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Efficient Genome Editing in Bacillus licheniformis Mediated by a Conditional CRISPR/Cas9 System.
Youran Li1,2,3, Hanrong Wang1,2,3, Liang Zhang1,2,3
1Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
Microorganisms
|May 21, 2020
Summary
Researchers developed a conditional CRISPR-Cas9 system for Bacillus licheniformis, significantly improving genetic manipulation efficiency. This breakthrough enhances its use as an industrial workhorse by overcoming low transformation rates.
Area of Science:
- Microbiology
- Genetic Engineering
- Industrial Biotechnology
Background:
- Bacillus licheniformis is a key industrial microorganism for enzyme and chemical production.
- Genetic manipulation of B. licheniformis is challenging due to extremely low transformation efficiency, limiting its applications.
Purpose of the Study:
- To develop an efficient genetic manipulation tool for Bacillus licheniformis.
- To overcome the limitations of low transformation efficiency in B. licheniformis.
Main Methods:
- Developed an all-in-one conditional clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 plasmid with a xylose-inducible promoter for B. licheniformis.
- Compared the conditional system with a constitutive overexpression system.
- Assessed genome editing efficiency targeting the α-amylase gene (amyL) using different protospacer-adjacent motif (PAM) sequences.
- Investigated the effect of temperature on CRISPR system function.
Main Results:
- The conditional CRISPR system significantly improved transformation efficiency from <0.1 cfu/μg to 2.42 cfu/μg DNA.
- A constitutive overexpression system caused significant growth retardation in bacterial cells.
- Genome editing efficiencies for amyL disruption reached up to 70.9%.
- Optimal performance of the CRISPR system was observed at 20 °C, with a maximum success rate of 97%.
Conclusions:
- The conditional CRISPR/Cas9 system with a xylose-inducible promoter greatly enhances genome editing success in B. licheniformis.
- This technology facilitates genetic manipulation, improving the utility of B. licheniformis as an industrial microorganism.
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