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

Tianyuan Su,Qian Wang,Qingsheng Qi,Pengfei Gu,Yizhao Chang,Quanfeng Liang,Haiying Jin,Fapeng Liu

doi:10.1038/srep37895

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.

Highlights

The ability to precisely manipulate the genomes of both eukaryotes and prokaryotes and efficiently is highly desirable in applications ranging from genetic analysis of functional genomic loci to metabolic engineering for intentional metabolic flux redistribution[1,2,3,4]
The novel strategy for homologous recombination (HR)-independent genome editing of prokaryotic cells presented in this study is the first reported E. coli engineering method employing a hetereologous non-homologous end-joining (NHEJ) mechanism
The single-guide RNA (sgRNA) plasmid involved in the Cas9 assisted non-homologous end-joining (CA-NHEJ) system is cured using an inducible sgRNA cassette targeting the p15A replicon, which further simplifies the genetic manipulation of the host

Summary

Introduction

The ability to precisely manipulate the genomes of both eukaryotes and prokaryotes and efficiently is highly desirable in applications ranging from genetic analysis of functional genomic loci to metabolic engineering for intentional metabolic flux redistribution[1,2,3,4]. The CRISPR-Cas[9] system, naturally responsible for the adaptive immunity of prokaryotes that is able to recognize and cleave invasive genetic elements, can generate double-stranded breaks (DSBs) at any genomic locus that existing a 5′-NGG-3′consensus sequence (so called PAM (“protospacer adjacent motif ”) sequence) immediately downstream of the target site via the reprogrammable DNA endonuclease activity of Cas[9] under the guidance of a engineered single-guide RNA (sgRNA) These DSBs can be repaired either by HR in the presence of the corresponding homologous template or by non-homologous end-joining (NHEJ) in the absence of DNA template accompanied with the modification of target genomic locus[10,13,14]. Bacterial Ku proteins are much smaller in size than their eukaryotic counterparts but protect damaged DNA by forming ring-like homodimer structures at the ends of the breaks[16] In certain species, such as Mycobacterium and Bacillus, the conserved prokaryotic NHEJ pathway safeguards the bacterial genomes against unexpected DSBs and promotes genetic variability[20,21,22]. We show that these DSBs can be successfully re-joined in the presence of a heterologously expressed NHEJ system with different nucleotides excision, and prove an efficient strategy for larger chromosomal fragments deletion

Methods

Results

Conclusion