Deleting essential genes from the genome is challenging due to their functional importance. Existing editing methods have drawbacks such as low efficiency and non-marker knockout. In this study, we present an efficient and traceless CRISPR/Cas9-based knockout method for essential genes in the Escherichia coli genome using metK disruption as an example. Firstly, we constructed plasmid pHL3 by cloning the metK gene into plasmid pTarget-metK. Through site-specific mutation, we made the metK gene synonymous, thereby removing the corresponding PAM sequence. Subsequently, we created a complement plasmid containing the same metK sequence as pHL3. The deletion of essential genes in the genome, in a highly efficient and marker manner, can be achieved by sequentially transforming pCas, pHL3, and the complement plasmid. The efficiency of metK deletion by this method can reach 100%. To investigate the impact of changes in the properties of proteins encoded by essential genes on host metabolism, we explored the replacement efficiency of the complement plasmid based on plasmid incompatibility and the Flp/FRT system. The Flp/FRT system proved capable of efficiently replacing the complement plasmid. Comparing the growth of genomic metK-deleted strains with wild-type metK or PAM site mutation to that of wild-type strains, no significant difference in growth between LB medium and M9 medium was observed. However, as expected, MetK mutants with reduced affinity for L-methionine exhibited a significant decrease in cell growth.
LIU Jinhui
,
HUANG Jianfeng
,
YOU Xiaoyan
,
ZHANG Yanfei
. CRISPR/Cas9 knockout strategy for essential genes in Escherichia coli and efficient plasmid replacement methods[J]. Food and Fermentation Industries, 2023
, 49(18)
: 17
-23
.
DOI: 10.13995/j.cnki.11-1802/ts.035910
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