title: Enhanced integration of large DNA into E. coli chromosome by CRISPR/Cas9.
abstract: Metabolic engineering often necessitates chromosomal integration of multiple genes but integration of large genes into Escherichia coli remains difficult. CRISPR/Cas9 is an RNA-guided system which enables site-specific induction of double strand break (DSB) and programmable genome editing. Here, we hypothesized that CRISPR/Cas9-triggered DSB could enhance homologous recombination and augment integration of large DNA into E. coli chromosome. We demonstrated that CRISPR/Cas9 system was able to trigger DSB in >98% of cells, leading to subsequent cell death, and identified that mutagenic SOS response played roles in the cell survival. By optimizing experimental conditions and combining the λ-Red proteins and linear dsDNA, CRISPR/Cas9-induced DSB enabled homologous recombination of the donor DNA and replacement of lacZ gene in the MG1655 strain at efficiencies up to 99%, and allowed high fidelity, scarless integration of 2.4, 3.9, 5.4, and 7.0 kb DNA at efficiencies approaching 91%, 92%, 71%, and 61%, respectively. The CRISPR/Cas9-assisted gene integration also functioned in different E. coli strains including BL21 (DE3) and W albeit at different efficiencies. Taken together, our methodology facilitated precise integration of dsDNA as large as 7 kb into E. coli with efficiencies exceeding 60%, thus significantly ameliorating the editing efficiency and overcoming the size limit of integration using the commonly adopted recombineering approach. Biotechnol. Bioeng. 2017;114: 172-183. © 2016 Wiley Periodicals, Inc.
authors.affiliation: Department of Chemical Engineering, National Tsing Hua University, 101, Sec 2, Kuang Fu Rd., Hsinchu, 300, Taiwan.
authors.forename: Mu-En
authors.initials: ME
authors.lastname: Chung
authors.affiliation: Department of Chemical Engineering, National Tsing Hua University, 101, Sec 2, Kuang Fu Rd., Hsinchu, 300, Taiwan.
authors.forename: I-Hsin
authors.initials: IH
authors.lastname: Yeh
authors.affiliation: Department of Chemical Engineering, National Tsing Hua University, 101, Sec 2, Kuang Fu Rd., Hsinchu, 300, Taiwan.
authors.forename: Li-Yu
authors.initials: LY
authors.lastname: Sung
authors.affiliation: Department of Chemical Engineering, National Tsing Hua University, 101, Sec 2, Kuang Fu Rd., Hsinchu, 300, Taiwan.
authors.forename: Meng-Ying
authors.initials: MY
authors.lastname: Wu
authors.affiliation: Department of Chemical Engineering, Feng Chia University, Taichung, Taiwan.
authors.forename: Yun-Peng
authors.initials: YP
authors.lastname: Chao
authors.affiliation: Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan.
authors.forename: I-Son
authors.initials: IS
authors.lastname: Ng
authors.affiliation: Department of Chemical Engineering, National Tsing Hua University, 101, Sec 2, Kuang Fu Rd., Hsinchu, 300, Taiwan.
authors.forename: Yu-Chen
authors.initials: YC
authors.lastname: Hu
mesh_terms: D064113:CRISPR-Cas Systems / Q000235:genetics*; D002470:Cell Survival; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D004926:Escherichia coli / Q000235:genetics*; D000072669:Gene Editing / Q000379:methods*; D060847:Metabolic Engineering / Q000379:methods*; D010957:Plasmids / Q000235:genetics; D013014:SOS Response, Genetics / Q000235:genetics
chemical_list: D004247:DNA
keywords: CRISPR/Cas9; E. coli; double strand break; genome editing; integration; metabolic engineering