Making point mutations in Escherichia coli BL21 genome using the CRISPR-Cas9 system

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated protein 9 (Cas9) system is an efficient and rapid tool for genome editing. However, its utilization in bacteria suffers challenges such as the risk of repeated recognition and cutting by Cas9. Here we established a two-step genome editing strategy using the Streptococcus pyogenes CRISPR-Cas9 system to achieve a clean mutation with only the target sites into the Escherichia coli genome. This strategy can avoid the risk of repeated cutting by guide RNA (gRNA)/Cas9 without altering the protospacer-adjacent motif or inserting additional silent mutations into the genome. The principles and approaches we developed in this study can be applied to modify coding and non-coding sequences in essential and non-essential genes and can also be used for precise genome editing in other microorganisms.