Journal
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
Volume 23, Issue 9, Pages -Publisher
MDPI
DOI: 10.3390/ijms23094853
Keywords
CRISPR-Cas9; Bacillus subtilis; genome engineering; long fragment deletion; metabolic engineering
Funding
- National Key R&D Program of China [2019YFA0904104, 2021YFC2100500]
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Bacillus subtilis is a versatile microbial cell factory that can produce valuable proteins and value-added chemicals. Researchers have developed an efficient CRISPR-Cas9 method for large-scale and scarless genome engineering in Bacillus subtilis. They were able to delete DNA fragments up to 134.3 kb, 3.5 times longer than previous reports, with a positivity rate of 100%. The study also investigated the effects of using a heterologous NHEJ system, linear donor DNA, and various donor DNA lengths on the engineering efficiencies.
Bacillus subtilis is a versatile microbial cell factory that can produce valuable proteins and value-added chemicals. Long fragment editing techniques are of great importance for accelerating bacterial genome engineering to obtain desirable and genetically stable host strains. Herein, we develop an efficient CRISPR-Cas9 method for large-scale and scarless genome engineering in the Bacillus subtilis genome, which can delete up to 134.3 kb DNA fragments, 3.5 times as long as the previous report, with a positivity rate of 100%. The effects of using a heterologous NHEJ system, linear donor DNA, and various donor DNA length on the engineering efficiencies were also investigated. The CRISPR-Cas9 method was then utilized for Bacillus subtilis genome simplification and construction of a series of individual and cumulative deletion mutants, which are further screened for overproducer of isobutanol, a new generation biofuel. These results suggest that the method is a powerful genome engineering tool for constructing and screening engineered host strains with enhanced capabilities, highlighting the potential for synthetic biology and metabolic engineering.
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