All-in-one Plasmid CRISPR-Cas9 System Allows Rapid Genome Editing of B. subtilis

The CRISPR-Cas9 system has previously been found suitable for Bacillus subtilis, a microorganism commonly used in industrial enzyme production, but is limited by the selection of knockout genes, long editing cycle, and instability. Scientists have overcome these with the construction of an all-in-one plasmid CRISPR-Cas9 system which significantly improved the bacterium's transformation efficiency.

The new system achieved 100% efficiency for single gene deletions and point mutations, 96% efficiency for gene insertions, and at least 90% efficiency for plasmid curing. The system also completed one round of genome editing in just 2.5 days, continuously knocking out two extracellular protease genes. The engineering strain was also used to express Douchi fibrinolytic enzyme DFE27, and its extracellular enzyme activity reached 159.5 FU/mL

This rapid all-in-one plasmid CRISPR-Cas9 system, which only requires one plasmid transformation and curing, is the fastest iterative genome editing system for B. subtilis to the best of the developers' knowledge.

Read the full paper in Microbial Cell Factories.