Lethal Endotoxin (ccdB) Based Counterselection Improved the Efficiency of Sequential Gene Editing in Escherichia coli

The CRISPR/Cas9 based technology has been used for sequential gene editing in E. coli . The plasmids carrying the sgRNA and/or Cas9 genes need to be cured after each round of editing. Curing of these plasmids, particularly the sgRNA plasmid, limits the efficiency of sequential gene editing. In this study, a lethal endotoxin ( ccd B) based counter-selection was established for improving the overall efficiency of sequential gene editing in E. coli . This approach was validated for sequential editing (deletion) of cst A and pps A genes. The experimental results showed that the transformation efficiency sgRNA plasmid (pTargetF- tcr -P _L - ccd B-N20) reached to 10 ⁸ -10 ⁹ cfu / µg _-DNA , resulting in a 90% of recombination rate for the target gene ( cst A and pps A). Upon completion of cst A gene editing, the sgRNA plasmid (pTargetF- tcr -P _L - ccd B-N20( cst A)) were effectively cured through ccd B based counterselection at 42°C, with a 43.75% efficiency. At the end of sequential editing of pps A gene, both Cas9 (25A) and sgRNA (pTargetF- tcr -P _L - ccd B-N20( pps A)) plasmids were cured simultaneously through the sac B and ccd B based counterselections by incubating the cells on LB-sucrose (5%) plate at 42°C, achieving a curing rate of 100% for Cas9 plasmid (25A), and 37.5% for sgRNA plasmid (pTargetF- tcr -P _L - ccd B-N20( pps A)). These results demonstrated that the endotoxin ( ccd B) based counterselection improved the transformation efficiency of sgRNA plasmid, the recombination rate of the editing target gene, the curing rate of sgRNA plasmid, and the overall efficiency of sequential gene editing.