title: In Vitro CRISPR/Cas9 System for Efficient Targeted DNA Editing.
abstract: The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system, an RNA-guided nuclease for specific genome editing in vivo, has been adopted in a wide variety of organisms. In contrast, the in vitro application of the CRISPR/Cas9 system has rarely been reported. We present here a highly efficient in vitro CRISPR/Cas9-mediated editing (ICE) system that allows specific refactoring of biosynthetic gene clusters in Streptomyces bacteria and other large DNA fragments. Cleavage by Cas9 of circular pUC18 DNA was investigated here as a simple model, revealing that the 3'→5' exonuclease activity of Cas9 generates errors with 5 to 14 nucleotides (nt) randomly missing at the editing joint. T4 DNA polymerase was then used to repair the Cas9-generated sticky ends, giving substantial improvement in editing accuracy. Plasmid pYH285 and cosmid 10A3, harboring a complete biosynthetic gene cluster for the antibiotics RK-682 and holomycin, respectively, were subjected to the ICE system to delete the rkD and homE genes in frame. Specific insertion of the ampicillin resistance gene (bla) into pYH285 was also successfully performed. These results reveal the ICE system to be a rapid, seamless, and highly efficient way to edit DNA fragments, and a powerful new tool for investigating and engineering biosynthetic gene clusters.
  
  
  OBJECTIVE
  Recent improvements in cloning strategies for biosynthetic gene clusters promise rapid advances in understanding and exploiting natural products in the environment. For manipulation of such biosynthetic gene clusters to generate valuable bioactive compounds, efficient and specific gene editing of these large DNA fragments is required. In this study, a highly efficient in vitro DNA editing system has been established. When combined with end repair using T4 DNA polymerase, Cas9 precisely and seamlessly catalyzes targeted editing, including in-frame deletion or insertion of the gene(s) of interest. This in vitro CRISPR editing (ICE) system promises a step forward in our ability to engineer biosynthetic pathways.
authors.affiliation: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, and Wuhan University School of Pharmaceutical Sciences, Wuhan, People's Republic of China.
authors.forename: Yunkun
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, and Wuhan University School of Pharmaceutical Sciences, Wuhan, People's Republic of China.
authors.forename: Weixin
authors.initials: W
authors.lastname: Tao
authors.affiliation: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, and Wuhan University School of Pharmaceutical Sciences, Wuhan, People's Republic of China.
authors.forename: Shishi
authors.initials: S
authors.lastname: Wen
authors.affiliation: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, and Wuhan University School of Pharmaceutical Sciences, Wuhan, People's Republic of China.
authors.forename: Zhengyuan
authors.initials: Z
authors.lastname: Li
authors.affiliation: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, and Wuhan University School of Pharmaceutical Sciences, Wuhan, People's Republic of China.
authors.forename: Anna
authors.initials: A
authors.lastname: Yang
authors.affiliation: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, and Wuhan University School of Pharmaceutical Sciences, Wuhan, People's Republic of China State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, People's Republic of China.
authors.forename: Zixin
authors.initials: Z
authors.lastname: Deng
authors.affiliation: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, and Wuhan University School of Pharmaceutical Sciences, Wuhan, People's Republic of China yhsun@whu.edu.cn.
authors.forename: Yuhui
authors.initials: Y
authors.lastname: Sun
mesh_terms: D064113:CRISPR-Cas Systems; D004247:DNA / Q000235:genetics*; D018390:Gene Targeting / Q000379:methods*; D010957:Plasmids; D013302:Streptomyces / Q000235:genetics
chemical_list: D004247:DNA