CN109825520B - A new method for gene assembly using improved CRISPR-Cpf1 - Google Patents

Abstract

The invention discloses a novel method for gene assembly by using improved CRISPR-Cpf1, which is a method for gene recombination cloning by using DNA gene editing enzyme FnCpf1 from Francisella tularemia NOVARIA NOVADATA subspecies U112 (gene sequence number: MF 193599). The method shortens the seed sequence of crRNA (RNA sequence transcribed by regularly clustered short palindromic repeats) from 23 nucleotides reported in literature to 18 nucleotides without affecting the FnCpf1 cleavage efficiency. Simultaneously selecting oligo (dN) ₆ The (oligonucleotide hexamer) is used as a cohesive end generated by cutting a target site, and the problem of non-uniform end after the FnCpf1 is cut by enzyme is solved, so that the assembly efficiency of the exogenous DNA fragment is obviously improved, and the assembly efficiency of the DNA fragment reaches about 85 percent. The gene-editing enzyme FnCpf1 may be replaced by other nucleic acid-editing enzymes Cpf1 such as AsCpf1 and LbCpf1.

Description

A new method for gene assembly using improved CRISPR-Cpf1

technical field

The invention belongs to the field of molecular biology or enzymology, in particular a new method for gene assembly using improved CRISPR-Cpf1.

Background technique

The CRISPR-Cas system (clustered regularly interspaced short palindromic repeats and associated protein complexes) is an acquired immune system widely found in bacteria and archaea. In 1987, scientists first discovered the CRISPR sequence. In 2010, researchers began to gradually elucidate the biological function of this sequence and apply it to gene editing. Based on this system, researchers have discovered a variety of nucleic acid editing enzymes and developed a large number of in vivo and in vitro gene editing technologies. In 2013, Zhang Feng et al first reported the application of CRISPR-Cas9 system in mammalian genome editing. In September 2015, the research team led by Zhang Feng found another nucleic acid editing enzyme Cpf1, which is also called Cas12a (regularly clustered interspaced short palindromic repeat sequence-related protein 12a), which belongs to class 25 Type CRISPR-Cas system, which is different from Cas9. Nucleic acid editing enzyme Cpf1, under the guidance of a single, 58-nucleotide crRNA (RNA sequence produced by the transcription of regularly clustered interspaced short palindromic repeats), utilizes the thymine-rich prespacer sequence adjacent gene The sequence recognizes and specifically cuts the complementary sequence of double-stranded DNA, and produces a 5' sticky end with 4-5 nucleotides protruding. Using this 5' sticky end cutting property, the 5' and 5' between the connected fragments can be designed The 5-base homology of the 3' produces a 4-5 nucleotide overhanging 5' complementary end for gene assembly after cleavage with the nucleic acid editing enzyme FnCpf1. Among them, the nucleic acid editing enzyme Cpf1 can be FnCpf1, AsCpf1 and LbCpf1 due to different sources.

In 2016, Zhao Guoping's research group at the Institute of Plant Physiology and Ecology, Chinese Academy of Sciences developed a C-Brick gene assembly method based on the nucleic acid editing enzyme FnCpf1 and bio-bricks, using the nucleic acid editing enzyme FnCpf1 instead of the traditional restriction endonuclease to cut nucleic acid fragments , but the nucleic acid editing enzyme FnCpf1 is inaccurate when cutting, and will produce uneven sticky ends of 5-8 bases, thereby reducing the ligation efficiency of the fragments. Therefore, the gene assembly efficiency in the C-Brick method can only reach about 20%. The recombination efficiency of this assembly method is low, and the probability of obtaining correct recombinants is small, and the sgRNA used in the CRISPR-Cpf1 method of the prior art is all sgRNA with a seed sequence of 24 nt, which is costly and more complicated to manipulate.

How to further improve the efficiency of reorganization, simplify operations, and reduce costs is an urgent problem to be solved.

references

[1] Li SY, Zhao GP, Wang J.C-Brick: A New Standard for Assembly of Biological Parts Using Cpf1.ACS Synth Biol.2016Dec16; 5(12):1383-1388.

[2] Li SY, Zhao GP, Wang J. Protocols for C-Brick DNA Standard Assembly Using Cpf1. J Vis Exp. 2017 Jun 15; (124).

[3]Lei C, Li SY, Liu JK, Zheng X, Zhao GP, Wang J.The CCTL(Cpf1-assistedCutting and Taq DNA ligase-assisted Ligation) method for efficient editing of large DNA constructs in vitro.Nucleic Acids Res.2017May 19;45(9):e74.

Contents of prior art 23nt crRNA

[4] Zetsche B, Gootenberg JS, Abudayyeh OO, et al. Cpf1 is a single RNA-guided endonuclease of a class 2CRISPR-Cas system. Cell. 2015; 163(3): 759-71

[5] Chen JS, Ma E, Harrington LB, Da Costa M, Tian X, Palefsky JM, Doudna JA. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity, Science. 2018Apr 27; 360(6387): 436-439.

Contents of the invention

The purpose of the present invention is to address the shortcomings of the existing Cpf1-based DNA assembly methods, and propose a new method for gene assembly using improved CRISPR-Cpf1, which simplifies operations, reduces costs, and achieves a recombination efficiency of 85%.

A new Cpf1-based cloning method using the programming enzyme Cpf1 derived from Francisella tularensis subsp. Uniform cleavage, resulting in oligo(dG) ₆ (deoxyguanine oligonucleotide hexamer) and 6nt oligo(dT) ₆ (deoxythymine oligonucleotide hexamer) at both ends Exogenous fragments, and oligo(dC) ₆ oligonucleotides (deoxycytosine oligonucleotide hexamers) with 6nt and oligo(dA) ₆ (deoxythymine oligonucleotide hexamers) with 6nt, respectively ) end of the vector backbone, followed by ligation with T ₄ DNA ligase, and the recombination efficiency after conventional transformation of Escherichia coli DH5α can reach more than 85%.

For realizing the purpose of the present invention, provide following technical scheme:

1. Construction, expression and purification of FnCpf1 nucleic acid editing enzyme

1) Construction of FnCpf1 expression vector: According to the cloning method mediated by T5 exonuclease, the gene fragment of the nucleic acid programming enzyme FnCpf1 and the expression vector pET23a were amplified by PCR. 8000 spectrophotometer was used to measure the concentration of nucleic acid, and the gene fragment and the carrier fragment were mixed according to the molar ratio of 3:1. After adding T5 exonuclease, they were reacted in the ice-water mixture for 5 minutes, and then the routine transformation of E. coli was carried out. Pick a single colony and put it into 3ml lysing broth liquid medium supplemented with ampicillin, incubate at 37°C for several hours, extract the plasmid DNA according to the alkaline lysis method, and then perform agarose gel electrophoresis detection, the size is correct and PCR identification contains The recombinant plasmid pet23a-FnCpf1 of the target fragment was sent to the company for sequencing identification. The expression vector pET23a, Cpf1 gene fragment and recombinant plasmid pet23a-FnCpf1 were detected by agarose gel electrophoresis.

The nucleic acid programming enzyme FnCpf1 is derived from Francisella tularensis novicida U112 strain (Francisella tularensis novicida subspecies U112, gene sequence number: MF193599, the coding sequence of the Cpf1 gene is 3903bp long, encoding 1301 amino acids.

2) Expression and identification of FnCpf1 nucleic acid editing enzyme in Escherichia coli: transform the sequenced correct recombinant plasmid pet23a-FnCpf1 into Escherichia coli BL21 (DE3) expression strain, pick a single colony and inoculate it into 100ml lysing broth supplemented with ampicillin for culture base, cultivated at 37°C until the OD ₆₀₀ was 0.6-0.8, added 1M IPTG (isopropyl-β-D-thiogalactoside) at 1% volume ratio, cultured overnight at 18°C with 200rpm shaking; collected the cells, and used Resuspend the bacteria in the lysis buffer, centrifuge after sonication, add 5× protein loading buffer to the centrifugation supernatant and the pellet, treat in a metal bath at 100°C for 10 minutes, and then perform SDS-PAGE dodecyl sulfonate Sodium acid-polyacrylamide gel electrophoresis detection.

3) Purification of FnCpf1 nucleic acid editing enzyme: Take a single colony containing the recombinant plasmid pet23a-FnCpf1 and inoculate it into 200 ml of lysing broth supplemented with ampicillin, and culture it at 37°C until the _OD600 reaches 0.6-0.8, adding 1% IPTG ( isopropyl-β-D-thiogalactoside), 18°C, induced overnight; collect E. coli cells, resuspend the cells with lysis buffer, then ultrasonically disrupt the cells, and mix the crude lysate with the affinity resin thoroughly , the target FnCpf1 protein is bound to nickel beads, and then the effluent obtained by elution with different concentrations of imidazole buffer and gradient elution is subjected to sodium dodecylsulfonate-polyacrylamide gel electrophoresis. Finally, the target nucleic acid editing enzyme FnCp1 is obtained.

2. For the synthesis of sgRNA (single-strand guide ribonucleic acid), two forward and reverse primers A and B were first synthesized by Shanghai Bioengineering Company (the primer sequence contains 41 primers including protective bases, T ₇ promoter and fixed region. bases and a 18-nucleotide seed sequence that completely matches the cutting point of the target sequence);

Primer A sequence:

5'-attaatacgactcactatagggaatttctactgttgtagatcatatgcatcaccatcacccttta-3'

Primer B sequence:

5'-taaagggtgatggtgatgcatatgatctacaacagtagaaattccctatagtgagtcgtattaat-3'

Then, the corresponding double-stranded DNA template was synthesized by primer annealing-polymerase chain amplification, and then the corresponding gRNA (guide ribonucleotide) was prepared with the in vitro _T7 transcription kit of Thermo Fisher. The reaction system (50 μl) of the transcription kit is shown in Table 1, reacted at 37°C for 2 hours, then added 15U of ribonuclease-free DNase I, and reacted for half an hour to remove the deoxyribonucleic acid template, and then use The Nanodrop 8000 spectrophotometer quantifies the RNA in the sample. The synthetic product is sgRNA (single-stranded guide ribonucleic acid) with a seed sequence of 18 nucleotides. The name was named sgRNA-18.

Table 1. gRNA (guide ribonucleotide) transcription system

3. Transform the target plasmid, select the plasmid pUC57-gfp (with green fluorescent marker protein) as the donor vector, and the two ends of the donor fragment GFP contain restriction sites for nucleic acid editing enzyme FnCpf1, and use conventional PCR methods to design primer pairs The gfp fragment was amplified to add a 6nt oligo(dG)6 (deoxyguanine oligonucleotide hexamer) and a 6nt oligo(dT)6 (deoxythymidine oligonucleotide hexamer), the transformed plasmid was named pUC57-gfp-6a6g;

At the same time, the plasmid pET23a-C2C1 was selected as the acceptor carrier, and the two ends of the acceptor vector pET23a respectively contained the restriction sites of the nucleic acid editing enzyme FnCpf1, and the primers were designed by conventional PCR method to amplify the acceptor vector pET23a, so that the acceptor vector pET23a Add 6nt oligo(dC)6 oligonucleotide (deoxycytosine oligonucleotide hexamer) and 6nt oligo(dA)6 (deoxythymus pyrimidine oligonucleotide hexamer); the above all use the T5 cloning method to obtain the recombinant acceptor plasmid and the carrier plasmid, and this operation makes the donor DNA fragment obtained after digesting the donor and acceptor plasmids with the nucleic acid editing enzyme FnCpf1 It is complementary to the cohesive end of the vector backbone, and the transformed plasmid is named pET23a-C2C1-6a6g.

4. The programming enzyme FnCpf1 purified in the first step and the second step seed sequence sgRNA-18 (single-stranded guide ribonucleic acid) form a complex, respectively, for the donor plasmid pUC57-gfp-6a6g and the acceptor plasmid pET23a-C2C1- 6a6g was digested at 45°C for 30 minutes, the reaction buffer was 50mM tris-hydrochloric acid, 40mM sodium chloride, and then the enzyme was inactivated at 80°C for 10 minutes.

FnCpf1 enzyme digestion reaction system

5. Use the conventional agarose gel electrophoresis method to recover the donor DNA fragment and the carrier skeleton after the reaction in step 4. 6. Mix the recovered donor DNA fragment and the carrier skeleton according to the molar ratio of 3:1, and pass through the T ₄ DNA Ligase ligation at 16°C for 4-5 hours.

7. The ligated product was transformed into Escherichia coli DH5α for expression. Escherichia coli DH5α competent and the linker were mixed according to the volume ratio of 1:100, ice-bathed for 30 minutes, heat-shocked at 42°C for 45 seconds, recovered at 37°C for 1 hour, coated with corresponding resistant plates, cultured at 37°C overnight, randomly selected Single colonies were identified for recombinants and sent to the company for sequencing. The recombinant was named pET23a-gfp. According to the statistics of the results of repeated experiments, the recombination rate can reach 85±0.3%.

The nucleic acid editing enzyme FnCpf1 in the present invention can be replaced by other nucleic acid editing enzymes Cpf1 such as AsCpf1 and LbCpf1.

In the present invention, we found that shortening the seed sequence in crRNA (the RNA sequence produced by the transcription of regularly clustered interspaced short palindromic repeat sequences) from the current 24 nucleotides to 18 does not affect the enzyme digestion of Cpf1 Efficiency, which significantly saves the synthesis cost and improves the synthesis efficiency, and since the nucleic acid editing enzyme FnCpf1 is at the 23rd position in the cleavage site of the complementary strand, it can be produced by transcribing a crRNA (regularly clustered interspaced short palindromic repeat sequence) RNA sequence) to cut multiple recognition sites, resulting in different cohesive ends. At the same time, in order to avoid the problem of uneven sticky ends after Cpf1 protein cleavage, the present invention proposes a method of adding 6 additional identical bases to the end of the target gene, so that the sticky ends produced after Cpf1 enzyme cleavage have better Consistency, thus significantly improving the subsequent connection efficiency.

Compared with the gene cloning method based on type 2 restriction endonuclease, the present invention has the advantages of longer sticky ends, no need to be restricted by the target fragment sequence, and more suitable for high-throughput cloning; compared with the prior Cpf1 in vitro cloning method, In the present invention, a crRNA (the RNA sequence produced by the transcription of regularly clustered interspaced short palindromic repeat sequences) can simultaneously pair with multiple sites of the DNA double strand and cut at a specific site, and produce different nucleotide hexamers Sticky ends, and no need to use special ligases and reaction conditions, significantly improving recombination efficiency and reproducibility.

The present invention has obvious advantages: we shorten the current 23-nucleotide sgRNA (guide ribonucleotide) to 18 nucleotides, which significantly saves the synthesis cost and improves the recombination efficiency.

Description of drawings

Figure 1 Schematic diagram of DNA fragment cloning assembly

Fig. 2 Agarose gel electrophoresis results of expression vector pET23a, FnCpf1 gene fragment and recombinant plasmid;

Among them: M: 1kb DNA molecular weight standard; lane 1: expression vector pET23a obtained by PCR amplification using pET23a-gfp plasmid as a template; lane 2: pET23a-gfp plasmid; lane 3: FnCpf1 gene amplified by PCR; lane 4: Recombinant plasmid pET23a-FnCpf1 containing FnCpf1 gene;

The electrophoresis results of the expression and purification of Fig. 3 FnCpf1 enzyme in Escherichia coli;

Among them, M: Pageruler prestained protein molecular weight standard; lane 1: whole bacteria; lane 2: supernatant of broken bacteria; lane 3: precipitate of broken bacteria;

Fig. 4 SDS-PAGE electrophoresis results of the effluent obtained by optimizing the elution of FnCpf1 enzyme;

Among them, M: Pageruler prestained protein molecular weight standard; lane 1: whole cells; lane 2: supernatant of broken bacteria; lane 3: flow-through; lanes 4-9: recombinant FnCpf1 purified by Ni beads and eluted with different concentrations of imidazole;

Fig. 5 schematic diagram of preparation of sgRNA;

Figure 6 Cloning plate diagram of a single-segment gene

Detailed ways

The present application is described in detail below in conjunction with the examples, but the present application is not limited to these examples.

Example 1 Construction of Nucleic Acid Editing Enzyme FnCpf1 Expression Vector

According to the T5 exonuclease-mediated cloning method, the nucleic acid programming enzyme FnCpf1 gene fragment and the expression vector pET23a were amplified by PCR. After the product was detected and recovered by agarose gel, the nucleic acid concentration was measured with a Nanodrop 8000 spectrometer. The fragment and the carrier fragment were mixed according to the molar ratio of 3:1, and after adding T5 exonuclease, they were reacted in ice-water mixture for 5 minutes, and then conventional transformation of Escherichia coli was carried out. Pick a single colony and put it into 3ml lysing broth liquid medium supplemented with ampicillin, incubate at 37°C for several hours, extract the plasmid DNA according to the alkaline lysis method, and then perform agarose gel electrophoresis detection, the size is correct and PCR identification contains The plasmid of the target fragment was sent to the company for sequencing identification. The agarose gel electrophoresis results of expression vector pET23a, FnCpf1 gene fragment and recombinant plasmid pET23a-FnCpf1 are shown in FIG. 2 .

The nucleic acid programming enzyme FnCpf1 is derived from Francisella tularensis novicida U112 strain (Francisella tularensis novicida subspecies U112, gene sequence number: MF193599, the coding sequence of the Cpf1 gene is 3903bp long, encoding 1301 amino acids.

Example 2 Expression and Identification of FnCpf1 Nucleic Acid Editing Enzyme in Escherichia coli

Transform the recombinant plasmid with correct sequencing into Escherichia coli BL21(DE3) expression strain, pick a single colony and inoculate it into 100ml lysing broth supplemented with ampicillin, cultivate it at 37°C until the _OD600 is 0.6-0.8, press 1% volume Add 1M IPTG (isopropyl-β-D-thiogalactopyranoside), culture overnight at 18°C with 200rpm shaking; collect the cells, resuspend the cells with lysis buffer, and centrifuge after sonication, and centrifuge upward 5× protein loading buffer was added to the supernatant and the precipitate respectively, treated in a metal bath at 100°C for 10 minutes, and then SDS-PAGE sodium dodecylsulfonate-polyacrylamide gel electrophoresis was performed. The results are shown in Figure 3.

Example 3 Nickel Bead Purification of Nucleic Acid Editing Enzyme FnCpf1

Get a single bacterium colony containing the recombinant plasmid and inoculate it into 200 ml of lysing broth supplemented with ampicillin, cultivate it at 37°C until the _OD600 reaches 0.6-0.8, add 1% IPTG (isopropyl-β-D-thiogalacto glucoside), 18°C, induced overnight; collect E. coli cells, resuspend the cells with lysis buffer, and then ultrasonically disrupt the cells, and mix the crude lysate with affinity resin to make the target protein bind to the nickel beads, and then use The recombinant protein was eluted with different concentrations of imidazole buffer, and the effluent obtained by gradient elution was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the results are shown in Figure 4.

The in vitro transcription of embodiment 4sgRNA

The sgRNA (single-stranded guide ribonucleic acid) in the present invention is relatively short. Firstly, the company synthesizes 41 bases including protective bases, _T7 promoter and fixed region, and 18 cores that completely match the cutting point of the target sequence. The primers A and B of the seed sequence of nucleotides, and then use the method of primer annealing-polymerase chain amplification to synthesize the corresponding double-stranded DNA template, and then prepare the corresponding gRNA with Thermo Fisher’s in vitro _T7 transcription kit (guide ribonucleotides). The reaction system (50 μl) of the transcription kit is shown in Table 1, reacted at 37°C for 2 hours, then added 15U of ribonuclease-free DNase I, and reacted for half an hour to remove the deoxyribonucleic acid template, and then use Nanodrop 8000 spectrophotometer was used to quantify the RNA in the sample, and the obtained product was named sgRNA-18.

Primer A sequence:

5'-attaatacgactcactatagggaatttctactgttgtagatcatatgcatcaccatcacccttta-3'

Primer B sequence:

5'-taaagggtgatggtgatgcatatgatctacaacagtagaaattccctatagtgagtcgtattaat-3'

Example 5 Target plasmid transformation

Transform the target plasmid, select the plasmid pUC57-gfp (with green fluorescent marker protein) as the donor vector, and the two ends of the donor fragment GFP contain restriction sites for the nucleic acid editing enzyme FnCpf1, and use conventional PCR methods to design primers for the gfp fragment Amplification was performed to add 6nt oligo(dG)6 (deoxyguanine oligonucleotide hexamer) and 6nt oligo( dT)6 (deoxythymine oligonucleotide hexamer), the transformed plasmid was named pUC57-gfp-6a6g;

At the same time, the plasmid pET23a-C2C1 was selected as the acceptor carrier, and the two ends of the acceptor vector pET23a respectively contained the restriction sites of the nucleic acid editing enzyme FnCpf1, and the primers were designed by conventional PCR method to amplify the acceptor vector pET23a, so that the acceptor vector pET23a Add 6nt oligo(dC)6 oligonucleotide (deoxycytosine oligonucleotide hexamer) and 6nt oligo(dA)6 (deoxythymus pyrimidine oligonucleotide hexamer); the above all use the T5 cloning method to obtain the recombinant acceptor plasmid and the carrier plasmid, and this operation makes the donor DNA fragment obtained after digesting the donor and acceptor plasmids with the nucleic acid editing enzyme FnCpf1 It is complementary to the cohesive end of the vector backbone, and the transformed plasmid is named pET23a-C2C1-6a6g. (see picture 1)

Obtaining of embodiment 6 restriction product

Using the Cpf1 enzyme purified in Example 3 and the sgRNA guide ribonucleic acid transcribed in Example 4 to work together to carry out enzyme digestion of the vectors pUC57-gfp-6a6g and pET23a-C2C1-6a6g obtained in Example 5, to obtain The GFP-encoding gene fragment and the pET23a vector at the cohesive ends of the polymer.

Assembly of embodiment 7 enzyme-cut fragments

Carry out agarose electrophoresis gel recovery of fragments and carriers, and mix the recovered carriers and fragments according to the fragment:carrier molar ratio = 3:1, wherein the carrier concentration is at least 40ng, and react with T ₄ DNA ligase at 16 degrees Celsius for 4-5h , and then transform Escherichia coli to be competent, coat a plate containing kalamycin, and culture overnight at 37°C. Since the inserted target fragment is the coding gene of green fluorescent protein, the preliminary judgment of the recombinant can be carried out by whether the colony emits green light under the blue light meter. The result shows that the colony that emits green light is the correct recombinant PET23a-gfp. The experiment was repeated for statistical results, and the data showed that the number of recombinants was 126±10, the number of non-recombinants was 19±2, and the recombination rate was 85±0.3% ( FIG. 6 ).

The above are only a few embodiments of the application, and do not limit the application in any form. Although the application is disclosed as above with preferred embodiments, it is not intended to limit the application. Any skilled person familiar with this field, Without departing from the scope of the technical solution of the present application, any changes or modifications made using the technical content disclosed above are equivalent to equivalent implementation cases, and all belong to the scope of the technical solution.

sequence listing

<110> Hubei University

<120> A new method for gene assembly using improved CRISPR-Cpf1

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tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

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ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240

attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300

tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360

tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt gcgatatcaa attccttgat 420

gtgaagagca ccatcatcat catcattctt cggatactta catatgcatc accatcacaa 480

aaaagccgtt cgctgtgtcg cacaattcgt ggacgtggga aaattgatcc cattctggtg 540

ccggatccct aagcccaatg tgtttttttc tagttggatt tgctcccccg ccgtcgttca 600

atgagaatgg ataagaggct cgtggggattg acgtgagggg gcagggatgg ctatatttct 660

gggagcgaac tccgggcgaa tacgaagcgc ttggatagct tcacttgtac agctcgtcca 720

tgccggtcga ctctagagga tcctctagat ttaagaagga gatatacata tgagtaaagg 780

agaagaactt ttcactggag ttgtcccaat tcttgttgaa ttagatggtg atgttaatgg 840

gcacaaattt tcagtcagtg gagagggtga aggtgatgca acatacggaa aacttaccct 900

taaatttatt tgcactactg gaaaactacc tgttccatgg ccaacacttg tcactacttt 960

cgcgtatggt cttcaatgct ttgcgagata cccagatcat atgaaacagc atgacttttt 1020

caagagtgcc atgcccgaag gttatgtaca ggaaagaacc atatttttca aagatgacgg 1080

gaactacaag acacgtgctg aagtcaagtt tgaaggtgat acccttgtta atagaatcga 1140

gttaaaaggt attgatttta aagaggatgg aaacattctt ggacacaaat tggaatacat 1200

ctataactca cacaatgtat acatcatggc agacaaacaa aagaatggaa tcaaagttaa 1260

cttcaaaatt agacacaaca ttgaagatgg aagcgttcaa ctagcagacc attatcaaca 1320

aaatactcca attggcgatg gccctgtcct tttaccagac aaccattacc tgtccacaca 1380

atctgccctt tcgaaagatc ccaacgaaaa gagagaccac atggtccttc ttgagtttgt 1440

aacagctgct gggattacac atggcatgga cgagctgtac aagtgagggg gggtgatggt 1500

gatgcatatg taaagtatcc tctagagcgc tcacaattgc tcttcttaat acgatatcga 1560

accagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca 1620

attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg 1680

agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg 1740

tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc 1800

tcatccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta 1860

tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag 1920

aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 1980

tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 2040

tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 2100

cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 2160

agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 2220

tccaagctgg gctgtgtgca cgaaccccccc gttcagcccg accgctgcgc cttatccggt 2280

aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact 2340

ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 2400

cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt 2460

accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt 2520

ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 2580

ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggatttg 2640

gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt 2700

aaatcaatct aaagtatata tgagtaaact tggtctgaca gttagaaaaa ctcatcgagc 2760

atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc 2820

cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg 2880

tatcggtctg cgattccgac tcgtccaaca tcaatacaac cctattaattt cccctcgtca 2940

aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc 3000

aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca 3060

aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat 3120

acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac 3180

actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat 3240

gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa 3300

tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct 3360

gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc 3420

ttcccataca atcgatagat tgtcgcacct gattgcccga catttatcgcg agccccatta 3480

tacccatata aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc 3540

cgttgaatat ggctcatact cttccttttt caatattatt gaagcattta tcagggttat 3600

tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg 3660

cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta 3720

acctataaaa ataggcgtat cacgaggccc tttcgtc 3757

<210> 4

<211> 4850

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atccggatat agttccctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa 60

ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt 120

tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt 180

cgacggagct cgaattcgga tccgcgaccc atttgctgtc caccagtcat gctagccata 240

tgtatatctc cttcttaaag ttaaacaaaa ttatttctag agggaaaccg ttgtggtctc 300

cctatagtga gtcgtattaa tttcgcggga tcgagatctc gggcagcgtt gggtcctggc 360

cacgggtgcg catgatcgtg ctcctgtcgt tgaggacccg gctaggctgg cggggttgcc 420

ttactggtta gcagaatgaa tcaccgatac gcgagcgaac gtgaagcgac tgctgctgca 480

aaacgtctgc gacctgagca acaacatgaa tggtcttcgg tttccgtgtt tcgtaaagtc 540

tggaaacgcg gaagtcagcg ccctgcacca ttatgttccg gatctgcatc gcaggatgct 600

gctggctacc ctttacatat gcatcaccat cacaaaaaaa cactgccgga tgcaaccgca 660

catccgattt ggacccgttt tgataaatta ggtggtaacc tgcaccagta cacctttctg 720

tttaatgaat ttggtgaacg tcgtcatgcc atccgttttc ataaactgct gaaagtggaa 780

aatggtgttg cacgtgaagt tgatgatgtt accgttccga ttagcatgag cgaacagctg 840

gataatctgc tgcctcgtga tccgaatgaa ccgattgcac tgtattttcg tgattatggt 900

gcagaacagc attttaccgg tgaatttggc ggtgcaaaaa ttcagtgtcg tcgtgatcag 960

ctggcccata tgcatcgtcg tcgtggtgca cgtgatgttt atctgaatgt tagcgttcgt 1020

gttcagagcc agagcgaagc acgtggcgaa cgtcgccctc cgtatgcagc cgtttttcgt 1080

ctggttggtg ataatcatcg tgcctttgtt cactttgata aactgagcga ttatctggcc 1140

gaacatcctg atgatggcaa actgggtagc gaaggtctgc tgagcggtct gcgtgttatg 1200

agcgttgatc tgggtctgcg taccagcgca agcattagcg tgtttcgtgttgcccgtaaa 1260

gatgaactga aaccgaatag caaaggtcgt gttccgtttt ttttcccgat taaaggcaat 1320

gataatctgg ttgcagtgca tgaacgtagt cagctgctga aattaccggg tgaaacagaa 1380

tcaaaagatc tgcgtgcgat tcgtgaagaa cgtcagcgta ccctgcgtca gctgcgcacc 1440

cagctggcat atctgcgtct gctggtgcgt tgtggtagtg aagatgttgg tcgtcgcgaa 1500

cgtagctggg caaaactgat tgagcagccg gttgatgcag ccaaccatat gacaccggat 1560

tggcgtgagg catttgaaaa tgaactgcag aaactgaaaa gcctgcatgg tatttgcagt 1620

gacaaagaat ggatggatgc cgtgtatgaa agcgttcgtc gtgtttggcg tcatatgggt 1680

aaacaggttc gtgattggcg caaagatgtt cgtagcggtg aacgccctaa aattcgtggt 1740

tatgcaaaag atgttgttgg tggcaatagg ggggggtgat ggtgatgcat atgtaagtgg 1800

aacacctaca tctgtattaa cgaagcgctg gcattgaccc tgagtgattt ttctctggtc 1860

ccgccgcatc cataccgcca gttgtttacc ctcacaacgt tccagtaacc gggcatgttc 1920

atcatcagta acccgtatcg tgagcatcct ctctcgtttc atcggtatca ttacccccat 1980

gaacagaaat cccccttaca cggaggcatc agtgaccaaa caggaaaaaa ccgcccttaa 2040

catggcccgc tttatcagaa gccagacatt aacgcttctg gagaaactca acgagctgga 2100

cgcggatgaa caggcagaca tctgtgaatc gcttcacgac cacgctgatg agctttaccg 2160

cagctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga 2220

gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc 2280

agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg atagcggagt 2340

gtatactggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatatgc 2400

ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc tcttccgctt 2460

cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact 2520

caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag 2580

caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata 2640

ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc 2700

cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg 2760

ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc 2820

tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg 2880

gctgtgtgca cgaaccccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc 2940

ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga 3000

ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg 3060

gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa 3120

aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg 3180

tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt 3240

ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggatttg gtcatgagat 3300

tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct 3360

aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta 3420

tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa 3480

ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac 3540

gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa 3600

gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag 3660

taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctgca ggcatcgtgg 3720

tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag 3780

ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg 3840

tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc 3900

ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat 3960

tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata 4020

ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa 4080

aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca 4140

actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc 4200

aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc 4260

tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg 4320

aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac 4380

ctgaaattgt aaacgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct 4440

cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg 4500

agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact 4560

ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac 4620

cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga 4680

gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga 4740

aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca 4800

ccaacacccgc cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca 4850

<210> 5

<211> 4722

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atccggatat agttccctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa 60

ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt 120

tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt 180

cgacggagct cgaattcgga tccgcgaccc atttgctgtc caccagtcat gctagccata 240

tgtatatctc cttcttaaag ttaaacaaaa ttatttctag agggaaaccg ttgtggtctc 300

cctatagtga gtcgtattaa tttcgcggga tcgagatctc gggcagcgtt gggtcctggc 360

cacgggtgcg catgatcgtg ctcctgtcgt tgaggacccg gctaggctgg cggggttgcc 420

ttactggtta gcagaatgaa tcaccgatac gcgagcgaac gtgaagcgac tgctgctgca 480

aaacgtctgc gacctgagca acaacatgaa tggtcttcgg tttccgtgtt tcgtaaagtc 540

tggaaacgcg gaagtcagcg ccctgcacca ttatgttccg gatctgcatc gcaggatgct 600

gctggctacc ctttacatat gcatcaccat cacaaaaaag ccgttcgctg tgtcgcacaa 660

ttcgtggacg tgggaaaatt gatccattc tggtgccgga tccctaagcc caatgtgttt 720

ttttctagtt ggatttgctc ccccgccgtc gttcaatgag aatggataag aggctcgtgg 780

gattgacgtg aggggggcagg gatggctata tttctgggag cgaactccgg gcgaatacga 840

agcgcttgga tagcttcact tgtacagctc gtccatgccg gtcgactcta gaggatcctc 900

tagatttaag aaggagatat acatatgagt aaaggagaag aacttttcac tggagttgtc 960

ccaattcttg ttgaattaga tggtgatgtt aatgggcaca aattttcagt cagtggagag 1020

ggtgaaggtg atgcaacata cggaaaactt acccttaaat ttatttgcac tactggaaaa 1080

ctacctgttc catggccaac acttgtcact actttcgcgt atggtcttca atgctttgcg 1140

agatacccag atcatatgaa acagcatgac tttttcaaga gtgccatgcc cgaaggttat 1200

gtacaggaaa gaaccatatt tttcaaagat gacgggaact acaagacacg tgctgaagtc 1260

aagtttgaag gtgataccct tgttaataga atcgagttaa aaggtattga ttttaaagag 1320

gatggaaaca ttcttggaca caaattggaa tacatctata actcacacaa tgtatacatc 1380

atggcagaca aacaaaagaa tggaatcaaa gttaacttca aaattagaca caacattgaa 1440

gatggaagcg ttcaactagc agaccattat caacaaaata ctccaattgg cgatggccct 1500

gtccttttac cagacaacca ttacctgtcc acacaatctg ccctttcgaa agatcccaac 1560

gaaaagagag accacatggt ccttcttgag tttgtaacag ctgctgggat tacacatggc 1620

atggacgagc tgtacaagtg agggggggtg atggtgatgc atatgtaagt ggaacaccta 1680

catctgtatt aacgaagcgc tggcattgac cctgagtgat ttttctctgg tcccgccgca 1740

tccataccgc cagttgttta ccctcacaac gttccagtaa ccgggcatgt tcatcatcag 1800

taacccgtat cgtgagcatc ctctctcgtt tcatcggtat cattaccccc atgaacagaa 1860

atccccctta cacggaggca tcagtgacca aacaggaaaa aaccgccctt aacatggccc 1920

gctttatcag aagccagaca ttaacgcttc tggagaaact caacgagctg gacgcggatg 1980

aacaggcaga catctgtgaa tcgcttcacg accacgctga tgagctttac cgcagctgcc 2040

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 2100

cagcttgtct gtaagcggat gccggggagca gacaagcccg tcagggcgcg tcagcgggtg 2160

ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtatactg 2220

gcttaactat gcggcatcag agcagattgt actgagagtg caccatatat gcggtgtgaa 2280

ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc 2340

actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg 2400

gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc 2460

cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc 2520

ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga 2580

ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc 2640

ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat 2700

agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg 2760

cacgaaccccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc 2820

aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga 2880

gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact 2940

agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt 3000

ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag 3060

cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg 3120

tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa 3180

aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata 3240

tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg 3300

atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata 3360

cgggaggggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg 3420

gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct 3480

gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt 3540

tcgccagtta atagtttgcg caacgttgtt gccattgctg caggcatcgt ggtgtcacgc 3600

tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga 3660

tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt 3720

aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc 3780

atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa 3840

tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca 3900

catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca 3960

aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct 4020

tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc 4080

gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa 4140

tattattgaa gcatttatca gggttatgt ctcatgagcg gatacatatt tgaatgtatt 4200

tagaaaaata aacaaatagg ggttccgcgc aatttcccc gaaaagtgcc acctgaaatt 4260

gtaaacgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt 4320

aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg 4380

ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc 4440

aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca 4500

agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga 4560

tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa 4620

ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc 4680

gccgcgctta atgcgccgct acagggcgcg tcccattcgc ca 4722

Claims (2)

1. A new method utilizing improved CRISPR-Cpf1 to carry out gene assembly, characterized in that the steps are: 1. Construction, expression and purification of nucleic acid editing enzyme FnCpf1 1) Construction of FnCpf1 expression vector: According to the cloning method mediated by T5 exonuclease, the gene fragment with the gene sequence number MF193599 and the expression vector pET23a were respectively amplified by PCR, and the products were detected and recovered by agarose gel. The final gene fragment and carrier fragment were mixed according to the molar ratio of 3:1, and T5 exonuclease was used, followed by conventional transformation culture of Escherichia coli, and the recombinant plasmid pet23a-FnCpf1 was extracted by alkaline lysis method and detected by agarose gel electrophoresis and sequencing identification; 2) Expression and purification of the nucleic acid editing enzyme FnCpf1 in Escherichia coli: Transform the recombinant plasmid pet23a-FnCpf1 with correct sequencing into the expression strain of Escherichia coli BL21(DE3) to induce expression culture, collect the Escherichia coli cells, and centrifuge after sonication. The crude lysate is fully mixed with the affinity resin to bind the target protein to the nickel beads, and the recombinant protein is eluted with imidazole buffer, detected by gel electrophoresis, and finally the target nucleic acid editing enzyme FnCp1 is obtained; 2. For the synthesis of single-stranded guiding ribonucleic acid sgRNA, two forward and reverse primers A and B were first synthesized by Shanghai Bioengineering Company; Primer A sequence: 5'-attaatacgactcactatagggaatttctactgttgtagatcatatgcatcaccatcacccttta-3' Primer B sequence: 5'-taaagggtgatggtgatgcatatgatctacaacagtagaaattccctatagtgagtcgtattaat-3' Then use the method of primer annealing-polymerase chain amplification to synthesize the corresponding double-stranded DNA template, and then use the in vitro _T7 transcription kit to transcribe and prepare the corresponding guide ribonucleotide gRNA, and use ribonuclease-free deoxyribonuclease 1 removes deoxyribonucleic acid template, obtains product and is the single strand guide ribonucleic acid sgRNA of 18 nucleotides in seed sequence, and name is called sgRNA-18; 3. Carry out target plasmid transformation, select the protein pUC57-gfp with green fluorescent marker as the donor vector, and the two ends of the donor fragment GFP contain restriction sites for the nucleic acid editing enzyme FnCpf1, and use conventional PCR methods to design primers for the gfp fragment Amplification was performed to add a 6nt deoxyguanine oligonucleotide hexamer oligo(dG)6 and a 6nt deoxythymine oligo between the fragment end of the donor fragment gfp and the cleavage site of the FnCpf1 nucleic acid editing enzyme Nucleotide hexamer oligo(dT)6, the transformed plasmid was named pUC57-gfp-6a6g; At the same time, the plasmid pET23a-C2C1 was selected as the acceptor carrier, and the two ends of the acceptor vector pET23a respectively contained the restriction sites of the nucleic acid editing enzyme FnCpf1, and the primers were designed by conventional PCR method to amplify the acceptor vector pET23a, so that the acceptor vector pET23a Add 6nt deoxycytosine oligonucleotide hexamer oligo(dC)6 oligonucleotide and 6nt deoxythymine oligonucleotide hexamer between the end of the vector and the restriction site of nucleic acid editing enzyme FnCpf1 oligo(dA)6; above all use the T5 cloning method to obtain the recombinant acceptor plasmid and carrier plasmid, this operation makes the donor DNA fragment and the carrier backbone obtained after digesting the donor and acceptor plasmid with the nucleic acid editing enzyme FnCpf1 The cohesive ends are complementary, and the transformed plasmid is named pET23a-C2C1-6a6g; 4. Composite the nucleic acid editing enzyme FnCpf1 purified in the first step and the seed sequence sgRNA-18 in the second step, and digest the donor plasmid pUC57-gfp-6a6g and the acceptor plasmid pET23a-C2C1-6a6g at 45°C 30 minutes, the reaction buffer is 50mM tris-hydrochloric acid, 40mM sodium chloride, then inactivate the enzyme at 80°C for 10 minutes; 5. Using conventional agarose gel electrophoresis method, recover the donor DNA fragment and carrier skeleton after the reaction in step 4 respectively; 6. Mix the recovered gfp-6a6g donor DNA fragment with the pET23a-6a6g vector backbone at a molar ratio of 3:1, and ligate with T ₄ DNA ligase at 16°C for 4-5 hours to carry out gene assembly; 7. The ligated product was transformed into Escherichia coli DH5α for expression culture; Escherichia coli DH5α competent and the linker were mixed at a volume ratio of 1:100, ice bathed for 30 minutes, heat-shocked at 42°C for 45 seconds, and recovered at 37°C for 1 hour. Spread the corresponding resistant plate, culture overnight at 37°C, randomly pick a single colony for recombinant identification, and send it to the company for sequencing. The name of the recombinant is pET23a-gfp, and the results of repeated experiments are counted. 85±0.3%. 2. A new method for gene assembly using improved CRISPR-Cpf1 according to claim 1, characterized in that the nucleic acid editing enzyme is FnCpf1, or AsCpf1, or LbCpf1.

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