title: An efficient system for deletion of large DNA fragments in Escherichia coli via introduction of both Cas9 and the non-homologous end joining system from Mycobacterium smegmatis.
abstract: Accompanied with the internal non-homologous end joining (NHEJ) system, Cas9 can be used to easily inactivate a gene or delete a fragment through introduction of DNA double-stranded breaks (DSBs) in eukaryotic cells. While in most prokaryotes (e.g. Escherichia coli), due to the lack of NHEJ, homologous recombination (HR) is required for repair of DSBs, which is less convenient. Here, a markerless system was developed for rapid gene inactivation or fragment deletion in E. coli via introduction of both Cas9 and a bacterial NHEJ system. Three bacterial NHEJ systems, i.e. Mycobacterium smegmatis (Msm), Mycobacterium tuberculosis (Mtb) and Bacillus subtilis (Bs), were tested in E. coli, and the MsmNHEJ system showed the best efficiency. With the employment of Cas9 and MsmNHEJ, we efficiently mutated lacZ gene, deleted glnALG operon and two large DNA fragments (67 kb and 123 kb) in E. coli, respectively. Moreover, the system was further designed to allow for continuous inactivation of genes or deletion of DNA fragments in E. coli. We envision this system can be extended to other bacteria, especially those with low HR efficiency.
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China; School of Life Science, Henan University, Kaifeng 475004, China.
authors.forename: Xuan
authors.initials: X
authors.lastname: Zheng
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China; University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Shi-Yuan
authors.initials: SY
authors.lastname: Li
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China; Department of Microbiology and Li KaShing Institute of Health Sciences, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR, China.
authors.forename: Guo-Ping
authors.initials: GP
authors.lastname: Zhao
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China. Electronic address: wangj01@hotmail.com.
authors.forename: Jin
authors.initials: J
authors.lastname: Wang
mesh_terms: D001412:Bacillus subtilis / Q000235:genetics; D001483:Base Sequence; D064113:CRISPR-Cas Systems; D059766:DNA End-Joining Repair; D004269:DNA, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods; D007763:Lac Operon / Q000235:genetics; D008957:Models, Genetic; D016296:Mutagenesis; D020102:Mycobacterium smegmatis / Q000235:genetics*; D009169:Mycobacterium tuberculosis / Q000235:genetics; D009876:Operon / Q000235:genetics; D015203:Reproducibility of Results; D017384:Sequence Deletion; D012689:Sequence Homology, Nucleic Acid
chemical_list: D004269:DNA, Bacterial
keywords: CRISPR; E. coli; Genome editing; Large fragment deletion; NHEJ