CRISPR/Cas9 nuclease cleavage enables marker-free genome editing in Escherichia coli: A sequential study

CRISPR/Cas9 is a new and powerful genome editing tool in the recent years. Although the CRISPR/Cas9 system has been demonstrated with characterizations of high efficiency and precise double-strand breaking in chromosomes, the accurate manipulation of this system in prokaryotes still remains difficult. The Escherichia coli, most important genetic strain, can be more easily manipulated with its chromosome by the assistance of lambda Red recombinase that relies on the insertion of antibiotic resistance for screening or selection. The aim of this study is to explore the possibility of using CRISPR/Cas9 only for the genome editing in E. coli. The results showed that the inappropriate function of Cas9 would cleave on the pCRISPR with sgRNA, and causing the SOS response. However, by combining the CRISPR/Cas9 system with lambda Red recombinase, the performance can be controlled by transforming pCRISPR with a dual-spacer and followed up by transforming pCas9 with donor DNA. This sequential strategy can allow marker-free in genomic editing of E. coli. Moreover, the efficiency of genomic editing is found over 90% at the optimal conditions, which are using a larger length (i.e., > 3000 bp) of donor DNA at 500 ng in CRISPR/Cas9 system with lambda Red assistance. (C) 2016 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.