe is provided with a fluorescent group and a fluorescence quenching group such as FAM and BHQ1, when a target sequence to be detected exists in a sample, the effector protein accessory cleavage activity is activated, and the probe is cut off to release fluorescence, so that the result is directly observed through a fluorometer or a colloidal gold test strip, and the detection of the target gene sequence is realized.
Original wild-type sgRNA sequence (SEQ ID NO: 50):
CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUUUCCUCUCCAAUUCUGCACAAgaaaGUUGCAGAACCCGAAUAGacgaaUGAAGGAAUGCAACCGUGCCCUGAUUGAAGUC CA。wherein the underlined moiety is the targeting sequence.
Optimal sgRNA sequence (SEQ ID NO: 51):
ACCGCUUCACUUAGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAGAAAGGAAUGCAACCGUGCCCUGAUUGAAGUCCAthe UUUAUUU comprises a fixed sequence shown as SEQ ID NO. 55, and a streak sequence is a target sequence, wherein the target sequence is designed according to the gene of a target sequence to be detected, the target sequence to be detected comprises a target sequence which can be in base complementary pairing with the target sequence in the sgRNA, and the 5' -end upstream of the target sequence is provided with a PAM sequence.
The beneficial effects of the invention are as follows: (1) The novel engineered CRISPR-Cas14a1 (Cas12f_g4.1) detection system can directly detect double-stranded DNA nucleic acid, avoids preparing complex single-stranded DNA, and is simple and quick to operate;
(2) The novel engineering CRISPR-Cas14a1 detection system has high detection sensitivity: by optimizing truncations of sgrnas of different lengths, the sequences are set forth in SEQ ID NOs: 51-54 shows that the sensitivity of the sgRNA (SEQ ID NO: 51) of Cas12f_g4.1 is 100 times higher than that of the sgRNA (SEQ ID NO: 50) of a natural system, and the sensitivity (5-10 copies/. Mu.l) close to Cas12 can be achieved;
(3) The single base resolution capability of the novel engineered CRISPR-Cas14a1 detection system is strong: by constructing a plasmid target with single base mismatch, the expansion of a core seed sequence is shown, so that the target selection of single base mutation is facilitated;
(4) The novel engineering CRISPR-Cas14a1 detection system has wide applicability: the novel engineering CRISPR-Cas14a1 detection system has high specificity, single base resolution and subsidiary cutting amplification effect for identifying target sequences to be detected, and combines the efficient amplification capability of isothermal amplification technology, so that a gene detection method for early and fast detecting viruses and bacteria and single base point mutation of tumors is established, a tool box of the CRISPR/Cas system detection system is expanded, and an accurate and reliable gene diagnosis means is provided for epidemic situation and accurate medical treatment of infectious diseases.
(5) The novel engineering CRISPR-Cas14a1 detection system provided by the invention is combined with an isothermal amplification technology, is simple and rapid to operate, does not need to rely on an expensive thermal cycler, can complete detection within 1 hour, and is convenient to use in areas with shortage of sanitary resources.
On the basis of the technical scheme, the invention can be improved as follows.
Further, the Cas14a1 protein, sgRNA to fluorescent probe species ratio by amount (1-8): (1-10): (4-64). The fluorescent probe is characterized in that the 5 'end of the fluorescent probe contains a fluorescent group, the 3' end of the fluorescent probe contains a quenching group, the fluorescent group and the quenching group are linked by a single-stranded DNA (deoxyribonucleic acid) of 6 to 21T, and the single-stranded DNA preferably contains a sequence shown as SEQ ID NO. 43-48.
Further, the PAM contains a sequence as set forth in SEQ ID NO:79 or SEQ ID NO: 80.
Further, the isothermal amplification system also comprises a primer and a buffer solution; the primer comprises a sequence shown as SEQ ID NO. 1-42.
Further, the target sequence to be detected is double-stranded DNA, RNA or single-stranded DNA.
A second object of the present invention is to provide a gene detection method by using the novel engineered CRISPR-Cas14a1 detection system as described in any one of the above.
Further, the method specifically comprises the following steps:
step 1: using a target sequence to be detected in an isothermal amplification system as a template, and performing isothermal amplification by using an isothermal amplification method to obtain an amplified target sequence to be detected;
step 2: and carrying out enzyme digestion reaction on the amplified target sequence to be detected and the Cas14a1-sgRNA complex system, and then carrying out signal detection and result interpretation. The fluorescent signal detection can be performed by a real-time fluorescent PCR instrument, a portable fluorescent instrument or a fluorescent reading plate, and also can be performed by rapid visual detection of colloidal gold.
Further, the isothermal amplification method in step 1 includes RPA isothermal amplification, LAMP isothermal amplification or ERA isothermal amplification. Other isothermal amplifications can also be used in the present invention, falling within the scope of the present invention. The temperature of the enzyme digestion reaction in the step 2 is 37-52 ℃, and the optimal cutting temperature is 42-52 ℃.
A third object of the present invention is to provide the use of a novel engineered CRISPR-Cas14a1 detection system as described by any of the above for the detection of mycoplasma pneumoniae, hepatitis b virus, chlamydophila psittaci or non-small cell lung cancer SNPs.
Further, when the SNP is detected for the non-small cell lung cancer, the 1 st position, the 3-4 rd position and the 7-12 th position of the target sequence to be detected are positioned behind the 3' -end of the PAM sequence, and the tolerance is lower.
Drawings
FIG. 1 is a schematic diagram of the present invention;
FIG. 2 is a graph showing comparison of the detection effect of sgRNA engineering according to the present invention: wherein Panel A is a graph comparing fluorescence values of all engineered system sgRNAs (SEQ ID NOS: 51-54); panel B is a graph comparing sensitivity of engineered sgRNA (SEQ ID NO: 51) to wild-type system sgRNA (SEQ ID NO: 50); * P <0.05;
FIG. 3 is a graph showing the effect of a fluorescent probe (ssDNA-reporter) of the present invention on the detection effect of CRISPR/Cas14a 1;
FIG. 4 is a graph showing the effect of different PAM motifs of the present invention on CRISPR/Cas14a1 detection effect;
fig. 5 is a CRISPR/Cas14a1 detection system optimization diagram of the present invention: wherein panel a is the optimization of fluorescent probe concentration; panel B shows the optimization of sgRNA concentration according to the present invention; panel C is Cas14a1 protein concentration optimization; FIG. D is an optimum cutting temperature probe;
FIG. 6 is a selection of mycoplasma pneumoniae genes by the CRISPR/Cas14a1 detection system of the invention; * P <0.001;
FIG. 7 shows the detection and clinical application of Mycoplasma pneumoniae of the present invention: wherein graph a is sensitivity verification; * P <0.05;
panel B is a specificity verification; panel C is a clinical sample validation;
FIG. 8 is a diagram of the detection and application of Chlamydia psittaci of the present invention, wherein panel A is a fluorescent method sensitivity verification of the CRISPR/Cas14a1 detection system; * P <0.01; FIG. B is a fluorescent method specificity verification of the CRISPR/Cas14a1 detection system; FIG. C is a test strip system detection method sensitivity verification; FIG. D is a test strip system detection method specificity verification; FIG. E is CRISPR/Cas14a1 system fluorescence biological sample validation;
FIG. 9 shows the detection and clinical application of the hepatitis B virus of the present invention, wherein FIG. A shows the sensitivity verification; * P <0.001;
panel B is a specificity verification; panel C is a clinical sample validation;
FIG. 10 is a schematic representation of the CRISPR/Cas14a1 system of the invention distinguishing EGFR tumor SNPs;
FIG. 11 is an EGFR mutation rate assay of the present invention;
FIG. 12 is an EGFR single base mismatching tolerance assay of the present invention;
fig. 13 is an EGFR clinical specimen validation of the present invention.
Detailed Description
The principles and features of the present invention are described below with examples given for the purpose of illustration only and are not intended to limit the scope of the invention. The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.
Example 1: construction of novel engineering CRISPR-Cas14a1 detection system and gene detection method
The novel engineered CRISPR-Cas14a1 detection system can directly detect double-stranded DNA in a PAM mode by combining isothermal amplification technologies such as (RPA, LAMP and the like), is effectively used for rapid detection of clinical pathogens, performs fluorescent signal detection through a real-time fluorescent PCR instrument, a portable fluorescent instrument or a fluorescent reading plate, and can also perform rapid visual detection through colloidal gold, as shown in figure 1.
The embodiment relates to the construction of a novel engineered CRISPR-Cas14a1 detection system, comprising the following steps:
expression and purification of Cas14a1 prokaryotic protein (SEQ ID NO: 49)
(1) Cas14a1 protein-expressing plasmids were purchased from addgene and transferred into BL21 (DE 3) competent cells, and LB plates (100. Mu.g/mL Amp) were incubated overnight at 37 ℃.
(2) The transformed bacteria were inoculated into 1L of liquid LB medium with ampicillin (final concentration: 100. Mu.g/mL), and placed in a shaking table at 37℃for shaking culture until OD600 value was approximately 0.6-0.8.
(3) IPTG (Isopropyl Thiogalactoside) inducer is added into the bacterial liquid to make the final concentration of the bacterial liquid be 0.3mM, and the bacterial liquid is placed in a shaking table at 16 ℃ and 180rpm, and is taken out after shaking culture for 16 hours.
(4) After induction, the cells were collected by centrifugation at 10000rpm for 5min.
(5) Lysates (50 mM Tris-HCl,500mM NaCl,5% (v/v) glycerol, 1mM TCEP,0.5mM PMSF and 0.25mg/mL lysozyme, pH 7.5) were added to the cells in an amount ten times the volume concentration before centrifugation to resuspend the cells.
(6) And carrying out ultrasonic crushing treatment on the treated thalli. Each cycle is 26 times, the ultrasonic treatment is carried out for 6s at intervals of 5s, and the ultrasonic probe is required to be cleaned by alcohol cotton after the ultrasonic treatment is finished for 2-4 cycles according to the crushing effect. The crushed thalli are centrifuged for 30min at 18000r/min at 4 ℃, and the supernatant is collected and placed at 4 ℃.
(7) The supernatant was filtered off with a 0.22 μm filter.
(8) The mixture was applied to a Ni-NTA agarose resin column (purchased from Bai Lai Bo technology Co., ltd.).
(9) Absorbance or SDS-PAGE analysis was measured by measurement at 280 nm.
(10) BCA (bicinchoninic acid) sodium salt the protein concentration was determined.
(11) The appropriate TEV enzyme (purchased from the next holy organism) was added according to the protein concentration. To 4mL of the sample, 0.5mL of TEV enzyme protein buffer (10X: 5mM EDTA and 10mM DTT and 500mM Tris-HCl, pH7.5, 25 ℃ C.) was added, and the mixture was made up to 5mL with ultrapure water. mu.L of TEV protease was added. Preparing an enzyme digestion system according to the system, uniformly mixing, and placing in a dialysis bag with the length of 8-14kDa to carry out dialysis enzyme digestion at the temperature of 4 ℃. The reaction time was set to 48h.
(12) After the reaction is completed, the sample is subjected to SDS-PAGE detection.
(13) The digested proteins were directly passed through MBP column (purchased from Ai Baisen Biotechnology Co.).
(14) 5mL of the sample was mixed in equal proportions with heparin column binding buffer (20 mM Tris-HCl,100mM NaCl,5% glycerol, 1mM TCEP, pH=7.5) to give 10mL of the sample.
(15) Samples were passed through a heparin column and analyzed by SDS-PAGE.
(16) Ultrafiltration concentration of the collected protein yields Cas14a1. Packaging and storing at-80 ℃.
sgRNA transcription and purification
(1) The DNA sequence corresponding to the sgRNA containing the T7 promoter at the proximal end is amplified by PCR, and the amplified product is subjected to gel recovery by using an agarose gel DNA recovery kit (TIANGEN, DP 219) to obtain a T7 transcription template, wherein the PCR reaction system is shown in Table 1 in detail.
TABLE 1PCR reaction System
(2) By usingKit(Thermo Scientific，AM 1334) kit for in vitro transcription, wherein the transcription system is detailed in table 2.
TABLE 2 transcription system
(3) Using MEGAclear TM Kit (Thermo Scientific, AM 1334) the sgrnas were purified.
(1) 1. Mu.L TURBO DNase, mixing well, 37℃for 15min. Then, 115. Mu.L of Nuclear-free Water and 15. Mu. L Ammonium Acetate Stop Solution (ammonium acetate) were added thereto to terminate and thoroughly mixed.
(2) Extracting with equal volume of phenol/chloroform (12000 rpm,10 min), and collecting the upper water phase into a new test tube; then extracted with an equal volume of chloroform at 12000rpm for 10min, the aqueous phase was recovered and transferred to a new tube.
(3) 1 volume of isopropanol (isopropanol) was added and mixed thoroughly.
(4) The mixture was cooled at-20℃for 2h. RNA was precipitated at 12000rpm for 15min at 4 ℃. The whole supernatant was carefully removed and discarded, and the primary particles were washed with 1ml of 70% ethanol (pre-chilled) and centrifuged again at 4℃and 12000rpm for 15min, and the supernatant discarded. Adding a proper amount of RNase-Free H 2 O is resuspended.
(5) And (3) marking the RNA with the measured concentration, adding RNase Inhibitor, subpackaging, and storing in a refrigerator at-80 ℃.
3. Selection of isothermal amplification template method
3.1 RPA amplification of fragments of interest
The Primer was designed using Primer Premier 5.0, see the TwistAmp assay design manual. The target fragment was amplified using TwistAmpTM Basic Kit (Twist DX) kit, wherein the RPA amplification system is shown in Table 3 and the RPA primers are shown in Table 4.
TABLE 3 RPA amplification System
TABLE 4RPA primers
3.2ERA amplification of fragments of interest
Primers were designed using Primer Premier 5.0, with reference to the design guidelines for the first gene. The fragment of interest was amplified using a first-come gene-based nucleic acid amplification kit, wherein the ERA amplification system is detailed in table 5.
TABLE 5ERA amplification System
4. Detection method
4.1CRISPR/Cas14a1 detection System fluorescence detection
(1) Constructing a Cas14a1-sgRNA complex, wherein the 5-x complex Buffer (5-x Assembly Buffer) comprises: 50mM Tris-HCl (pH 8.0), 500mM NaCl, 5mM EDTA, pH7.5. Wherein the complex construction system is as shown in Table 6.
TABLE 6 fluorescent detection Cas14a1-sgRNA complex building System
/>
(2) The CRISPR/Cas14a1 system cleaves the detection target gene, wherein the 5 x Cleavage Buffer (5 x Cleavage Buffer) comprises: 25mM Tris-HCl (pH 8.0), 250mM NaCl, 2.5mM DTT, 25mM MgCl 2 pH7.5. Wherein the cleavage detection system is as shown in Table 7.
TABLE 7 fluorescent detection cleavage detection System
Reagent(s) Volume (mu L)
Cas14a1-sgRNA complexes 5
ssDNA-reporter(10μM) 2
5ⅹ Cleavage Buffer 10
Nuclease-free Water 32
RPA amplification products 1
Total 50
The components of the system are added into a clean eight-joint tube, mixed uniformly, placed into a fluorometer for incubation at 46 ℃ and observed in real time.
4.2 CRISPR/Cas14a1 test strip system detection
(1) Constructing a Cas14a1-sgRNA complex, wherein the 5-x complex Buffer (5-x Assembly Buffer) comprises: 50mM Tris-HCl (pH 8.0), 500mM NaCl, 5mM EDTA, pH7.5. The test strip system detects the complex construction system as shown in Table 8.
Table 8 test strip System detection Cas14a1-sgRNA Complex construction System
(2) The CRISPR/Cas14a1 system cleaves a target gene, wherein the 5 x Cleavage Buffer (5 x Cleavage Buffer) comprises: 25mM Tris-HCl (pH 8.0), 250mM NaCl, 2.5mM DTT, 25mM MgCl 2 pH7.5. The test strip system detection cleavage detection system is shown in Table 9.
Table 9 test strip system detection cutting detection system
The components of the system are added into a clean eight-joint tube, mixed evenly and put into a PCR instrument for incubation at 46 ℃.
(3) And detecting and observing a result by using colloidal gold, uniformly mixing 10 mu L of reaction products and 90 mu L of dilution buffer solution, putting the mixture into a test strip, and judging the result after 5-7 min.
Example 2: optimization of novel engineered CRISPR-Cas14a1 detection system
The embodiment relates to optimization of a novel engineered CRISPR-Cas14a1 detection system, comprising the following steps:
(1) And (3) comparing the sgRNA engineering detection effects: editing and processing are carried out on the wild type Cas12f1 system sgRNA (SEQ ID NO: 50) to obtain engineering systems respectively including Cas12f_g4.1 (SEQ ID NO: 51), cas12f_g4.0 (SEQ ID NO: 52), cas12f_g3.0 (SEQ ID NO: 53) and Cas12f_g2.0 (SEQ ID NO: 54), and CRISPR/Cas14a1 cutting fluorescence detection is carried out by taking the CARDS gene of mycoplasma pneumoniae (SEQ ID NO: 74) as a target gene, wherein the specific steps are described in example 1. The results show that Cas12 f_g4.1 (SEQ ID NO: 51) has the best effect, compared with the wild Cas12f1 system (SEQ ID NO: 50), the fluorescence effect is greatly improved by 1.6 times (FIG. 2A), the sensitivity is improved by 100 times (FIG. 2B), and the detection effect of other 3 engineered sgRNAs is not obviously improved. The engineered CRISPR/Cas14a1 system (Cas12f_g4.1) (SEQ ID NO: 51) can significantly improve detection performance.
(2) Influence of different PAM motifs on detection effect: the mycoplasma pneumoniae P1 and CARDS genes are respectively designed into sgRNA according to the PAM motif of 'TTTA, TTTG, TTTC' before the spacer, wherein the mycoplasma pneumoniae P1 is respectively designed into the sgRNA with the sequence shown as SEQ ID NO:56-58 wherein the CARDS gene is designed with a sgRNA sequence as set forth in SEQ ID NO according to the spacer pre-PAM motif "TTTA, TTTG, TTTC", respectively: 59-61, and fluorescence detection is performed according to the method described in example 1. When the PAM motif is TTTC, the fluorescence value is almost the same as that of a negative control, and the detection effect is poor; when the PAM motif is TTTG/TTTA, the fluorescence curve has obvious increasing trend, and the detection effect is different according to the position. Thus, when designing sgrnas, PAM avoids the sequence of TTTC due to the selection of TTTA/G (fig. 3).
(3) Optimization of ssDNA reporter probes of different lengths: ssDNA reporter probes with the sequences of 6T, 9T, 12T, 18T and 21T are constructed as shown in Table 10, and the sequences are shown in SEQ ID NO:43-48, a CRISPR/Cas14a1 detection system was constructed to perform fluorescence detection of a target gene as described in example 1. The length of the fluorescent probe is found to be increased, the fluorescent value is correspondingly increased, but the background fluorescent value is correspondingly increased. Therefore, in the detection system of mycoplasma pneumoniae CARDS gene, a ssDNA-reporter with 9T being optimal was selected (FIG. 4); the optimal ssDNA-reporter in the detection system of the hepatitis B virus is 12T; the optimal ssDNA-reporter in the EGFR L858R detection system is 12T.
TABLE 10 fluorescent probe sequence listing
(4) ssDNA-reporter concentration effect: the target genes were fluorescently detected as described in example 1 for ssDNA-reporters with final concentrations of 100nM, 200nM, 400nM, 800nM, 1600nM, respectively. The optimal ssDNA-reporter concentrations for mycoplasma pneumoniae CARDS gene assay (FIG. 5A), HBV assay, EGFR L858R assay were all 400nM.
(5) sgRNA concentration optimization: the target genes were detected by fluorescence as described in example 1 for sgrnas with final concentrations of 25nM, 50nM,100nM, 150nM, 200nM, 250nM, respectively. Mycoplasma pneumoniae CARDS gene detection system (FIG. 5B) and HBV detection system with optimal sgRNA concentration of 50nM and 150nM respectively; the optimal sgRNA concentration for EGFR L858R WT and MT detection systems was 50nM.
(6) Cas14a1 protein concentration effect: the target gene was fluorescently detected as described in example 1 for Cas14a1 proteins with final concentrations of 25nM, 50nM,100nM, 150nM, 200nM, respectively. Mycoplasma pneumoniae CARDS gene detection system (FIG. 5C), HBV detection system with optimal Cas14a1 protein concentration of 100nM and 100nM respectively; the optimal Cas14a1 protein concentrations for EGFR L858R WT and MT detection systems were 50nM,100nM, respectively.
(7) Optimum cutting temperature probe: the cleavage temperatures of 25℃at 30℃at 37℃at 42℃at 46℃and 52℃were measured by fluorescence using the Mycoplasma pneumoniae CARDS gene as a target gene as described in example 1. The CRISPR/Cas14a1 detection system can cut in the environment of 25-52 ℃, and the optimal cutting temperature is 42-52 ℃ (figure 5D).
Embodiment 3: engineered CRISPR/Cas14a1 detection system for detecting mycoplasma pneumoniae and application
(1) Construction of engineered CRISPR/Cas14a1 detection System for detection of Mycoplasma pneumoniae
Specific construction of the engineered CRISPR/Cas14a1 detection system for detection of mycoplasma pneumoniae of this embodiment reference example 1, wherein the nucleotide sequence of sgRNA for the P1 gene is as set forth in SEQ ID NO:57, the nucleotide sequence of sgRNA for CARDS gene is shown in SEQ ID NO:59, any one of the following; the optimization procedure of the system was described in example 2, wherein in the detection system of Mycoplasma pneumoniae CARDS gene, ssDNA-reporters with 9T being optimal were selected, ssDNA-reporter concentrations were 400nM, sgRNA concentration was 50nM, cas14a1 protein concentration was 100nM, and the optimal cutting temperature was 42-52 ℃.
(2) Selection of genes
The concentration of plasmids containing the P1 gene of Mycoplasma pneumoniae (SEQ ID NO: 73) and the CARDS gene sequence (SEQ ID NO: 74) were 10, respectively 5 、10 4 、10 3 、10 2 、10 1 、10 0 The copies/. Mu.L was subjected to gradient dilution, fluorescence detection was performed on the plasmids with the optimal sgRNA screened from the respective genes, and negative control was performed using enzyme-free water as a reaction template. The sensitivity of the two groups of genes was similar, but the CARDS gene reached the plateau earlier and tended to stabilize within 30min, so the CARDS gene (SEQ ID NO: 74) was selected as the target gene in the CRISPR/Cas14a1 system fluorescence detection of Mycoplasma pneumoniae (FIG. 6).
(3) Sensitivity verification
The concentration of the plasmid containing the CARDS gene sequence was 10 5 、10 4 、10 3 、10 2 、10 1 、10 0 The copies/. Mu.L is subjected to gradient dilution, the plasmid is subjected to fluorescence detection by using an optimized detection system, and meanwhile, the negative control is performed by using enzyme-free water as a reaction template, so that the sensitivity of the method is evaluated. When the plasmid concentration was 10 1 When the peptides/mu L and above are compared with a negative control, the CRISPR/Cas14a1 system fluorescence detection lower limit of the mycoplasma pneumoniae is 10 1 COPIES/. Mu.L (FIG. 7A).
(4) Specificity verification
In a mycoplasma pneumoniae CARDS gene detection system, the established CRISPR/Cas14a1 system fluorescence detection method is used for respectively carrying out specific detection on klebsiella pneumoniae, chlamydia psittaci, pseudomonas aeruginosa, streptococcus pneumoniae, haemophilus influenzae, mycobacterium tuberculosis, acinetobacter baumannii, mycoplasma penetrative, mycoplasma hominis, influenza virus and mycoplasma pneumoniae, and the result shows that the method has good specificity for detecting mycoplasma pneumoniae and has no cross reaction with other pathogens (figure 7B).
(5) Clinical sample validation
105 cases of throat swab samples from mycoplasma pneumoniae infected persons and healthy persons belonging to the clinical laboratory of south China university are collected, and simultaneously detected by a CRISPR/Cas14a1 detection system fluorescence detection method and a mycoplasma pneumoniae nucleic acid detection kit (PCR-fluorescence probe method), and enzyme-free water is used as a reagent control. The real-time fluorescence PCR method is used as a standard to evaluate the clinical sample for detecting mycoplasma pneumoniae. The clinical diagnostic sensitivity of the method was 93.4% and the specificity was 100% (FIG. 7C).
Embodiment 4: engineered CRISPR/Cas14a1 detection system for detecting Chlamydia psittaci and application
(1) Construction of engineered CRISPR/Cas14a1 detection System for detection of Chlamydia psittaci
Specific construction of the engineered CRISPR/Cas14a1 detection system for detecting chlamydia psittaci of this embodiment reference example 1, wherein the nucleotide sequence of the sgRNA for chlamydia psittaci is set forth in SEQ ID NO:66 to 70; the optimization steps of the system are shown in example 2, in a detection system of the Chlamydia psittaci gene, the nucleotide sequence of sgRNA is shown in SEQ ID NO:70, selecting the ssDNA-reporter with 9T as the optimal, wherein the ssDNA-reporter concentration is 400nM, the sgRNA concentration is 150nM, the Cas14a1 protein concentration is 100nM, and the optimal cutting temperature is 42-52 ℃.
(2) Sensitivity verification
The concentration of the plasmid containing the MOMP gene sequence (SEQ ID NO: 76) was 10 5 、10 4 、10 3 、10 2 、10 1 、10 0 The copies/. Mu.L is subjected to gradient dilution, the plasmid is subjected to fluorescence detection by using an optimized detection system, and meanwhile, the negative control is performed by using enzyme-free water as a reaction template, so that the sensitivity of the method is evaluated. In CRISPR/Cas14a1 system fluorescence detection, plasmid concentration was 10 1 When the copies/mu L and above are compared with a negative control, the CRISPR/Cas14a1 system fluorescence detection lower limit of the Chlamydia psittaci is 10 1 COPIES/. Mu.L (FIG. 8A). In CRISPR/Cas14a1 test strip system detection, plasmid concentration was 10 2 When the copies/mu L and above are adopted, the T/C ratio of the copies/mu L is obviously different from that of a negative control, and the CRISPR/Cas14a1 test strip system detects the psittacosis clothesThe lower limit of detection of the original substances is 10 2 COPIES/. Mu.L (FIG. 8C).
(3) Specificity verification
In the MOMP gene detection system of the Chlamydia psittaci, the established CRISPR/Cas14a1 system fluorescence detection method and the test strip system detection method are respectively used for specifically detecting the Chlamydia trachomatis, the mycoplasma pneumoniae, the staphylococcus aureus, the pseudomonas aeruginosa, the klebsiella pneumoniae, the mycobacterium tuberculosis, the haemophilus influenzae, the streptococcus pneumoniae and the Chlamydia psittaci, and the results show that the CRISPR/Cas14a1 system fluorescence detection method (figure 8B) and the test strip system detection method (figure 8D) are good in the specificity of detecting the Chlamydia psittaci and have no cross reaction with other pathogens.
(4) Biological sample validation
93 cases of lung samples from the parrot market of Heng-yang city bird are collected, and meanwhile, a CRISPR/Cas14a1 system fluorescence detection method, a test strip system detection method and a fluorescence PCR probe method are used for detection, and enzyme-free water is used as a reagent control. The method is used for evaluating the biological sample for detecting the Chlamydia psittaci by taking the result of the fluorescent PCR method as a standard. The diagnostic sensitivity of the CRISPR/Cas14a1 system fluorescence assay was 95.4% and the specificity was 100% (fig. 8E).
Embodiment case 5: hepatitis B virus detection engineering CRISPR/Cas14a1 detection system and clinical application
(1) Construction of engineering CRISPR/Cas14a1 detection system for detecting hepatitis B virus
Specific construction of the engineered CRISPR/Cas14a1 detection system for detecting hepatitis b virus of this embodiment refers to example 1, in which the nucleotide sequence of sgRNA for hepatitis b virus is as set forth in SEQ ID NO:62 to 65; the optimization steps of the system are shown in example 2, in a hepatitis B virus detection system, the nucleotide sequence of sgRNA is shown as SEQ ID NO:64, a 12T optimum ssDNA-reporter was selected, the ssDNA-reporter concentrations were 400nM, the sgRNA concentration was 150nM, the cas14a1 protein concentration was 100nM, and the optimum cleavage temperature was 42-52 ℃.
(2) Sensitivity verification
The concentration of plasmid containing coding region sequence of hepatitis B virus gene (SEQ ID NO: 75) polymerase is as follows10 5 、10 4 、10 3 、10 2 、10 1 、10 0 The copies/. Mu.L is subjected to gradient dilution, the plasmid is subjected to fluorescence detection by using an optimized detection system, and meanwhile, the negative control is performed by using enzyme-free water as a reaction template, so that the sensitivity of the method is evaluated. When the plasmid concentration was 10 1 When the peptides/mu L and above are compared with negative control, the CRISPR/Cas14a1 system has a detection lower limit of 10 when fluorescence detection of hepatitis B virus is carried out 1 COPIES/. Mu.L (FIG. 9A).
(3) Specificity verification
In the hepatitis B virus detection system, the established CRISPR/Cas14a1 system fluorescence detection method is used for respectively detecting hepatitis A virus, hepatitis C virus, EB virus, cytomegalovirus and hepatitis B virus. The discovery that the method can not generate fluorescence amplification curves for other viruses except the hepatitis B virus shows that the method has good specificity for detecting the hepatitis B virus and has no cross reaction with other viruses (figure 9B).
(4) Clinical sample validation
Serum samples from hepatitis B virus infected persons and healthy persons belonging to the clinical laboratory of south China university were collected for 76 cases, and simultaneously detected by using a CRISPR/Cas14a1 system fluorescence detection method and a hepatitis B virus nucleic acid detection kit (PCR-fluorescence probe method), and enzyme-free water was used as a reagent control. The result of the real-time fluorescence PCR method is used as a standard to evaluate the clinical sample for detecting the hepatitis B virus. The clinical diagnostic sensitivity of the method was 92.16% and the specificity was 100% (FIG. 9C).
Embodiment 6: engineered CRISPR/Cas14a1 detection system for detecting non-small cell lung tumor EGFR SNP (single nucleotide polymorphism) and application
(1) Construction of engineered CRISPR/Cas14a1 detection system for detecting EGFR SNP of non-small cell lung tumor
Specific construction of the engineered CRISPR/Cas14a1 detection system for detecting non-small cell lung tumor EGFR SNPs of this example reference example 1, wherein the nucleotide sequence of the wild strain sgRNA for the non-small cell lung tumor EGFR SNPs is as set forth in SEQ ID NO:71 and mutant sgRNA as shown in SEQ ID NO:72; the optimization steps of the system are shown in example 2, in a non-small cell lung tumor EGFR SNP detection system, ssDNA-reporter with 12T being optimal is selected, the ssDNA-reporter concentrations are 400nM, the wild strain sgRNA and the mutant strain sgRNA are 50nM, the Cas14a1 protein concentration is 50nM and 100nM respectively, and the optimal cutting temperature is 42-52 ℃.
(2) The CRISPR/Cas14a1 system is capable of distinguishing single base variants (SNPs)
A negative control was performed using EGFR L858R wild strain plasmid (SEQ ID NO: 77), mutant strain plasmid (SEQ ID NO: 78) and MIX mixed at equal concentrations as a template, and enzyme-free water as a reaction template, and fluorescence detection was performed using wild strain sgRNA (SEQ ID NO: 71) and mutant strain sgRNA (SEQ ID NO: 72), respectively (see FIG. 10). In the detection of EGFR L858R by the CRISPR/Cas14a1 system, the fluorescent signal of mutant sgRNA detection containing mutant genes was significantly higher than that of the negative control group and the wild strain. Wherein the mutant sgRNA detects a mutant template signal 5-fold greater than the wild-type template. The fluorescent signal of the wild strain sgRNA containing the wild strain gene is obviously higher than that of a negative control group and a mutant strain. Wherein the signal of the wild strain template detected by the sgRNA of the wild strain is 8 times that of the mutant strain template. The CRISPR/Cas14a1 system is demonstrated to distinguish single bases with high resolution.
(3) Mutation rate detection
EGFR L858R wild strain plasmid templates and mutant strain plasmid templates were mixed in different proportions to construct different mutation rate templates, with mutation rates of 100%, 50%, 25%, 10%, 1%, 0.1%, 0.01% and 0.001%, respectively. The plasmids with different mutation rates are used as templates, meanwhile, enzyme-free water is used as a reaction template to carry out negative control, and mutant sgRNA is used for carrying out fluorescence detection to evaluate the mutation rate of the method. In the CRISPR/Cas14a1 system for EGFR L858R, 1% and above showed significant differences from the wild strain, so the lower mutation rate detection limit was 1% (fig. 11).
(4) Single base mismatching tolerance analysis
The wild strain sgRNA forms continuous single base mismatch with the 1 st to 20 th positions after the 3' -end of the target site PAM sequence. Taking 20 single-base mismatched plasmids as templates, simultaneously taking enzyme-free water as a reaction template to carry out negative control, and carrying out single-base mismatched fluorescent detection by using wild strain sgRNA. The single base mismatch tolerance of this method was evaluated (FIG. 12). In the detection of EGFR L858R by the CRISPR/Cas14a1 system, the fluorescent signal of single base mismatches at positions 1, 3-4 and 7-12 after the 3' -end of the PAM sequence is significantly reduced or even not generated. The Cas14a1 shows lower tolerance to 1 st, 3-4 rd and 7-12 th positions after the 3' -end of the PAM sequence, and the positions are seed sequences of the CRISPR/Cas14a1 system core and have high conservation. The CRISPR/Cas14a1 system detects that SNP seed regions are further enlarged, and genome editing specificity is improved.
(5) Clinical sample validation
44 cases of EGFR gene L858R mutation and normal FFPE samples of non-small cell lung cancer from the Pathology department of south China Hospital affiliated to the university of south China were collected, and simultaneously detected by CRISPR/Cas14a1 system fluorescence detection method and gene sequencing, and enzyme-free water was used as a reagent control. The detection of the EGFR L858R mutation from non-small cell lung cancer by the present method was evaluated using the results of gene sequencing as a standard. The clinical diagnostic sensitivity of the method was 84.62% and the specificity was 100% (FIG. 13).
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.

Claims (10)

1. A novel engineered CRISPR-Cas14a1 detection system, characterized in that it comprises a Cas14a1-sgRNA complex system comprising Cas14a1 protein, sgRNA and a fluorescent probe and an isothermal amplification system comprising a target sequence to be detected; the Cas14a1 protein contains a sequence shown as SEQ ID NO. 49, the sgRNA contains a sequence shown as SEQ ID NO. 55 and a target sequence, the target sequence to be detected contains a target sequence capable of complementary pairing with the target sequence in the sgRNA, and the 5' -end upstream of the target sequence is provided with a PAM sequence.
2. The novel engineered CRISPR-Cas14a1 detection system of claim 1, wherein the Cas14a1 protein, sgRNA to fluorescent probe species ratio is (1-8): (1-10): (4-64).
3. The novel engineered CRISPR-Cas14a1 detection system of claim 1, wherein said PAM comprises the amino acid sequence set forth in SEQ ID NO:79 or SEQ ID NO: 80.
4. The novel engineered CRISPR-Cas14a1 detection system of claim 1, wherein the isothermal amplification system further comprises primers and buffers; the primer comprises a sequence shown as SEQ ID NO. 1-42.
5. The novel engineered CRISPR-Cas14a1 detection system of claim 1, wherein the target sequence to be detected is double-stranded DNA, RNA or single-stranded DNA.
6. A method of gene detection, characterized by gene detection by using the novel engineered CRISPR-Cas14a1 detection system as defined in any one of claims 1 to 5.
7. The method for gene assaying according to claim 6, comprising the steps of:
step 1: using a target sequence to be detected in an isothermal amplification system as a template, and performing isothermal amplification by using an isothermal amplification method to obtain an amplified target sequence to be detected;
step 2: and carrying out enzyme digestion reaction on the amplified target sequence to be detected and the Cas14a1-sgRNA complex system, and then carrying out signal detection and result interpretation.
8. The method according to claim 7, wherein the isothermal amplification method in step 1 comprises RPA isothermal amplification, LAMP isothermal amplification or ERA isothermal amplification; the temperature of the enzyme digestion reaction in the step 2 is 37-52 ℃; and detecting the signal of the fluorescent or colloidal gold lateral flow chromatography test strip.
9. Use of a novel engineered CRISPR-Cas14a1 detection system, characterized in that the novel engineered CRISPR-Cas14a1 detection system of any one of claims 1 to 5 is used for detection of mycoplasma pneumoniae, hepatitis b virus, chlamydophila psittaci or non-small cell lung cancer SNPs.
10. The use of a novel engineered CRISPR-Cas14a1 detection system according to claim 9, characterized in that, when detecting SNPs against non-small cell lung cancer, the 1 st, 3-4 rd and 7-12 th positions of the target sequence to be detected, which are located after the 3' end of the PAM sequence, show lower tolerance.
CN202310311799.5A 2023-01-10 2023-03-28 Novel engineering CRISPR-Cas14a1 detection system, method and application Pending CN116622810A (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202310035106 2023-01-10
CN2023100351064 2023-01-10

Publications (1)

Publication Number Publication Date
CN116622810A true CN116622810A (en) 2023-08-22

Family

ID=87625463

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202310311799.5A Pending CN116622810A (en) 2023-01-10 2023-03-28 Novel engineering CRISPR-Cas14a1 detection system, method and application

Country Status (1)

Country Link
CN (1) CN116622810A (en)

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020142739A1 (en) * 2019-01-04 2020-07-09 Mammoth Biosciences, Inc. COMPOSITIONS AND METHODS FOR DETECTING MODIFIED NUCLEIC ACIDS AND AMPLIFYING ssDNA
CN112176035A (en) * 2020-10-14 2021-01-05 杭州优思达生物技术有限公司 Novel CRISPR nucleic acid detection method and application
WO2021243276A1 (en) * 2020-05-29 2021-12-02 University Of Florida Research Foundation Crispr/cas chain reaction systems and methods for amplifying the detection sensitivity of crispr-based target detection
CN114015674A (en) * 2021-11-02 2022-02-08 辉二(上海)生物科技有限公司 Novel CRISPR-Cas12i system
KR20220047502A (en) * 2020-10-08 2022-04-18 주식회사 진코어 An engineered guide RNA for the optimized CRISPR/Cas12f1(Cas14a1) system and use thereof
WO2022092317A1 (en) * 2020-10-30 2022-05-05 国立大学法人東京大学 ENGINEERED Cas12f PROTEIN
WO2022132955A2 (en) * 2020-12-16 2022-06-23 Proof Diagnostics, Inc. Coronavirus rapid diagnostics
CN114846146A (en) * 2019-10-29 2022-08-02 基恩科雷有限责任公司 Engineered guide RNAs for increasing efficiency of CRISPR/Cas12f1 system and uses thereof
CN115210370A (en) * 2020-02-28 2022-10-18 亥姆霍兹传染研究中心 RNA detection and transcription-dependent editing using reprogrammed tracrRNA

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020142739A1 (en) * 2019-01-04 2020-07-09 Mammoth Biosciences, Inc. COMPOSITIONS AND METHODS FOR DETECTING MODIFIED NUCLEIC ACIDS AND AMPLIFYING ssDNA
CN114846146A (en) * 2019-10-29 2022-08-02 基恩科雷有限责任公司 Engineered guide RNAs for increasing efficiency of CRISPR/Cas12f1 system and uses thereof
CN115210370A (en) * 2020-02-28 2022-10-18 亥姆霍兹传染研究中心 RNA detection and transcription-dependent editing using reprogrammed tracrRNA
WO2021243276A1 (en) * 2020-05-29 2021-12-02 University Of Florida Research Foundation Crispr/cas chain reaction systems and methods for amplifying the detection sensitivity of crispr-based target detection
KR20220047502A (en) * 2020-10-08 2022-04-18 주식회사 진코어 An engineered guide RNA for the optimized CRISPR/Cas12f1(Cas14a1) system and use thereof
CN112176035A (en) * 2020-10-14 2021-01-05 杭州优思达生物技术有限公司 Novel CRISPR nucleic acid detection method and application
WO2022092317A1 (en) * 2020-10-30 2022-05-05 国立大学法人東京大学 ENGINEERED Cas12f PROTEIN
WO2022132955A2 (en) * 2020-12-16 2022-06-23 Proof Diagnostics, Inc. Coronavirus rapid diagnostics
CN114015674A (en) * 2021-11-02 2022-02-08 辉二(上海)生物科技有限公司 Novel CRISPR-Cas12i system

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
RENJIAN XIAO ET AL.: "Structural basis for substrate recognition and cleavage by the dimerization-dependent CRISPR-Cas12f nuclease", NUCLEIC ACIDS RES ., vol. 49, no. 7, 25 March 2021 (2021-03-25), pages 4120 - 4128, XP055926469, DOI: 10.1093/nar/gkab179 *

Similar Documents

Publication Publication Date Title
AU2021282536B2 (en) Polynucleotide enrichment using CRISPR-Cas systems
CN110541022B (en) Mycobacterium tuberculosis complex detection kit based on CRISPR-Cas12a system
Zhou et al. The applications of CRISPR/Cas system in molecular detection
CN109666662A (en) Application of the novel ScCas12a in terms of detection of nucleic acids
CN109680053A (en) Application of the novel SsCas12a albumen in terms of detection of nucleic acids
US20140038241A1 (en) Genomic enrichment method, composition, and reagent kit
EP4159853A1 (en) Genome editing system and method
Kouprina et al. Selective isolation of large segments from individual microbial genomes and environmental DNA samples using transformation-associated recombination cloning in yeast
WO2022228351A1 (en) Representation and application of novel high temperature argonaute protein tpsago
US20210147915A1 (en) Crispr/cas and transposase based amplification compositions, systems and methods
CN110218802A (en) A method of detection respiratory pathogen nucleic acid
WO2021170877A1 (en) Rna detection and transcription-dependent editing with reprogrammed tracrrnas
JP2023537850A (en) Sequence-specific targeted transposition and selection and selection of nucleic acids
Nguyen et al. Harnessing noncanonical crRNAs to improve functionality of Cas12a orthologs
Dong et al. A single digestion, single-stranded oligonucleotide mediated PCR-independent site-directed mutagenesis method
CN116622810A (en) Novel engineering CRISPR-Cas14a1 detection system, method and application
WO2023148235A1 (en) Methods of enriching nucleic acids
US20220162620A1 (en) Applications of Streptococcus-derived Cas9 nucleases on minimal Adenine-rich PAM targets
Žedaveinytė et al. Antagonistic conflict between transposon-encoded introns and guide RNAs
Osakabe et al. Genome editing in mammals and plants using CRISPR type ID nuclease
WO2022210748A1 (en) Novel polypeptide having ability to form complex with guide rna
CN112941051A (en) FENM protein mutant and application thereof and kit containing mutant
WO2023109887A1 (en) Method for detecting integration site
EP4095243A1 (en) System for hybridization-based precision genome cleavage and editing, and uses thereof
Yu et al. CRISPR-Cas Approaches for Diagnostic Applications

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination