Development and application of a rapid all-in-one plasmid CRISPR-Cas9 system for iterative genome editing in Bacillus subtilis.
Microb Cell Fact
; 21(1): 173, 2022 Aug 23.
Article
em En
| MEDLINE
| ID: mdl-35999638
ABSTRACT
BACKGROUND:
Bacillus subtilis, an important industrial microorganism, is commonly used in the production of industrial enzymes. Genome modification is often necessary to improve the production performance of cell. The dual-plasmid CRISPR-Cas9 system suitable for iterative genome editing has been applied in Bacillus subtilis. However, it is limited by the selection of knockout genes, long editing cycle and instability.RESULTS:
To address these problems, we constructed an all-in-one plasmid CRISPR-Cas9 system, which was suitable for iterative genome editing of B. subtilis. The PEG4000-assisted monomer plasmid ligation (PAMPL) method greatly improved the transformation efficiency of B. subtilis SCK6. Self-targeting sgRNArep transcription was tightly controlled by rigorous promoter PacoR, which could induce the elimination of plasmids after genome editing and prepare for next round of genome editing. Our system achieved 100% efficiency for single gene deletions and point mutations, 96% efficiency for gene insertions, and at least 90% efficiency for plasmid curing. As a proof of concept, two extracellular protease genes epr and bpr were continuously knocked out using this system, and it only took 2.5 days to complete one round of genome editing. The engineering strain was used to express Douchi fibrinolytic enzyme DFE27, and its extracellular enzyme activity reached 159.5 FU/mL.CONCLUSIONS:
We developed and applied a rapid all-in-one plasmid CRISPR-Cas9 system for iterative genome editing in B. subtilis, which required only one plasmid transformation and curing, and accelerated the cycle of genome editing. To the best of our knowledge, this is the rapidest iterative genome editing system for B. subtilis. We hope that the system can be used to reconstruct the B. subtilis cell factory for the production of various biological molecules.Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Bacillus subtilis
/
Edição de Genes
Idioma:
En
Revista:
Microb Cell Fact
Assunto da revista:
BIOTECNOLOGIA
/
MICROBIOLOGIA
Ano de publicação:
2022
Tipo de documento:
Article