Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system-based bacillus licheniformis genome editing vector and preparation method thereof

A Bacillus licheniformis genome editing technology, applied in the genetic field, can solve problems such as the inability of Bacillus licheniformis to develop

Active Publication Date: 2017-11-28
JIANGNAN UNIV
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Problems solved by technology

[0005] However, the current Bacillus licheniformis genome editing system based on the CRISPR-Cas9 system has not yet been constructed, making it impossible for this advanced technology to be used in Bacillus licheniformis, an important industrial microorganism.

Method used

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  • Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system-based bacillus licheniformis genome editing vector and preparation method thereof
  • Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system-based bacillus licheniformis genome editing vector and preparation method thereof
  • Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system-based bacillus licheniformis genome editing vector and preparation method thereof

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Embodiment 1

[0037] A preparation method of a Bacillus licheniformis genome editing vector based on the CRISPR-Cas9 system, the preparation method comprising the steps of:

[0038] (1) Design and synthesize Cas9 protein expression cassette (SEQ ID NO.1), sgRNA scaffold expression cassette (SEQ ID NO.2) and multiple sgRNA fragments (SEQ ID NO.3, SEQ ID NO. ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8);

[0039] Genomic DNA of Bacillus licheniformis 9945a was extracted and amplified using pfu DNA polymerase and primers P1 (5'-GCCAAGCTTTTACGACCTTTTATGATTTAG-3') and P2 (5'-CGAAAGCTTATTTGCAACCGAGCTGTCGC-3') to obtain the partial open reading frame truncamyL of the amylase coding gene;

[0040] Reaction conditions: Pre-denaturation at 94°C for 5 min, then enter the next cycle: denaturation at 95°C for 10 s, annealing at 54°C for 10 s, extension at 72°C for 1 min, 30 cycles; extension at 72°C for 10 min, incubation at 4°C.

[0041] After purification, the amplified product was T-A...

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Abstract

The invention discloses a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system-based bacillus licheniformis genome editing vector. A preparation method of the bacillus licheniformis genome editing vector comprises the steps of integrating a Cas9 protein expression cassette and a small guide ribonucleic acid (sgRNA) scaffold expression cassette into a starting vector pHY xyl to obtain pHY-Cas9-sgRNA scaffold; then, integrating any one of multiple sgRNA fragments, which are designed and synthesized by taking amyL as a target site, into the pHY-Cas9-sgRNA scaffold to obtain the bacillus licheniformis genome editing vector. The CRISPR-Cas9 system-based bacillus licheniformis genome editing vector can greatly improve the current situation that at present, the gene editing of bacillus licheniformis is difficult, and obviously increases the gene editing efficiency.

Description

technical field [0001] The invention relates to the field of gene technology, in particular to a Bacillus licheniformis genome editing vector based on a CRISPR-Cas9 system inserted into a specific sgRNA fragment. Background technique [0002] Bacillus licheniformis is a kind of Gram-positive bacillus, which has only a single layer of cell membrane, and can generate endospores under extremely harsh conditions such as dryness and lack of nutrition, and resume growth again when the conditions are suitable. Bacillus licheniformis is a widely used industrial microorganism. It is not only the main producer of bulk industrial enzyme preparations - amylase and protease; at the same time, due to its excellent secretion ability, simple fermentation conditions and food safety characteristics, it is also used as the expression host of foreign genes to produce a variety of Enzymes for food. With the complete analysis of the genome of its model strain ATCC14580, people's understanding o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/75C12N15/66C12N1/21C12R1/10
CPCC12N15/75C12N2800/22C12N2800/80C12N2810/10C12N2830/34
Inventor 石贵阳李由然张梁丁重阳顾正华
Owner JIANGNAN UNIV
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