Bacillus subtilis polygene editing and expression adjusting and controlling system based on CRISPR Cpf1

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

Active Publication Date: 2020-04-03
JIANGNAN UNIV
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  • 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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  • Bacillus subtilis polygene editing and expression adjusting and controlling system based on CRISPR Cpf1
  • Bacillus subtilis polygene editing and expression adjusting and controlling system based on CRISPR Cpf1
  • Bacillus subtilis polygene editing and expression adjusting and controlling system based on CRISPR Cpf1

Examples

Experimental program
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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 bacillus subtilis polygene editing and expression adjusting and controlling system based on CRISPR Cpf1. Through a constructed Cpf1 expression vector pHT-XCR6 and a constructed crRNA array expression vector pcrF11, complete knockout of 2 genes, basic group modification of 6 genes and knock-in of 1 gene can be completed at one time. The vector pHT-XCR6 contains NgAgo protein, and is used for promoting recA mediated homologous recombination. Besides, a vector pLCg6-dCpf1-remA (used for integral expression of a Cpf1 mutant dCpf1 inactivated by deoxyribonuclease and fusion protein of an activating transcription factor remA on a bacillus subtilis genome) and a vector pcra3 (used for integral expression of a crRNA array on the bacillus subtilis genome) are constructed,and can be used for performing transcription inhibition and activation on different genes. The invention further establishes an economic efficient crRNA array assembling method of which the name is SOMACA (Synthetic Oligos Mediated Assembly of crRNA Array), and through synthesizing short-single-strand DNA (60nt ), a needed crRNA array is inserted in the vector pcrF11 or pcra3 and is used for guiding Cpf1 or dCpf1-remA for gene editing and expression adjustment and control.

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 Applications(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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