Abstract
A miniature CRISPR-Cas12f has been demonstrated to serve as an effective genome editing tool in gram negative bacteria as well as human cells. Here, we developed an alternative method to edit the genome of Bacillus anthracis based on the AsCas12f1 nuclease from Acidibacillus sulfuroxidans. When the htrA gene on the chromosome and the lef gene on the plasmid pXO1 were selected as targets, the CRISPR-AsCas12f1 system showed very high efficiency (100%). At the same time, a high efficiency was observed for large-fragment deletion. Our results also indicated that the length of the homologous arms of the donor DNA had a close relationship with the editing efficiency. Furthermore, a two-plasmid CRISPR-AsCas12f1 system was also constructed and combined with the endonuclease I-SceI for potential multi-gene modification. This represents a novel tool for mutant strain construction and gene function analyses in B. anthracis and other Bacillus cereus group bacteria.
Highlights
Clustered regularly interspaced short palindromic repeats (CRISPRs) are part of the bacterial immune system that defends against invading viruses (Westra et al, 2014)
To explore the feasibility of CRISPR-Cas12f system in B. anthracis, the htrA gene on the B. anthracis chromosome and the lef gene on the plasmid pXO1 were selected as targets
The htrA gene on the B. anthracis chromosome and the lef gene on the plasmid pXO1 were selected as targets, and alternative mutants were detected by colony PCR
Summary
Clustered regularly interspaced short palindromic repeats (CRISPRs) are part of the bacterial immune system that defends against invading viruses (Westra et al, 2014) They are made up of repeating sequences of genetic code that are interrupted by pieces of genetic code from previous invaders, which allows a cell to detect and destroy returning invaders. The miniature CRISPR effectors, such as CasΦ, Cas, and Cas, have been developed into functional genome editors (Aquino-Jarquin, 2019; Savage, 2019; Pausch et al, 2020; Awan et al, 2021) Of these Cas nucleases, Cas12f has been shown to be superior as an editing system due to its small molecular weight and ease of cellular delivery (Awan et al, 2021). Cas12f has been demonstrated to serve as an effective genome editing tool in bacteria as well as human cells
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