Chung Mu-En, Yeh I-Hsin, Sung Li-Yu, Wu Meng-Ying, Chao Yun-Peng, Ng I-Son, Hu Yu-Chen
Department of Chemical Engineering, National Tsing Hua University, 101, Sec 2, Kuang Fu Rd., Hsinchu, 300, Taiwan.
Department of Chemical Engineering, Feng Chia University, Taichung, Taiwan.
Biotechnol Bioeng. 2017 Jan;114(1):172-183. doi: 10.1002/bit.26056. Epub 2016 Aug 5.
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.
代谢工程通常需要多个基因的染色体整合，但将大基因整合到大肠杆菌中仍然困难。CRISPR/Cas9是一种RNA引导系统，能够实现双链断裂（DSB）的位点特异性诱导和可编程基因组编辑。在此，我们假设CRISPR/Cas9触发的DSB可以增强同源重组并增加大DNA整合到大肠杆菌染色体中的效率。我们证明CRISPR/Cas9系统能够在超过98%的细胞中触发DSB，导致随后的细胞死亡，并确定诱变的SOS反应在细胞存活中起作用。通过优化实验条件并结合λ-Red蛋白和线性双链DNA，CRISPR/Cas9诱导的DSB使供体DNA在MG1655菌株中实现同源重组并替换lacZ基因，效率高达99%，并允许分别以接近91%、92%、71%和61%的效率对2.4、3.9、5.4和7.0 kb的DNA进行高保真、无疤痕整合。CRISPR/Cas9辅助的基因整合在包括BL21(DE3)和W在内的不同大肠杆菌菌株中也起作用，尽管效率不同。综上所述，我们的方法促进了长达7 kb的双链DNA精确整合到大肠杆菌中，效率超过60%，从而显著提高了编辑效率并克服了使用常用重组工程方法的整合大小限制。《生物技术与生物工程》2017年；第114卷：172 - 183页。© 2016威利期刊公司
