CRISPR-Cas9-assisted native end-joining editing offers a simple strategy for efficient genetic engineering in Escherichia coli

Unlike eukaryotes, prokaryotes are less proficient in homologous recombination (HR) and non-homologous end-joining (NHEJ). All existing genomic editing methods for Escherichia coli (E. coli) rely on exogenous HR or NHEJ systems to repair DNA double-strand breaks (DSBs). Although an E. coli native end-joining (ENEJ) system has been reported, its potential in genetic engineering has not yet been explored. Here, we present a CRISPR-Cas9-assisted native end-joining editing and show that ENEJ-dependent DNA repair can be used to conduct rapid and efficient deletion of chromosome fragments up to 83 kb or gene inactivation. Moreover, the positive rate and editing efficiency are independent of high-efficiency competent cells. The method requires neither exogenous DNA repair systems nor introduced editing template. The Cas9-sgRNA complex is the only foreign element in this method. This study is the first successful engineering effort to utilize ENEJ mechanism in genomic editing and provides an effective strategy for genetic engineering in bacteria that are inefficient in HR and NHEJ.