A CRISPR-Cas9 Assisted Non-Homologous End-Joining Strategy for One-step Engineering of Bacterial Genome

Abstract

Homologous recombination-mediated genome engineering has been broadly applied in prokaryotes with high efficiency and accuracy. However, this method is limited in realizing larger-scale genome editing with numerous genes or large DNA fragments because of the relatively complicated procedure for DNA editing template construction. Here, we describe a CRISPR-Cas9 assisted non-homologous end-joining (CA-NHEJ) strategy for the rapid and efficient inactivation of bacterial gene (s) in a homologous recombination-independent manner and without the use of selective marker. Our study show that CA-NHEJ can be used to delete large chromosomal DNA fragments in a single step that does not require homologous DNA template. It is thus a novel and powerful tool for bacterial genomes reducing and possesses the potential for accelerating the genome evolution.

Strategies for Developing CRISPR‐Based Gene Editing Methods in Bacteria

“…Su et al attempted to co‐express Ku and LigD from M. tuberculosis along with a typical SpCas9 targeting machine in E. coli , in order to achieve genome editing without introducing a repair template. The study showed that MtKu and MtLigD efficiently rescued the bacteria from fatal genome targeting, which created deletions surrounding the target site ranging from ten to hundreds of base pairs . Moreover, by simultaneously using a pair of sgRNA at distant locations, large genomic fragment deletions up to 17 kB are manageable, which is promising in manipulating large gene cluster deletion in bacteria.…”

“…To exploit the usage of NHEJ, it is a good idea to reconstitute exogenous Ku and LigD into the bacteria without intrinsic NHEJ machines. Su et al attempted to co‐express Ku and LigD from M. tuberculosis along with a typical SpCas9 targeting machine in E. coli , in order to achieve genome editing without introducing a repair template. The study showed that MtKu and MtLigD efficiently rescued the bacteria from fatal genome targeting, which created deletions surrounding the target site ranging from ten to hundreds of base pairs .…”

“…Moreover, reconstitution of exogenous NHEJ process in strains without NHEJ machines is also promising for gene disruption proposes, owning to its relatively simple design. However, current attempts in this field suffer from the high occurrence of large fragment deletions . Although NHEJ machines are rare in bacterial species, the actual abundance of NHEJ is vast, owning to the diversity of bacteria .…”

Serine integrase recombinational engineering (SIRE): A versatile toolbox for genome editing

“…Li et al (2015) stated a 14% efficiency for the insertion of an 8 kb fragment while others achieved a 50% efficiency for the insertion of a 10 kb construct (Bassalo et al, 2016). Operon deletions of 10 kb were reported with an efficiency of 25% (Su et al, 2016). All reported efficiencies dropped significantly when fragment size was increased and varied greatly between the different methods.…”

“…Non-recombined cells will die as a consequence of the double-stranded break. Operon deletions of 10 kb were reported with an efficiency of 25% (Su et al, 2016). However, these methods apply homologous recombination for DNA repair and hence suffer from the same size limitations for introducing DNA fragments as stated above.…”