Construction method and application of gene editing vector regulated by hot promoter

Abstract

The invention relates to a construction method and application of a gene editing vector regulated and controlled by a hot promoter. The method comprises the following steps: providing three plasmids, namely pAAV-U6-sgRNA-CMV-GFP, pSQT1665-Lbcpf1-NLS-3xHA and pHSP70-MCS-P2A-EGFP, as a skeleton; and constructing a final vector pHSP70-Lbcpf1-2cr, wherein the Lbcpf1- And confirming that the final vector is successfully constructed through enzyme digestion identification and sequencing. The invention provides the gene editing pHSP70-Lbcpf1-2cr plasmid which is regulated and controlled by the hot promoter and is constructed by using the construction method disclosed by the invention. The invention also provides a pHSP70-Lbcpf1-2cr FASN carrier constructed by using the pHSP70-Lbcpf1-2cr plasmid, and the pHSP70-Lbcpf1-2cr FASN carrier has the capability of carrying out targeted cutting on the FASN cancer gene before and after thermal induction in a prostate cancer cell 22RV1. According to the plasmid provided by the invention, any gene can be edited theoretically after the crRNA region is replaced.

Description

Technical Field

[0001] The invention belongs to the fields of life science and biotechnology, and relates to a method for constructing a gene editing vector pHSP70-Lbcpf1-2cr plasmid regulated by a hot promoter and an application thereof. Background Art

[0002] Plasmids are smaller DNA molecules that are attached to non-cellular chromosomes or nuclear DNA in cells and can replicate autonomously. The tool that delivers a useful target DNA fragment into a recipient cell for reproduction and expression through recombinant DNA technology is called a vector. Bacterial plasmids are commonly used vectors in recombinant DNA technology. The plasmid molecule itself is a genetic structure with replication function. Plasmids also carry certain genetic information, so they will give the host cell some genetic traits.

[0003] Since its inception, the CRISPR gene editing system has become a powerful tool for studying cell functions. Since its discovery, the CRISPR / Cas system has set off a huge research boom in the biological field. Currently, the most in-depth research is the CRISPR / Cas9 system. With the continuous expansion of applications, the fixed PAM motif and some restrictions of the characteristics of the Cas9 protein itself, such as protein size, limit the application of the CRISPR / Cas system in gene editing. Therefore, more and more Cas9 variants and new nucleases are being discovered to expand the Cas enzyme series research tools.

[0004] Cas12a belongs to the Cas protein family. Under the guidance of crRNA, it can find the target site with a PAM sequence of TTTN (N is an arbitrary base), achieve specific binding to the target double-stranded DNA through complementary pairing of crRNA and the target chain, and use the RuvC subunit to cut the double-stranded DNA to produce sticky ends, and then complete the insertion and deletion of DNA through the DNA repair pathway in the body, thereby performing the function of gene editing.

[0005] The current method of directly transferring ribonucleoprotein (RNP) formed by Cas12a (Cpf1) and crRNA into cells has the advantages of being safer, faster, having lower off-target effects, and having higher editing efficiency. It has had a significant impact on our understanding of cancer biology, and is constantly driving new discoveries and accelerating the development of cancer diagnostics and treatments. Using CRISPR gene editing to directly target genetic mutations that drive cancer is an attractive and difficult challenge. In theory, downregulating oncogenes through CRISPR gene editing, or killing cancer cells that produce specific genetic mutations, can achieve the effect of inhibiting tumor growth. However, this strategy needs to overcome multiple obstacles, including expression at specific tumor sites and efficient gene editing at specific tumor sites.

[0006] In tumor gene therapy, the promoter can achieve the purpose of tumor targeted therapy by controlling the time and level of therapeutic gene expression. HSP70, as a molecular chaperone, participates in protein synthesis, folding, assembly and transport, is expressed in tumor cells, and participates in tumor immune protection; and the HSP70 promoter also exhibits unique thermal inducibility. It will not be expressed in normal cells and tissues, but only in cells induced by high temperature. This unique biological feature makes it of great significance in the study of genetic engineering therapy for tumors.

[0007] Therefore, through homologous recombination, the CRISPR-Cas12a and target gene crRNA expression vector pHSP70-Lbcpf1-2cr regulated by the HSP70 hot promoter was constructed. Through the heat source induction of specific parts (such as tumors), the dual effects of Cas12a enzyme and target gene crRNA expression can be achieved, thereby realizing the editing of specific genes in specific parts. It provides a good foundation for studying gene editing therapy for tumors.

[0008] At present, there is no report on the construction method of CRISPR-Cas12a plasmid vector regulated by hot promoter in the world. Summary of the invention

[0009] The present invention is to solve the above-mentioned problems and defects and provides a method for constructing and applying a gene editing vector regulated by a hot promoter.

[0010] Based on the problems existing in the prior art, the first purpose of the present invention is to provide a method for constructing a pHSP70-Lbcpf1-2cr vector regulated by a hot promoter; the second purpose of the present invention is to provide an application of a plasmid vector constructed by the construction method in tumor gene editing. Taking the oncogene FASN as an example, the present invention obtains a pHSP70-Lbcpf1-2crFASN plasmid that specifically regulates the FASN gene by screening crRNA targeting the FASN gene and constructing it into the pHSP70-Lbcpf1-2cr vector. The targeted regulation of the FASN gene in the cell is verified by plasmid transfection and heat induction. In theory, any target gene can be gene edited after replacing the crRNA region.

[0011] The present invention is implemented by the following technical solution.

[0012] On the one hand, the present invention provides a method for constructing a pHSP70-Lbcpf1-2cr plasmid vector controlled by a hot promoter.

[0013] Select pAAV-U6-sgRNA-CMV-GFP, pSQT1665-Lbcpf1-NLS-3xHA, and pHSP70-MCS-P2A-EGFP plasmids as the backbone to construct the target plasmid: pHSP70-Lbcpf1-2cr.

[0014] In the above construction method, the amount of pAAV-U6-sgRNA-CMV-GFP, pSQT1665-Lbcpf1-NLS-3xHA, and pHSP70-MCS-P2A-EGFP vectors is selected according to the actual operation routine.

[0015] In the above construction method, the steps of constructing the pHSP70-Lbcpf1-2cr plasmid vector controlled by a hot promoter include:

[0016] pHSP70-MCS-P2A-EGFP provides the HSP70 promoter,

[0017] pAAV-U6-sgRNA-CMV-GFP provides crRNA transcription-related elements,

[0018] pSQT1665-Lbcpf1-NLS-3xHA provides Lbcpf1 (Cas12a) protein expression-related elements.

[0019] Homologous recombination primers were designed based on pSQT1665-Lbcpf1-NLS-3xHA and pHSP70-MCS-P2A-EGFP, and the intermediate vector pHSP70-Lbcpf1 was preferentially constructed;

[0020] The hot-start CRISPR vector pHSP70-Lbcpf1-U6 was constructed with the intermediate vector pHSP70-Lbcpf1 and pAAV-U6-sgRNA-CMV-GFP as the main components;

[0021] pHSP70-Lbcpf1-U6 was used as the main body, and the scaffold and target gene target required by Lbcpf1 were inserted to obtain the final vector pHSP70-Lbcpf1-2cr

[0022] The successful construction of the final vector was confirmed by enzyme digestion identification and sequencing.

[0023] The primer sequences for constructing the intermediate vector pHSP70-Lbcpf1 homologous fragment are as follows:

[0024] Insert-Lbcpf1-fw:GCTTACTGGCTTATCGAAATGAATTCTGCAGTCGACGGTACC

[0025] Insert-Lbcpf1-rev: TCACGGGTTTTAAACGGGCCGGGCATTGGCCACACCAGC

[0026] pHSP-fw:GGCCCGTTTAAACCCGCT

[0027] pHSP-rev:ATTTCGATAAGCCAGTAAGCAGTG.

[0028] The primer sequences for constructing the hot start CRISPR vector pHSP70-Lbcpf1-U6 homology fragment are as follows:

[0029] pHSP-Lb-Line-fw:AAAAGGCCAGGAACCGTAAAA

[0030] pHSP-Lb-Line-rev:GGCGTCGCTTGGTCGGTC

[0031] U6-Insert-fw: atgaccgaccaagcgacgccGAGGGCCTATTTCCCATGATTC

[0032] U6-Insert-rev:tttacggttcctggccttttAAGGACAGGGAAGGGAGCAG.

[0033] The primer sequences for constructing the final vector pHSP70-Lbcpf1-2cr homologous fragment are as follows:

[0034] Scaffold-infu-fw:TAATTTCTACTAAGTGTAGATttacggggtcattagttcat

[0035] Scaffold-infu-rev: ATCTACACTTAGTAGAAATTAagaagagctcgctcttcc

[0036] Du-cr-fw:AGCCCATATATGGAGTTCCG

[0037] Du-cr-rev:ATGAACTAATGACCCCGTAA

[0038] Du-seq-fw:ttacggggtcattagttcatGAGGGCCTATTTCCCATGATTC

[0039] Du-seq-rev: cggaactccatatgggctATCTACACTTAGTAGAAATTAAGAAGAGCTCG.

[0040] In the above construction method, the amplified plasmid strains used were all DH-5α. The culture medium was prepared with ddH2O and sterilized at 121°C for 15-20 minutes.

[0041] LB liquid medium (g / L): yeast powder 5.0, peptone 10.0, NaCl 10.0.

[0042] LB plate (g / L): yeast powder 5.0, tryptone 10.0, NaCl 10.0, agar powder 15.0.

[0043] On the other hand, the method provides a functional application of the plasmid vector constructed by the construction method in cells. In the present invention, the target gene selected for knockout is human fatty acid synthase (FASN), which is a recognized oncogene. It includes the following steps:

[0044] Identify crRNA targets.

[0045] crRNA1: AGGATGTGGTGCTGCACCAGGCCAAAAAA

[0046] crRNA2:ACCCCAGGCTCTTCGACCACCCGAAAAAA

[0047] The above-mentioned crRNA1, the crRNA2 and the Cas12a nuclease are transcribed into mRNA in vitro to obtain active crRNA and Cas12a protein; in the above-mentioned construction method, the amount of crRNA1, crRNA2, and Cas12a protein is selected according to the actual operation routine.

[0048] Taking pHSP70-Lbcpf1-U6 as the main body, the scaffold required by Lbcpf1 and the crRNA of the target gene FASN were inserted to obtain the final vector pHSP70-Lbcpf1-2crFASN.

[0049] In the above construction method, the targeting strategy is: tumor cells are heat-induced after being transfected with plasmids, and the FASN gene is down-regulated.

[0050] 22RV1 cells were cultured in six-well plates for 24 hours, and the cell confluence reached 60%-70%. The vector plasmid was transfected into the cells using lipo3000 liposomes, and heat-induced at 43°C for 30 minutes after 24 hours and 48 hours, respectively. The target gene editing efficiency was verified by Western Blot and qPCR.

[0051] The beneficial effect of the present invention is that the present invention provides the ability of the pHSP70-Lbcpf1-2cr plasmid constructed by the above construction method to target and cut genes in prostate cancer cell 22RV1. On the other hand, the present invention also provides the ability of the pHSP70-Lbcpf1-2cr plasmid constructed by the above construction method to target and cut genes before and after heat induction. On the other hand, after the crRNA region is replaced, the plasmid provided by the present invention can theoretically edit any gene.

[0052] The present invention will be further explained below in conjunction with the accompanying drawings and specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 To verify the in vitro function of FASN gene crRNA. The crRNA, Cas12a protein, FASN template DNA, and fluorescent probe F5Q were mixed. After the CRISPR-Cas12a system worked, the F5Q probe was cut and the fluorescence intensity increased, indicating that crRNA can target the FASN gene and activate CRISPR-Cas12a activity.

[0054] Figure 2A This is a map of plasmid pSQT1665-LbCpf1-NLS-3×HA.

[0055] Figure 2B This is the map of plasmid pHSP70-MCS-P2A-EGFP.

[0056] Figure 2C This is a map of plasmid pAAV-U6-sgRNA-CMV-GFP.

[0057] Figure 2D This is a map of plasmid pHSP70-Lbcpf1.

[0058] Figure 2E Plasmid pHSP70-Lbcpf1-U6-EGFP-Amp del Atlas.

[0059] Figure 2F This is the map of the final plasmid pHSP70-Lbcpf1-2crFASN.

[0060] Figure 2GSchematic diagram of the final plasmid synthesis for the three plasmids.

[0061] Figure 3 The final plasmid pHSP70-Lbcpf1-2crFASN nucleic acid electrophoresis diagram is 10.9kb in size. There is a HindIII restriction site on the plasmid. After restriction digestion and electrophoresis verification, the electrophoresis band is consistent with the expectation (Band 2), indicating that the final plasmid was successfully synthesized.

[0062] Figure 4 This is the Western blot detection result of FASN protein after pHSP70-Lbcpf1-2crFASN was transfected into 22RV1 cells and subjected to heat induction at 43°C for 30 min at 24 h and 48 h, respectively. After continuous culture for 48 h, the cells were cultured continuously.

[0063] Figure 5 This is the qPCR detection result of FASN after pHSP70-Lbcpf1-2crFASN vector was transfected into 22RV1 cells and heat induced at 43°C for 30 min.

[0064] Figure 6 It is a flowchart of the steps of the present invention.

[0065] Figure 2F The nucleic acid sequence of pHSP70-Lbcpf1-2crFASN was verified by sequencing as follows:

[0066] CTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTAAATTAAAAATGAAGTTT

[0067] TAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGCACATTTCCCCGAAAAGTGCAAAGTATATATGAGTAAACTTGGTCTGACAGC

[0068] ACATTTTCCCCGAAAAGTGATATATGAGTAAACTTGGTCTGACAGCACATTTCCCCGAAAAGTGCAAGTATATATGAGTAAACTTGGTCTGACAG

[0069] CACATTTCCCCGAAAAGTGCAGTATATATGAGTAAACTTGGTCTGACAGCACATTTCCCCGAAAAGTGCCACCTGACGTCGACGGATCGGGAG

[0070] ATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCG

[0071] CTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGC

[0072] TGCTTCGCGATGTACGGGCCAGATATACGCGTGGTAGATCTCACTCTGGCCTCTGATTGGTCCAAGGAAGGCTGGGGGGCAGGACGGGAGGCG

[0073] AAACCCCTGGAATATTCCCGACCTGGCAGCCTCATCGAGCTCGGTGATTGGCTCAGAAGGGAAAAGGCGGGTCTCCGTGACGACTTATAAAAG

[0074] CCCAGGGGCAAGCGGTCCGGATAACGGCTAGCCTGAGGAGCTGCTGCGACAGTCCACTACCTTTTTCGAGAGTGACTCCCGTTGTCCCAAGGCT

[0075] TCCCAGAGCGAACCTGTGCGGCTGCAGGCACCGGCGCGTCGAGTTCCCGGCGTCCGGAAGGACCGAGCTCTTCTCGCCTCTGGCTAACTAGAG

[0076] AACCCACTGCTTACTGGCTTATCGAAATGAATTCTGCAGTCGACGGTACCGCGGGCCCGGGATCCACCGGTCGCCACCATGAGCAAGCTGGAG

[0077] AAGTTTACAAACTGCTACTCCCTGTCTAAGACCCTGAGGTTCAAGGCCATCCCTGTGGGCAAGACCCAGGAGAACATCGACAATAAGCGGCTG

[0078] CTGGTGGAGGACGAGAAGAGAGCCGAGGATTATAAGGGCGTGAAGAAGCTGCTGGATCGCTACTATCTGTCTTTTATCAACGACGTGCTGCAC

[0079] AGCATCAAGCTGAAGAATCTGAACAATTACATCAGCCTGTTCCGGAAGAAAACCAGAACCGAGAAGGAGAATAAGGAGCTGGAGAACCTGGA

[0080] GATCAATCTGCGGAAGGAGATCGCCAAGGCCTTCAAGGGCAACGAGGGCTACAAGTCCCTGTTTAAGAAGGATATCATCGAGACAATCCTGCC

[0081] AGAGTTCCTGGACGATAAGGACGAGATCGCCCTGGTGAACAGCTTCAATGGCTTTACCACAGCCTTCACCGGCTTCTTTGATAACAGAGAGAAT

[0082] ATGTTTTCCGAGGAGGCCAAGAGCACATCCATCGCCTTCAGGTGTATCAACGAGAATCTGACCCGCTACATCTCTAATATGGACATCTTCGAGA

[0083] AGGTGGACGCCATCTTTGATAAGCACGAGGTGCAGGAGATCAAGGAGAAGATCCTGAACAGCGACTATGATGTGGAGGATTTCTTTGAGGGCG

[0084] AGTTCTTTAACTTTGTGCTGACACAGGAGGGCATCGACGTGTATAACGCCATCATCGGCGGCTTCGTGACCGAGAGCGGCGAGAAGATCAAGG

[0085] GCCTGAACGAGTACATCAACCTGTATAATCAGAAAACCAAGCAGAAGCTGCCTAAGTTTAAGCCACTGTATAAGCAGGTGCTGAGCGATCGGG

[0086] AGTCTCTGAGCTTCTACGGCGAGGGCTATACATCCGATGAGGAGGTGCTGGAGGTGTTTAGAAACACCCTGAACAAGAACAGCGAGATCTTCA

[0087] GCTCCATCAAGAAGCTGGAGAAGCTGTTCAAGAATTTTGACGAGTACTCTAGCGCCGGCATCTTTGTGAAGAACGGCCCCGCCATCAGCACAA

[0088] TCTCCAAGGATATCTTCGGCGAGTGGAACGTGATCCGGGACAAGTGGAATGCCGAGTATGACGATATCCACCTGAAGAAGAAGGCCGTGGTGA

[0089] CCGAGAAGTACGAGGACGATCGGAGAAAGTCCTTCAAGAAGATCGGCTCCTTTTCTCTGGAGCAGCTGCAGGAGTACGCCGACGCCGATCTGT

[0090] CTGTGGTGGAGAAGCTGAAGGAGATCATCATCCAGAAGGTGGATGAGATCTACAAGGTGTATGGCTCCTCTGAGAAGCTGTTCGACGCCGATT

[0091] TTGTGCTGGAGAAGAGCCTGAAGAAGAACGACGCCGTGGTGGCCATCATGAAGGACCTGCTGGATTCTGTGAAGAGCTTCGAGAATTACATCA

[0092] AGGCCTTCTTTGGCGAGGGCAAGGAGACAAACAGGGACGAGTCCTTCTATGGCGATTTTGTGCTGGCCTACGACATCCTGCTGAAGGTGGACC

[0093] ACATCTACGATGCCATCCGCAATTATGTGACCCAGAAGCCCTACTCTAAGGATAAGTTCAAGCTGTATTTTCAGAACCCTCAGTTCATGGGCGG

[0094] CTGGGACAAGGATAAGGAGACAGACTATCGGGCCACCATCCTGAGATACGGCTCCAAGTACTATCTGGCCATCATGGATAAGAAGTACGCCAA

[0095] GTGCCTGCAGAAGATCGACAAGGACGATGTGAACGGCAATTACGAGAAGATCAACTATAAGCTGCTGCCCGGCCCTAATAAGATGCTGCCAAA

[0096] GGTGTTCTTTTCTAAGAAGTGGATGGCCTACTATAACCCCAGCGAGGACATCCAGAAGATCTACAAGAATGGCACATTCAAGAAGGGCGATAT

[0097] GTTTAACCTGAATGACTGTCACAAGCTGATCGACTTCTTTAAGGATAGCATCTCCCGGTATCCAAAGTGGTCCAATGCCTACGATTTCAACTTTT

[0098] CTGAGACAGAGAAGTATAAGGACATCGCCGGCTTTTACAGAGAGGTGGAGGAGCAGGGCTATAAGGTGAGCTTCGAGTCTGCCAGCAAGAAG

[0099] GAGGTGGATAAGCTGGTGGAGGAGGGCAAGCTGTATATGTTCCAGATCTATAACAAGGACTTTTCCGATAAGTCTCACGGCACACCCAATCTG

[0100] CACACCATGTACTTCAAGCTGCTGTTTGACGAGAACAATCACGGACAGATCAGGCTGAGCGGAGGAGCAGAGCTGTTCATGAGGCGCGCCTCC

[0101] CTGAAGAAGGAGGAGCTGGTGGTGCACCCAGCCAACTCCCCTATCGCCAACAAGAATCCAGATAATCCCAAGAAAACCACAACCCTGTCCTAC

[0102] GACGTGTATAAGGATAAGAGGTTTTCTGAGGACCAGTACGAGCTGCACATCCCAATCGCCATCAATAAGTGCCCCAAGAACATCTTCAAGATC

[0103] AATACAGAGGTGCGCGTGCTGCTGAAGCACGACGATAACCCCTATGTGATCGGCATCGATAGGGGCGAGCGCAATCTGCTGTATATCGTGGTG

[0104] GTGGACGGCAAGGGCAACATCGTGGAGCAGTATTCCCTGAACGAGATCATCAACAACTTCAACGGCATCAGGATCAAGACAGATTACCACTCT

[0105] CTGCTGGACAAGAAGGAGAAGGAGAGGTTCGAGGCCCGCCAGAACTGGACCTCCATCGAGAATATCAAGGAGCTGAAGGCCGGCTATATCTC

[0106] TCAGGTGGTGCACAAGATCTGCGAGCTGGTGGAGAAGTACGATGCCGTGATCGCCCTGGAGGACCTGAACTCTGGCTTTAAGAATAGCCGCGT

[0107] GAAGGTGGAGAAGCAGGTGTATCAGAAGTTCGAGAAGATGCTGATCGATAAGCTGAACTACATGGTGGACAAGAAGTCTAATCCTTGTGCAAC

[0108] AGGCGGCGCCCTGAAGGGCTATCAGATCACCAATAAGTTCGAGAGCTTTAAGTCCATGTCTACCCAGAACGGCTTCATCTTTTACATCCCTGCC

[0109] TGGCTGACATCCAAGATCGATCCATCTACCGGCTTTGTGAACCTGCTGAAAACCAAGTATACCAGCATCGCCGATTCCAAGAAGTTCATCAGCT

[0110] CCTTTGACAGGATCATGTACGTGCCCGAGGAGGATCTGTTCGAGTTTGCCCTGGACTATAAGAACTTCTCTCGCACAGACGCCGATTACATCAA

[0111] GAAGTGGAAGCTGTACTCCTACGGCAACCGGATCAGAATCTTCCGGAATCCTAAGAAGAACAACGTGTTCGACTGGGAGGAGGTGTGCCTGAC

[0112] CAGCGCCTATAAGGAGCTGTTCAACAAGTACGGCATCAATTATCAGCAGGGCGATATCAGAGCCCTGCTGTGCGAGCAGTCCGACAAGGCCTT

[0113] CTACTCTAGCTTTATGGCCCTGATGAGCCTGATGCTGCAGATGCGGAACAGCATCACAGGCCGCACCGACGTGGATTTTCTGATCAGCCCTGTG

[0114] AAGAACTCCGACGGCATCTTCTACGATAGCCGGAACTATGAGGCCCAGGAGAATGCCATCCTGCCAAAGAACGCCGACGCCAATGGCGCCTAT

[0115] AACATCGCCAGAAAGGTGCTGTGGGCCATCGGCCAGTTCAAGAAGGCCGAGGACGAGAAGCTGGATAAGGTGAAGATCGCCATCTCTAACAA

[0116] GGAGTGGCTGGAGTACGCCCAGACCAGCGTGAAGCACAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGGGATCC

[0117] TACCCATACGATGTTCCAGATTACGCTTATCCCTACGACGTGCCTGATTATGCATACCCATATGATGTCCCCGACTATGCCTAAAGCGGCCGCAC

[0118] TCCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTA

[0119] GTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATT

[0120] GCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCAT

[0121] GCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTA

[0122] AGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGC

[0123] CACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTG

[0124] ATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTG

[0125] TTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCT

[0126] GATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATG

[0127] CAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAG

[0128] TCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTT

[0129] ATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCC

[0130] CGGGAGCTTGTATTATCCCTCCAGTGCAGAGAAAATCGGCCAGTTTTCTCTGCCTGCAGTCCGCATGCCGTATCGGGCCTTGGGTTCTAACCTGT

[0131] TGCGTAGATTTATGCAGCGGACTGCCTTTCTCCCAAAGTGATAAACCGGACAGTATCATGGACCGGTTTTCCCGGTAATCCGTATTTGCAAGGT

[0132] TGGTTTCACTATGGAACATGAACTTCATTATATCGGTATCGACACCGCTAAAGAGAAACTGGATGTCGATGTGTTGCGTCCTGATGGTCGTCAT

[0133] CGCACCAAAAAATTCGCTAACACCACTAAAGGGCACGATGAGCTGGTGAGCTGGCTGAAAGGTCACAAGATTGACCATGCGCATATCTGCATC

[0134] GAAGCGACCGGCACCTATATGGAACCTGTCGCTGAGTGCCTTTACGATGCTGGCTACATAGTGTCAGTCATTAATCCTGCGCTGGGTAAAGCTT

[0135] TCGCTCAGAGTGAAGGACTGCGTAACAAGACTGATACCGTGGATGCGCGCATGCTGGCAGAGTTCTGTCGTCAGAAGCGCCCTGCAGCCTGGG

[0136] AAGCGCCTCACCCGCTTGAACGCGCGTTGCGTGCCCTGGTAGTCCGCCACCAGGCGCTGACAGATATGCACACGCAGGAACTGAATCGCACTG

[0137] AAACGGCGCGGGAAGTCCAGAGACCGAGCATTGATGCTCACCTTCTGTGGCTTGAAGCAGAGCTGAAGCGTCTTGAGAAGCAGATAAAAGACC

[0138] TGACAGACGATGATCCGGATATGAAACACCGCAGGAAACTGCTGGAAAGCATCCCGGGTATCGGAGAGAAAACATCTGCGGTATTGCTGGCTT

[0139] ATATCGGTCTGAAGGACCGCTTCGCCCATGCCAGACAGTTCGCCGCTTTTGCGGGTCTGACACCACGGCGTTATGAATCAGGTAGCAGTGTGAG

[0140] AGGGGCGAGCCGGATGAGTAAGGCCGGACATGTGTCGCTTCGCAGGGCGTTGTATATGCCCGCAATGGTAGCCACCAGTAAGACTGAGTGGGG

[0141] ACGGGCGTTCCGCGACCGTCTGGCGGCTAATGGCAAGAAAGGAAAGGTGATTCTCGGCGCGATGATGCGCAAGCTGGCATAGGTGGCGTATGG

[0142] AGTGCTGAAGTCAGGCGTGCCGTTCGATGCGTCACGGCATAATCCGGTAGCGGCGTAAAAATCGCGGAAGGGATGAAAAAAACAGCGCCTGA

[0143] CGGCGCTGTGTCTGGCATGCCTGCAATCCGGGAAACCGGACCAGGAAAAAACTTGCAGGCCATAACAGTATCTTGTATATCCATTTTCGGATCT

[0144] GATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGRGAGGCTATTCGGC

[0145] TATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACC

[0146] TGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGT

[0147] CACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATC

[0148] ATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACT

[0149] CGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGC

[0150] ATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACT

[0151] GTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCT

[0152] TCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCG

[0153] AAATGACCGACCAAGCGACGCCGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATT

[0154] AATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTA

[0155] AAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGGAAGAGCGAG

[0156] CTCTTCTTAATTTCTACTAAGTGTAGATAGGATGTGGTGCTGCACCAGgccaaaaaaGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACG

[0157] ATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCT

[0158] TGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTT

[0159] GTGGAAAGGACTAATTTCTACTAAGTGTAGATaccccAGGCTCTTCGACCACCCGaaaaaaGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGAT

[0160] AGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATG

[0161] TCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGAT

[0162] CCGCTAGCGCTACCGGTCGCCACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTA

[0163] AACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTG

[0164] CCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCA

[0165] AGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGG

[0166] GCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAAC

[0167] AGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCA

[0168] GCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAG

[0169] CAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTC

[0170] CGGACTCAGATCTCGATAACCTGCAGCGAATTCGATATCAAGCTTATCGATACCGAGCGCTGCTCGAGAGATCTACGGGTGGCATCCCTGTGAC

[0171] CCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGTCCTAATAAAATTAAGTTGCATCATTTTGTCTGACTA

[0172] GGTGTCCTTCTATAATATTATGGGGTGGAGGGGGGTGGTATGGAGCAAGGGGCAAGTTGGGAAGACAACCTGTAGGGCCTGCGGGGTCTATTG

[0173] GGAACCAAGCTGGAGTGCAGTGGCACAATCTTGGCTCACTGCAATCTCCGCCTCCTGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGAGTTG

[0174] TTGGGATTCCAGGCATGCATGACCAGGCTCAGCTAATTTTTGTTTTTTTGGTAGAGACGGGGTTTCACCATATTGGCCAGGCTGGTCTCCAACTC

[0175] CTAATCTCAGGTGATCTACCCACCTTGGCCTCCCAAATTGCTGGGATTACAGGCGTGAACCACTGCTCCCTTCCCTGTCCTTAAAAGGCCAGGA

[0176] ACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAA

[0177] ACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCC

[0178] GCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGT

[0179] GCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCA

[0180] GCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGA

[0181] ACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCG

[0182] GTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATC. DETAILED DESCRIPTION

[0183] The following examples are only a part of the technical solution of the present invention, and are not intended to limit the entire technical solution of the present invention. The examples of the present invention are provided to further explain and illustrate the details of the technical solution of the present invention.

[0184] In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present invention, the technical solution of the present invention is now described in detail below, but it should not be understood as limiting the scope of the present invention. The product information not specified in the text is commercially available in the art, and the experimental methods for which the specific conditions are not specified in the text are generally conventional methods and conditions in the art. Unless otherwise defined, all professional and scientific terms used in the text have the same meaning as those familiar to those skilled in the art.

[0185] Embodiment 1:

[0186] This embodiment provides a method for constructing a pHSP70-Lbcpf1-2cr plasmid vector regulated by a hot promoter. The specific steps are as follows: Construction of pHSP70-Lbcpf1-2cr expression vector

[0187] The present invention selected three plasmid backbones: pAAV-U6-sgRNA-CMV-GFP, pSQT1665-Lbcpf1-NLS-3xHA, pHSP70-MCS-P2A-EGFP, to construct the target plasmid: pHSP70-Lbcpf1-U6. The structures of the three plasmids are shown in Figure 2.

[0188] 1. Construction of the intermediate vector pHSP70-Lbcpf1

[0189] 1. PCR amplification

[0190] Using pHSP70-MCS-P2A-EGFP as a template, a linear fragment was obtained by PCR;

[0191] The insert fragment was obtained by PCR using pSQT1665-Lbcpf1-NLS-3xHA as a template.

[0192] PCR reaction primers:

[0193]

[0194] PCR reaction system:

[0195] Element Volume (μL) DNA template 1 primer-F 1 primer-R 1 2x Phanta max Buffer 25 dNTP Mix 1 DNA Polymerase 1 <![CDATA[ddH2O]]> 20

[0196] PCR reaction procedure:

[0197]

[0198] PCR amplification product detection: Prepare a 1.5% agarose gel of appropriate size according to the amount of each experiment, draw 1.5μL of Loading buffer into a PCR microcentrifuge tube, mix with an equal amount of PCR amplification product, draw and drop into the well of agarose gel after sufficient mixing, soak the agarose gel in TBE buffer, set the electrophoresis instrument to 120V constant voltage, and the electrophoresis time is 30min. Record the results after electrophoresis with a UV gel imager, and select a suitable marker to estimate the size of the PCR product fragment.

[0199] 2. Use Tiangen Agarose Gel DNA Recovery Kit Enhanced-DP219 to recover the target fragment.

[0200] Cut out a single target DNA band from the agarose gel (try to remove excess parts) and place it in a clean centrifuge tube and weigh it.

[0201] Add 3 times the volume of sol solution PE to the gel block (if the gel weight is 0.1g, its volume can be regarded as 100μL, then add 300μL of sol solution PE. Use a gel cutter to cut 1% agarose gel, the weight of a single block is about 0.06g, and the actual gel block weight is related to the gel concentration and thickness). Sol at room temperature 15-25℃ for 5-10min, during which time the centrifuge tube is gently turned upside down to ensure that the gel block is fully dissolved.).

[0202] Add the solution obtained in the previous step to an adsorption column CA5 (the adsorption column is placed in a collection tube), place at room temperature for 2 minutes, centrifuge at 12,000 rpm (~13,400×g) for 30-60 seconds, pour out the waste liquid in the collection tube, and place the adsorption column CA5 in the collection tube.

[0203] Add 600 μL of rinsing solution PW to the adsorption column CA5 (please check whether anhydrous ethanol has been added before use), centrifuge at 12,000 rpm (~13,400×g) for 30-60 seconds, pour out the waste liquid in the collection tube, and place the adsorption column CA5 in the collection tube.

[0204] Place the adsorption column CA5 back into the collection tube and centrifuge at 12,000 rpm (~13,400 × g) for 2 min to remove as much rinsing solution as possible. Place the adsorption column CA5 at room temperature for several minutes to dry thoroughly to prevent residual rinsing solution from affecting the next step of the experiment.

[0205] Place the adsorption column CA5 in a clean centrifuge tube, add an appropriate amount of elution buffer TB to the middle of the adsorption membrane, and place it at room temperature for 2 minutes. Centrifuge at 12,000 rpm (~13,400×g) for 2 minutes to collect the DNA solution.

[0206] 3. Perform homologous recombination between the collected pHSP70 linearized vector and the inserted fragment Lbcpf1

[0207] Calculate the amount of DNA required for the recombination reaction according to the formula.

[0208] Optimal amount of cloning vector used = [0.02 × 5000 (number of base pairs of cloning vector)] ng (0.03 pmol) = 100 ng

[0209] Optimal amount of insert fragment = [0.04 × 4000 (number of base pairs of insert fragment] ng (0.06 pmol) = 160 ng

[0210] Set up the following reaction system on ice:

[0211] Element Recombination reaction (μL) Linearized vector 1 primer-F 2 primer-R 2 2xPhanta max Buffer 25 dNTP Mix 1 DNA Polymerase 1 <![CDATA[ddH2O]]> 19

[0212] Use a pipette to gently mix, and centrifuge briefly to collect the reaction solution at the bottom of the tube.

[0213] Incubate at 37°C for 30 min; cool to 4°C or immediately place on ice.

[0214] 4. Transformation of recombinant products

[0215] Thaw cloning competent cells on ice

[0216] Take 10 μl of the recombinant product and add it to 100 μl of competent cells, flick the tube wall to mix, and let it stand on ice for 30 min.

[0217] After heat shock in a 42°C water bath for 45 seconds, immediately cool on ice for 2-3 minutes.

[0218] Add 900 μl of SOC or LB medium (without antibiotics) and shake at 37°C for 1 h (speed 200-250 rpm).

[0219] Pre-warm the LB solid medium plates of the corresponding resistance in a 37°C incubator.

[0220] Centrifuge at 5,000 rpm (2,400 × g) for 5 min, discard 900 μl of supernatant, resuspend the cells in the remaining medium, and spread evenly on a plate containing the correct resistance using a sterile spreader.

[0221] Incubate inverted in a 37°C incubator for 12-16 hours.

[0222] 5. Sequencing and identification of recombinant products

[0223] 2. Construction of pHSP70-LbCpf1-U6 intermediate vector

[0224] 1. PCR reaction, the specific method is the same, the construction of the intermediate vector pHSP70-Lbcpf1, 1

[0225] PCR reaction primers:

[0226]

[0227] 2. Then, homologous recombination was performed, and the specific method was the same as that of the construction of the intermediate vector pHSP70-Lbcpf1.

[0228] 3. Construction of pHSP70-LbCpf1-2crFASN

[0229] 1. PCR reaction, the specific method is the same, the construction of the intermediate vector pHSP70-Lbcpf1,

[0230] PCR reaction primers:

[0231]

[0232] 2. Then, homologous recombination was performed, and the specific method was the same as that of the construction of the intermediate vector pHSP70-Lbcpf1.

[0233] Embodiment 2:

[0234] This embodiment provides a detailed DNA sequence of a CRISPR-Cas12a plasmid vector (pHSP70-Lbcpf1-2crFASN) controlled by a hot promoter, and the specific sequence is as follows:

[0235] GGCGTCGCTTGGTCGGTCATTTCGAACCCCAGAGTCCCGCTCAGAAGAACTCGTCAAGAAGGCGATAGAAGGCGATGCGCTGCGAATCGGGAG

[0236] CGGCGATACCGTAAAGCACGAGGAAGCGGTCAGCCCATTCGCCGCCAAGCTCTTCAGCAATATCACGGGTAGCCAACGCTATGTCCTGATAGC

[0237] GGTCCGCCACACCCAGCCGGCCACAGTCGATGAATCCAGAAAAGCGGCCATTTTCCACCATGATATTCGGCAAGCAGGCATCGCCATGGGTCA

[0238] CGACGAGATCCTCGCCGTCGGGCATGCGCGCCTTGAGCCTGGCGAACAGTTCGGCTGGCGCGAGCCCCTGATGCTCTTCGTCCAGATCATCCTG

[0239] ATCGACAAGACCGGCTTCCATCCGAGTACGTGCTCGCTCGATGCGATGTTTCGCTTGGTGGTCGAATGGGCAGGTAGCCGGATCAAGCGTATGC

[0240] AGCCGCCGCATTGCATCAGCCATGATGGATACTTTCTCGGCAGGAGCAAGGTGAGATGACAGGAGATCCTGCCCCGGCACTTCGCCCAATAGC

[0241] AGCCAGTCCCTTCCCGCTTCAGTGACAACGTCGAGCACAGCTGCGCAAGGAACGCCCGTCGTGGCCAGCCACGATAGCCGCGCTGCCTCGTCCT

[0242] GCAGTTCATTCAGGGCACCGGACAGGTCGGTCTTGACAAAAAGAACCGGGCGCCCCTGCGCTGACAGCCGGAACACGGCGGCATCAGAGCAG

[0243] CCGATTGTCTGTTGTGCCCAGTCATAGCCGAATAGCCTCCCCACCCAAGCGGCCGGAGAACCTGCGTGCAATCCATCTTGTTCAATCATGCGAA

[0244] ACGATCCTCATCCTGTCTCTTGATCAGATCCGAAAATGGATATACAAGATACTGTTATGGCCTGCAAGTTTTTTCCTGGTCCGGTTTCCCGGATT

[0245] GCAGGCATGCCAGACACAGCGCCGTCAGGCGCTGTTTTTTTCATCCCTTCCGCGATTTTTACGCCGCTACCGGATTATGCCGTGACGCATCGAAC

[0246] GGCACGCCTGACTTCAGCACTCCATACGCCACCTATGCCAGCTTGCGCATCATCGCGCCGAGAATCACCTTTCCTTTCTTGCCATTAGCCGCCAG

[0247] ACGGTCGCGGAACGCCCGTCCCCACTCAGTCTTACTGGTGGCTACCATTGCGGGCATATACAACGCCCTGCGAAGCGACACATGTCCGGCCTTA

[0248] CTCATCCGGCTCGCCCCTCTCACACTGCTACCTGATTCATAACGCCGTGGTGTCAGACCCGCAAAAGCGGCGAACTGTCTGGCATGGGCGAAGC

[0249] GGTCCTTCAGACCGATATAAGCCAGCAATACCGCAGATGTTTTCTCTCCGATACCCGGGATGCTTTCCAGCAGTTTCCTGCGGTGTTTCATATCC

[0250] GGATCATCGTCTGTCAGGTCTTTTATCTGCTTCTCAAGACGCTTCAGCTCTGCTTCAAGCCACAGAAGGTGAGCATCAATGCTCGGTCTCTGGAC

[0251] TTCCCGCGCCGTTTCAGTGCGATTCAGTTCCTGCGTGTGCATATCTGTCAGCGCCTGGTGGCGGACTACCAGGGCACGCAACGCGCGTTCAAGC

[0252] GGGTGAGGCGCTTCCCAGGCTGCAGGGCGCTTCTGACGACAGAACTCTGCCAGCATGCGCGCATCCACGGTATCAGTCTTGTTACGCAGTCCTT

[0253] CACTCTGAGCGAAAGCTTTACCCAGCGCAGGATTAATGACTGACACTATGTAGCCAGCATCGTAAAGGCACTCAGCGACAGGTTCCATATAGG

[0254] TGCCGGTCGCTTCGATGCAGATATGCGCATGGTCAATCTTGTGACCTTTCAGCCAGCTCACCAGCTCATCGTGCCCTTTAGTGGTGTTAGCGAAT

[0255] TTTTTGGTGCGATGACGACCATCAGGACGCAACACATCGACATCCAGTTTCTCTTTAGCGGTGTCGATACCGATATAATGAAGTTCATGTTCCAT

[0256] AGTGAAACCAACCTTGCAAATACGGATTACCGGGAAAACCGGTCCATGATACTGTCCGGTTTATCACTTTGGGAGAAAGGCAGTCCGCTGCAT

[0257] AAATCTACGCAACAGGTTAGAACCCAAGGCCCGATACGGCATGCGGACTGCAGGCAGAGAAAACTGGCCGATTTTCTCTGCACTGGAGGGATA

[0258] ATACAAGCTCCCGGGAGCTTTTTGCAAAAGCCTAGGCCTCCAAAAAAGCCTCCTCACTACTTCTGGAATAGCTCAGAGGCAGAGGCGGCCTCG

[0259] GCCTCTGCATAAATAAAAAAAATTAGTCAGCCATGGGGCGGAGAATGGGCGGAACTGGGCGGAGTTAGGGGCGGGATGGGCGGAGTTAGGGG

[0260] CGGGACTATGGTTGCTGACTAATTGAGATGCATGCTTTGCATACTTCTGCCTGCTGGGGAGCCTGGGGACTTTCCACACCTGGTTGCTGACTAAT

[0261] TGAGATGCATGCTTTGCATACTTCTGCCTGCTGGGGAGCCTGGGGACTTTCCACACCCTAACTGACACACATTCCACAGAATTAATTCGCGTTA

[0262] AATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTG

[0263] TTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTAC

[0264] GTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGAC

[0265] GGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCG

[0266] CGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTGGGGATACCCCCTAGAGCCCCAGCTGGTTCTTTCCGCCTCAGAAGCC

[0267] ATAGAGCCCACCGCATCCCCAGCATGCCTGCTATTGTCTTCCCAATCCTCCCCCTTGCTGTCCTGCCCCACCCCACCCCCCAGAATAGAATGACA

[0268] CCTACTCAGACAATGCGATGCAATTTCCTCATTTTATTAGGAAAGGACAGTGGGAGTGGCACCTTCCAGGGTCAAGGAAGGCACGGGGGAGGG

[0269] GCAAACAACAGATGGCTGGCAACTAGAAGGCACAGTCGAGGCTGATCAGCGGGTTTAAACGGGCCGGCATTGGCCACACCAGCCACCACCTTC

[0270] TGATAGGCAGCCTGCACCTGAGGAGTGCGGCCGCTTTAGGCATAGTCGGGGACATCATATGGGTATGCATAATCAGGCACGTCGTAGGGATAA

[0271] GCGTAATCTGGAACATCGTATGGGTAGGATCCCTTTTTCTTTTTTGCCTGGCCGGCCTTTTTCGTGGCCGCCGGCCTTTTGTGCTTCACGCTGGTC

[0272] TGGGCGTACTCCAGCCACTCCTTGTTAGAGATGGCGATCTTCACCTTATCCAGCTTCTCGTCCTCGGCCTTCTTGAACTGGCCGATGGCCCACAG

[0273] CACCTTTCTGGCGATGTTATAGGCGCCATTGGCGTCGGCGTTCTTTGGCAGGATGGCATTCTCCTGGGCCTCATAGTTCCGGCTATCGTAGAAGA

[0274] TGCCGTCGGAGTTCTTCACAGGGCTGATCAGAAAATCCACGTCGGTGCGGCCTGTGATGCTGTTCCGCATCTGCAGCATCAGGCTCATCAGGGC

[0275] CATAAAGCTAGAGTAGAAGGCCTTGTCGGACTGCTCGCACAGCAGGGCTCTGATATCGCCCTGCTGATAATTGATGCCGTACTTGTTGAACAGC

[0276] TCCTTATAGGCGCTGGTCAGGCACACCTCCTCCCAGTCGAACACGTTGTTCTTCTTAGGATTCCGGAAGATTCTGATCCGGTTGCCGTAGGAGTA

[0277] CAGCTTCCACTTCTTGATGTAATCGGCGTCTGTGCGAGAGAAGTTCTTATAGTCCAGGGCAAACTCGAACAGATCCTCCTCGGGCACGTACATG

[0278] ATCCTGTCAAAGGAGCTGATGAACTTCTTGGAATCGGCGATGCTGGTATACTTGGTTTTCAGCAGGTTCACAAAGCCGGTAGATGGATCGATCT

[0279] TGGATGTCAGCCAGGCAGGGATGTAAAAGATGAAGCCGTTCTGGGTAGACATGGACTTAAAGCTCTCGAACTTATTGGTGATCTGATAGCCCTT

[0280] CAGGGCGCCGCCTGTTGCACAAGGATTAGACTTCTTGTCCACCATGTAGTTCAGCTTATCGATCAGCATCTTCTCGAACTTCTGATACACCTGCT

[0281] TCTCCACCTTCACGCGGCTATTCTTAAAGCCAGAGTTCAGGTCCTCCAGGGCGATCACGGCATCGTACTTCTCCACCAGCTCGCAGATCTTGTGC

[0282] ACCACCTGAGAGATATAGCCGGCCTTCAGCTCCTTGATATTCTCGATGGAGGTCCAGTTCTGGCGGGCCTCGAACCTCTCCTTCTCCTTCTTGTC

[0283] CAGCAGAGAGTGGTAATCTGTCTTGATCCTGATGCCGTTGAAGTTGTTGATGATCTCGTTCAGGGAATACTGCTCCACGATGTTGCCCTTGCCGT

[0284] CCACCACCACGATATACAGCAGATTGCGCTCGCCCCTATCGATGCCGATCACATAGGGGTTATCGTCGTGCTTCAGCAGCACGCGCACCTCTGT

[0285] ATTGATCTTGAAGATGTTCTTGGGGCACTTATTGATGGCGATTGGGATGTGCAGCTCGTACTGGTCCTCAGAAAACCTCTTATCCTTATACACGT

[0286] CGTAGGACAGGGTTGTGGTTTTCTTGGGATTATCTGGATTCTTGTTGGCGATAGGGGAGTTGGCTGGGTGCACCACCAGCTCCTCCTTCTTCAGG

[0287] GAGGCGCGCCTCATGAACAGCTCTGCTCCTCCGCTCAGCCTGATCTGTCCGTGATTGTTCTCGTCAAACAGCAGCTTGAAGTACATGGTGTGCA

[0288] GATTGGGTGTGCCGTGAGACTTATCGGAAAAGTCCTTGTTATAGATCTGGAACATATACAGCTTGCCCTCCTCCACCAGCTTATCCACCTCCTTC

[0289] TTGCTGGCAGACTCGAAGCTCACCTTATAGCCCTGCTCCTCCACCTCTCTGTAAAAGCCGGCGATGTCCTTATACTTCTCTGTCTCAGAAAAGTT

[0290] GAAATCGTAGGCATTGGACCACTTTGGATACCGGGAGATGCTATCCTTAAAGAAGTCGATCAGCTTGTGACAGTCATTCAGGTTAAACATATCG

[0291] CCCTTCTTGAATGTGCCATTCTTGTAGATCTTCTGGATGTCCTCGCTGGGGTTATAGTAGGCCATCCACTTCTTAGAAAAGAACACCTTTGGCAG

[0292] CATCTTATTAGGGCCGGGCAGCAGCTTATAGTTGATCTTCTCGTAATTGCCGTTCACATCGTCCTTGTCGATCTTCTGCAGGCACTTGGCGTACT

[0293] TCTTATCCATGATGGCCAGATAGTACTTGGAGCCGTATCTCAGGATGGTGGCCCGATAGTCTGTCTCCTTATCCTTGTCCCAGCCGCCCATGAAC

[0294] TGAGGGTTCTGAAAATACAGCTTGAACTTATCCTTAGAGTAGGGCTTCTGGGTCACATAATTGCGGATGGCATCGTAGATGTGGTCCACCTTCA

[0295] GCAGGATGTCGTAGGCCAGCACAAAATCGCCATAGAAGGACTCGTCCCTGTTTGTCTCCTTGCCCTCGCCAAAGAAGGCCTTGATGTAATTCTC

[0296] GAAGCTCTTCACAGAATCCAGCAGGTCCTTCATGATGGCCACCACGGCGTCGTTCTTCTTCAGGCTCTTCTCCAGCACAAAATCGGCGTCGAAC

[0297] AGCTTCTCAGAGGAGCCATACACCTTGTAGATCTCATCCACCTTCTGGATGATGATCTCCTTCAGCTTCTCCACCACAGACAGATCGGCGTCGGC

[0298] GTACTCCTGCAGCTGCTCCAGAGAAAAGGAGCCGATCTTCTTGAAGGACTTTCTCCGATCGTCCTCGTACTTCTCGGTCACCACGGCCTTCTTCT

[0299] TCAGGTGGATATCGTCATACTCGGCATTCCACTTGTCCCGGATCACGTTCCACTCGCCGAAGATATCCTTGGAGATTGTGCTGATGGCGGGGCC

[0300] GTTCTTCACAAAGATGCCGGCGCTAGAGTACTCGTCAAAATTCTTGAACAGCTTCTCCAGCTTCTTGATGGAGCTGAAGATCTCGCTGTTCTTGT

[0301] TCAGGGTGTTTCTAAACACCTCCAGCACCTCCTCATCGGATGTATAGCCCTCGCCGTAGAAGCTCAGAGACTCCCGATCGCTCAGCACCTGCTT

[0302] ATACAGTGGCTTAAACTTAGGCAGCTTCTGCTTGGTTTTCTGATTATACAGGTTGATGTACTCGTTCAGGCCCTTGATCTTCTCGCCGCTCTCGGT

[0303] CACGAAGCCGCCGATGATGGCGTTATACACGTCGATGCCCTCCTGTGTCAGCACAAAGTTAAAGAACTCGCCCTCAAAGAAATCCTCCACATCA

[0304] TAGTCGCTGTTCAGGATCTTCTCCTTGATCTCCTGCACCTCGTGCTTATCAAAGATGGCGTCCACCTTCTCGAAGATGTCCATATTAGAGATGTA

[0305] GCGGGTCAGATTCTCGTTGATACACCTGAAGGCGATGGATGTGCTCTTGGCCTCCTCGGAAAACATATTCTCTCTGTTATCAAAGAAGCCGGTG

[0306] AAGGCTGTGGTAAAGCCATTGAAGCTGTTCACCAGGGCGATCTCGTCCTTATCGTCCAGGAACTCTGGCAGGATTGTCTCGATGATATCCTTCTT

[0307] AAACAGGGACTTGTAGCCCTCGTTGCCCTTGAAGGCCTTGGCGATCTCCTTCCGCAGATTGATCTCCAGGTTCTCCAGCTCCTTATTCTCCTTCTC

[0308] GGTTCTGGTTTTCTTCCGGAACAGGCTGATGTAATTGTTCAGATTCTTCAGCTTGATGCTGTGCAGCACGTCGTTGATAAAAGACAGATAGTAGC

[0309] GATCCAGCAGCTTCTTCACGCCCTTATAATCCTCGGCTCTCTTCTCGTCCTCCACCAGCAGCCGCTTATTGTCGATGTTCTCCTGGGTCTTGCCCA

[0310] CAGGGATGGCCTTGAACCTCAGGGTCTTAGACAGGGAGTAGCAGTTTGTAAACTTCTCCAGCTTGCTCATGGTGGCGACCGGTGGATCCCGGGC

[0311] CCGCGGTACCGTCGACTGCAGAATTCATTTCGATAAGCCAGTAAGCAGTGGGTTCTCTAGTTAGCCAGAGGCGAGAAGAGCTCGGTCCTTCCGG

[0312] ACGCCGGGAACTCGACGCGCCGGTGCCTGCAGCCGCACAGGTTCGCTCTGGGAAGCCTTGGGACAACGGGAGTCACTCTCGAAAAAGGTAGTG

[0313] GACTGTCGCAGCAGCTCCTCAGGCTAGCCGTTATCCGGACCGCTTGCCCCTGGGCTTTTATAAGTCGTCACGGAGACCCGCCTTTTCCCTTCTGA

[0314] GCCAATCACCGAGCTCGATGAGGCTGCCAGGTCGGGAATATTCCAGGGGTTTCGCCTCCCGTCCTGCCCCCCAGCCTTCCTTGGACCAATCAGA

[0315] GGCCAGAGTGAGATCTACCACGCGTATATCTGGCCCGTACATCGCGAAGCAGCGCAAAACGCCTAACCCTAAGCAGATTCTTCATGCAATTGTC

[0316] GGTCAAGCCTTGCCTTGTTGTAGCTTAAATTTTGCTCGCGCACTACTCAGCGACCTCCAACACACAAGCAGGGAGCAGATACTGGCTTAACTAT

[0317] GCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATAGGGGATCGGGAGATCTCCCGATCCGTCGACGTCAGGTGGCACTTTTCGGGGAAATG

[0318] TGCTGTCAGACCAAGTTTACTCATATATACTGCACTTTTCGGGGAAATGTGCTGTCAGACCAAGTTTACTCATATATACTTGCACTTTTCGGGGA

[0319] AATGTGCTGTCAGACCAAGTTTACTCATATATCACTTTTCGGGGAAATGTGCTGTCAGACCAAGTTTACTCATATATACTTTGCACTTTTCGGGG

[0320] AAATGTGCTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTG

[0321] ATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTT

[0322] TTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGA

[0323] AGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCC

[0324] TACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCG

[0325] GATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCG

[0326] TGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGG

[0327] GAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGG

[0328] GCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTAAGGACAGGGAAGGGAGCAGTGGTTCACGCCTGTAAT

[0329] CCCAGCAATTTGGGAGGCCAAGGTGGGTAGATCACCTGAGATTAGGAGTTGGAGACCAGCCTGGCCAATATGGTGAAACCCCGTCTCTACCAA

[0330] AAAAACAAAAATTAGCTGAGCCTGGTCATGCATGCCTGGAATCCCAACAACTCGGGAGGCTGAGGCAGGAGAATCGCTTGAACCCAGGAGGC

[0331] GGAGATTGCAGTGAGCCAAGATTGTGCCACTGCACTCCAGCTTGGTTCCCAATAGACCCCGCAGGCCCTACAGGTTGTCTTCCCAACTTGCCCC

[0332] TTGCTCCATACCACCCCCCTCCACCCCATAATATTATAGAAGGACACCTAGTCAGACAAAATGATGCAACTTAATTTTATTAGGACAAGGCTGG

[0333] TGGGCACTGGAGTGGCAACTTCCAGGGCCAGGAGAGGCACTGGGGAGGGGTCACAGGGATGCCACCCGTAGATCTCTCGAGCAGCGCTCGGT

[0334] ATCGATAAGCTTGATATCGAATTCGCTGCAGGTTATCGAGATCTGAGTCCGGACTTGTACAGCTCGTCCATGCCGAGAGTGATCCCGGCGGCGG

[0335] TCACGAACTCCAGCAGGACCATGTGATCGCGCTTCTCGTTGGGGTCTTTGCTCAGGGCGGACTGGGTGCTCAGGTAGTGGTTGTCGGGCAGCAG

[0336] CACGGGGCCGTCGCCGATGGGGGTGTTCTGCTGGTAGTGGTCGGCGAGCTGCACGCTGCCGTCCTCGATGTTGTGGCGGATCTTGAAGTTCACC

[0337] TTGATGCCGTTCTTCTGCTTGTCGGCCATGATATAGACGTTGTGGCTGTTGTAGTTGTACTCCAGCTTGTGCCCCAGGATGTTGCCGTCCTCCTTG

[0338] AAGTCGATGCCCTTCAGCTCGATGCGGTTCACCAGGGTGTCGCCCTCGAACTTCACCTCGGCGCGGGTCTTGTAGTTGCCGTCGTCCTTGAAGA

[0339] AGATGGTGCGCTCCTGGACGTAGCCTTCGGGCATGGCGGACTTGAAGAAGTCGTGCTGCTTCATGTGGTCGGGGTAGCGGCTGAAGCACTGCA

[0340] CGCCGTAGGTCAGGGTGGTCACGAGGGTGGGCCAGGGCACGGGCAGCTTGCCGGTGGTGCAGATGAACTTCAGGGTCAGCTTGCCGTAGGTGG

[0341] CATCGCCCTCGCCCTCGCCGGACACGCTGAACTTGTGGCCGTTTACGTCGCCGTCCAGCTCGACCAGGATGGGCACCACCCCGGTGAACAGCTC

[0342] CTCGCCCTTGCTCACCATGGTGGCGACCGGTAGCGCTAGCGGATCTGACGGTTCACTAAACCAGCTCTGCTTATATAGACCTCCCACCGTACAC

[0343] GCCTACCGCCCATTTGCGTCAATGGGGCGGAGTTGTTACGACATTTTGGAAAGTCCCGTTGATTTTGGTGCCAAAACAAACTCCCATTGACGTC

[0344] AATGGGGTGGAGACTTGGAAATCCCCGTGAGTCAAACCGCTATCCACGCCCATTGATGTACTGCCAAAACCGCATCACTTTTTTCGGGTGGTCG

[0345] AAGAGCCTGGGGTATCTACACTTAGTAGAAATTAGTCCTTTCCACAAGATATATAAAGCCAAGAAATCGAAATACTTTCAAGTTACGGTAAGC

[0346] ATATGATAGTCCATTTTAAAACATAATTTTAAAACTGCAAACTACCCAAGAAATTATTACTTTCTACGTCACGTATTTTGTACTAATATCTTTGT

[0347] GTTTACAGTCAAATTAATTCCAATTATCTCTCTAACAGCCTTGTATCGTATATGCAAATATGAAGGAATCATGGGAAATAGGCCCTCTTTTTTGG

[0348] CCTGGTGCAGCACCACATCCTATCTACACTTAGTAGAAATTAAGAAGAGCTCGCTCTTCCGGTGTTTCGTCCTTTCCACAAGATATATAAAGCC

[0349] AAGAAATCGAAATACTTTCAAGTTACGGTAAGCATATGATAGCCATTTTAAAACATAATTTTAAAACTGCAAACTACCCAAGAAATTATTACT

[0350] TTCTACGTCACGTATTTTGTACTAATATCTTTGTGTTTACAGTCAAATTAATTCCAATTATCTCTCTAACAGCCTTGTATCGTATATGCAAATATG

[0351] AAGGAATCATGGGAAATAGGCCCTC

[0352] Embodiment 3:

[0353] This embodiment provides a method for down-regulating the key gene FASN of prostate cancer cell 22RV1 under heat induction using the pHSP70-Lbcpf1-2cr vector of the present invention.

[0354] 22RV1 cells were plated into 6-well culture plates one night in advance. The plated volume should be about 80% during transfection.

[0355] Transfect cells. Prepare two EP tubes, add 125μL opti-MEM, 7.5μL P3000 and the plasmid to be transfected to one tube, and add 125μL opti-MEM and 7.5μL Lipo3000 transfection reagent to the other tube, shake and mix the two tubes on a vortex mixer, and let stand for 5 minutes;

[0356] Mix the liquids in the two EP tubes into one tube, shake again, and let stand at room temperature for 15 minutes;

[0357] In the clean bench, the transfection system was added to 22RV1 cells, the culture medium and transfection system were mixed, and the cells were placed in the incubator for continued culture. Fresh culture medium was replaced after 12 hours. Heat induction was performed at 43°C for 30 minutes after 24 and 48 hours. Gene expression was detected after 48 hours. The test results showed that FASN was significantly downregulated. Figure 3 (Protein Level) Figure 4 (mRNA level).

[0358] The key points and protection points of the present invention are as follows: For the first time, a method for constructing a hot promoter-regulated CRISPR-Cas12a plasmid pHSP70-Lbcpf1-2cr was constructed, and its working efficiency was verified.

[0359] There have been reports on in vivo editing plasmids for CRISPR-Cas9, but there are fewer for CRISPR-Cas12a. The technical point of the present invention is to use hot start to control the spatial temporality of the CRISPR-Cas12a system, and the constructed plasmid can theoretically target any target gene after replacing the crRNA region, and compared to the Cas9 system, the inserted fragment (crRNA) is shorter and more convenient.

[0360]

[0361]

[0362] The above are only some specific embodiments of the present invention. The known specific contents or common sense in the scheme are not described in detail here (including but not limited to abbreviations, abbreviations, and units commonly used in the art). It should be pointed out that the above embodiments do not limit the present invention in any way. For those skilled in the art, all technical solutions obtained by equivalent replacement or equivalent transformation fall within the protection scope of the present invention. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A method for constructing a gene editing vector regulated by a hot promoter, characterized in that: The method comprises the following steps: a) providing three plasmids, pAAV-U6-sgRNA-CMV-GFP, pSQT1665-Lbcpf1-NLS-3xHA, and pHSP70-MCS-P2A-EGFP, as backbones; b) Using pHSP70-MCS-P2A-EGFP as a template, PCR amplified a linear fragment containing the HSP70 promoter; Using pSQT1665-Lbcpf1-NLS-3xHA as a template, PCR was performed to amplify the insert containing the elements related to Lbcpf1 protein expression; c) homologously recombining the linear fragment obtained in step b) with the inserted fragment to construct an intermediate vector pHSP70-Lbcpf1; d) Using the intermediate vector pHSP70-Lbcpf1 and pAAV-U6-sgRNA-CMV-GFP as the main body, homologous recombination was performed to construct the hot start CRISPR vector pHSP70-Lbcpf1-U6; e) Using pHSP70-Lbcpf1-U6 as the main body, inserting the scaffold required by Lbcpf1 and the crRNA of the target gene target to construct the final vector pHSP70-Lbcpf1-2cr; f) Confirm the successful construction of the final vector through enzyme digestion identification and sequencing.

2. The method for constructing a gene editing vector regulated by a hot promoter according to claim 1, characterized in that: The construction method also includes the steps of transfecting the pHSP70-Lbcpf1-2cr vector into cells and regulating gene editing through heat induction.

3. The method for constructing a gene editing vector regulated by a hot promoter according to claim 1, characterized in that: The crRNA of the target gene target is replaced for any target gene to achieve multi-target gene editing.

4. The method for constructing a gene editing vector regulated by a hot promoter according to claim 1, characterized in that: The vector can specifically express Lbcpf1 protein and crRNA of the target gene under heat-induced conditions in tumor cells, thereby achieving editing of specific genes.

5. The method for constructing a gene editing vector regulated by a hot promoter according to claim 1, characterized in that: The vector can significantly down-regulate the expression of FASN gene in prostate cancer cell 22RV1 through heat induction.

6. The method for constructing a gene editing vector regulated by a hot promoter according to claim 1, characterized in that: The amplified plasmid strain used in the construction method is DH-5α, and the culture medium is LB liquid culture medium or LB plate culture medium.

7. The method for constructing a gene editing vector regulated by a hot promoter according to claim 1, characterized in that: The homologous recombination primer sequences include but are not limited to: Insert-Lbcpf1-fw, Insert-Lbcpf1-rev, pHSP-fw, pHSP-rev, pHSP-Lb-Line-fw, pHSP-Lb-Line-rev, U6-Inser t-fw, U6-Insert-rev, Scaffold-infu-fw, Scaffold-infu-rev, Du-cr-fw, Du-cr-rev, Du-seq-fw, Du-seq-rev.

8. The pHSP70-Lbcpf1-2cr plasmid vector constructed according to the method described in any one of claims 1 to 7.

9. Use of the pHSP70-Lbcpf1-2cr plasmid vector according to claim 8 in tumor gene editing.

10. The use according to claim 9, characterized in that: The application includes transfecting the vector into tumor cells and regulating the editing of specific genes by heat induction.

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