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A Crispr Cpf1-based multiple gene editing and expression regulation system in Bacillus subtilis

A Bacillus subtilis, expression regulation technology, applied in the field of genetic engineering, can solve the problems of increasing the complexity of operations, reducing the stability of plasmids, increasing the difficulty of plasmid construction, etc.

Active Publication Date: 2021-11-02
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there have been many studies on the construction and application of gene editing and transcriptional regulation systems based on CRISPR / Cas9 in Bacillus subtilis, but there are no reports on gene editing and expression regulation using the CRISPR / Cpf1 system
When editing or regulating multiple sites, the CRISPR / Cas9-based gene editing system needs to express multiple sgRNAs, which increases the complexity of the operation; There are many repetitive sequences, which increases the difficulty of plasmid construction and reduces the stability of the plasmid

Method used

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  • A Crispr Cpf1-based multiple gene editing and expression regulation system in Bacillus subtilis
  • A Crispr Cpf1-based multiple gene editing and expression regulation system in Bacillus subtilis
  • A Crispr Cpf1-based multiple gene editing and expression regulation system in Bacillus subtilis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Construction of Cpf1 expression vector pHT-XCR6

[0044] Firstly, the plasmid pHT01 was linearized by PCR using primers with sequences such as 5'-ctgcagaacgctcggttgccgccgggcgttttttcgtcattcctctagagtcgacgtcc-3' and 5'-cgctcccttttccgttag-3' and the template DNA was digested with endonuclease DpnI; the plasmid pLCx-dCas9 was used as a template to amplify Xylose promoter PxylA; using PY001 as a template (from literature: Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, MakarovaKS, Essletzbichler P, Volz SE, Joung J, Van Der Oost J, Regev A, Koonin E V. Amplification FnCpf1 gene; finally, PxylA and FnCpf1 can be connected to the vector pHT01 to obtain the vector pHT-XC through the seamless cloning kit.

[0045] Then use primers with sequences such as 5'-ctaatgtcggattcctctaatcctctagagtcgacgtcc-3' and 5'-atgacgaaaaaacgcccg-3' to linearize the vector pHT-XC by PCR and digest the template DNA with endonuclease DpnI; amplify with plasmid pHT00 as a template IPTG-...

Embodiment 2

[0046] Example 2: Construction of crRNA array expression vector pcrF11

[0047] First, the synthetic DNA was used as a template to amplify the DNA fragment composed of the crRNA insertion region and the Bacillus subtilis replicon repF; then the kanamycin resistance gene was amplified by using the plasmid pUC57-Kan as a template; The DNA sequence was used as a template to amplify the fragment composed of the Bacillus subtilis single-stranded replicon ssoU and the promoter Pbs, and finally the plasmid pP43-egfp was used as a template to amplify the E. coli replicon ori; finally, the above-mentioned The four fragments were spliced ​​to obtain the vector pcrF11 (sequence shown in SEQ ID NO: 2).

Embodiment 3

[0048] Example 3: Multiple gene editing in Bacillus subtilis based on CRISPR / Cpf1 system

[0049] During gene editing, pHT-XCR6 was first transformed into Bacillus subtilis, and the required crRNA array and homology arms were designed according to the needs of genome editing and added to the plasmid pcrF11 respectively. Here, the main six extracellular protease genes aprE, epr, nprE, bpr, mpr and nprB in Bacillus subtilis were selected as target genes for verification. It has been verified that if the homology arm is integrated into pcrF11, the complete deletion of two genes, the partial base mutation of six sites, or the insertion of a gene can be achieved at one time; and if the homology arm fragment is not inserted into pcrF11 , but when co-transforming with pcrF11 inserted with crRNA, it may be possible that due to the reduced transformation efficiency, only one gene can only be completely deleted at one time, or two sites can be partially mutated, and one gene can also be...

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Abstract

The invention discloses a CRISPR Cpf1-based multi-gene editing and expression regulation system of Bacillus subtilis. Through the constructed Cpf1 expression vector pHT‑XCR6 and the crRNA array expression vector pcrF11, the present invention can complete the complete knockout of two genes, the base modification of six genes and the knock-in of one gene at one time. The vector pHT‑XCR6 contains NgAgo protein to promote recA-mediated homologous recombination. At the same time, the vectors pLCg6-dCpf1-remA (used for the integrated expression of the fusion protein of DNase inactivated Cpf1 mutant dCpf1 and transcription activator remA on the Bacillus subtilis genome) and pcra3 (used for crRNA array in Bacillus subtilis integrated expression on the Bacillus genome), which can be used for simultaneous transcriptional repression and activation of different genes. The present invention also establishes a cost-effective crRNA array assembly method called SOMACA (Synthetic Oligos Mediated Assembly of crRNA Array), which can insert the required crRNA array into the carrier pcrF11 or pcra3 by synthesizing short single-stranded DNA (<60nt) above, used to guide Cpf1 or dCpf1‑remA for gene editing and expression regulation.

Description

technical field [0001] The invention relates to a CRISPR Cpf1-based multi-gene editing and expression regulation system of Bacillus subtilis, which belongs to the technical field of genetic engineering. Background technique [0002] Bacillus subtilis (Bacillus subtilis) is widely used as a production host for food enzyme preparations and important nutritional chemicals, and its products are certified by the FDA as "generally regarded as safe" (GRAS) safety level; in addition, its It is also a Gram-positive model microorganism and is often used in the study of microbial mechanisms. In order to prevent the invasion of phages, microorganisms have evolved many defense systems, and the CRISPR-Cas (clustered regularly interspacedshort palindromic repeats and CRISPR-associated proteins) system is one of the more common acquired immune systems. Taking the most widely studied CRISPR / Cas9 system of Streptococcus pyogenes as an example, its mechanism of action is as follows: when a ne...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/75C12N15/90
CPCC12N15/113C12N15/75C12N15/902C12N2310/20
Inventor 刘龙武耀康堵国成李江华陈坚
Owner JIANGNAN UNIV
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