Construction of CRISPR-Cpf1-based efficient gene editing system

A gene editing and gene technology, applied in the field of high-efficiency gene editing system construction, can solve the problems that the efficiency of gene knock-in and large fragment deletion has not been verified, there is no strategy for multi-gene deletion, and the deletion efficiency needs to be improved. The effect of efficient knockout efficiency

Pending Publication Date: 2022-03-29
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the deletion efficiency of this system needs to be improved, and the gene knock-in and deletion efficiency of large fragments have not been verified
More importantly, the system also does not provide a strategy for multiple gene deletion

Method used

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  • Construction of CRISPR-Cpf1-based efficient gene editing system
  • Construction of CRISPR-Cpf1-based efficient gene editing system
  • Construction of CRISPR-Cpf1-based efficient gene editing system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Construction of CRISPR-Cpf1 system and its application in single gene knockout

[0034] Select the Cpf1 gene (nucleotide sequence as shown in SEQ ID NO.1) from F.novicida, the Cpf1 gene is cloned to the downstream of the xylose promoter of the Bacillus subtilis integration vector pAX01 (the recombinant vector is integrated into the Bacillus subtilis 168 genome lacA site), making it regulated by xylose.

[0035]In order to fully express crRNA, the high-copy vector pB-Pveg-sfGFP (pBSG03) was selected as the expression vector of crRNA, and the crRNA targeting sacA, ligDV and pps genes were respectively cloned into pB-Pveg-sfGFP and replaced The sfGFP gene was constructed to obtain pB-Pveg-crRNA, forming pB-Pveg-sacAcrRNA, pB-Pveg-ligDVcrRNA and pB-Pveg-ppscrRNA.

[0036] Three different recombinant plasmids were transformed into Bacillus subtilis in which the Cpf1 gene was integrated at the lacA site of the genome ( figure 1 ), relevant primers are provided (s...

Embodiment 2

[0048] Example 2: Application of CRISPR-Cpf1 system in single gene knock-in

[0049] In order to reflect the knock-in efficiency of this system, the sfGFP gene was knocked into the sacA locus. The crRNA design of sacA is the same as that of sacA knockout. The sfGFP gene was connected to pB-Pveg-sacAcrRNA by seamless cloning, and the primers required for the connection were as follows (Table 3), and the recombinant plasmid pB-Pveg-sacAcrRNA-sfGFP was constructed. The recombinant Bacillus subtilis obtained by transforming the recombinant plasmid pB-Pveg-sacAcrRNA-sfGFP into the lacA site of the genome and integrating the Cpf1 gene in Bacillus subtilis was cultured in LB medium at 37°C and 200rpm at OD 600 After reaching about 0.4, xylose with a final concentration of 1% (1g / 100mL) was added to induce the expression of Cpf1 for 12 hours, and whether the target gene was knocked in was judged by colony PCR.

[0050] Through verification, the knock-in efficiency of sfGFP is as hig...

Embodiment 3

[0053] Example 3: Application of CRISPR-Cpf1 system in multiple gene knockout

[0054] Two genes, sacA and aprE, were selected as target genes and deleted at the same time. The primers for constructing crRNA are shown in Table 2. The crRNA expression cassette of aprE was cloned into pB-Pveg-sacAcrRNA to form pB-sacA-aprEKO. Then the plasmid was transformed into the Bacillus subtilis with Cpf1 integrated in the genome, and the obtained recombinant Bacillus subtilis was induced by colony PCR in LB medium at 37°C, 200rpm, and 1% xylose (1g / 100mL) for 20h. Verify the knockout efficiency of double genes. The results showed that the double gene knockout efficiency of integrated CRISPR-Cpf1 was as high as 58.3%.

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Abstract

The invention discloses construction of an efficient gene editing system based on CRISPR-Cpf1, and belongs to the technical field of gene engineering. The method comprises the following steps: firstly, representing a Cas protein Cpf1 from F.novicida, and integrating FnCpf1 to a lacA site of a bacillus subtilis genome; the crRNA targeting different genes is placed at the downstream of a strong constitutive promoter Pveg from bacillus subtilis and is used for high-intensity expression of the corresponding crRNA. By selecting crRNA of different target genes, the genome editing method is verified. A bacterial colony PCR (Polymerase Chain Reaction) result shows that the sacA gene, the ligDV gene and the pps gene cluster can be efficiently knocked out. Meanwhile, in order to verify the knock-in efficiency of the system, the exogenous gene sfGFP is efficiently knocked out to the sacA site. In order to further verify the polygene knockout efficiency of the system, sacA and aprE are selected as targets, and a bacterial colony PCR result shows high knockout efficiency.

Description

technical field [0001] The invention relates to the construction of a high-efficiency gene editing system based on CRISPR-Cpf1, belonging to the technical field of genetic engineering. Background technique [0002] Bacillus subtilis is a Gram-positive strain widely used in exogenous protein expression, and it is also the best chassis microorganism for basic research, so it has a wide range of applications in the fields of metabolic engineering and synthetic biology. Currently, Cre / loxP, a tool widely used for genome editing, has been established in Bacillus subtilis. However, such editing tools belong to the introduction of exogenous resistance genes as a sign of positive selection, which does not meet the requirements of the food industry. Although it has been possible to eliminate the resistance gene by introducing flippase using this system, this operation process is cumbersome. Therefore, constructing an efficient and portable genome editing tool is of great significan...

Claims

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Application Information

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IPC IPC(8): C12N15/75C12N15/55C12N1/21C12R1/125
CPCC12N15/75C12N9/22
Inventor 周哲敏崔文璟郝文亮韩来闯程中一刘中美周丽郭军玲叶乘锋
Owner JIANGNAN UNIV
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