Combining CRISPR-Cpf1 and Recombineering Facilitates Fast and Efficient Genome Editing in Escherichia coli.
Combining CRISPR-Cpf1 and Recombineering Facilitates Fast and Efficient Genome Editing in Escherichia coli.
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DOI：
10.1021/acssynbio.2c00041
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发表时间：
2022-04
影响因子：
4.7
通讯作者：
Xuewen Zhu;Yaokang Wu;Xueqin Lv;Yanfeng Liu;G. Du;Jianghua Li;Long Liu
中科院分区：
文献类型：
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作者：
Xuewen Zhu;Yaokang Wu;Xueqin Lv;Yanfeng Liu;G. Du;Jianghua Li;Long Liu
基于成簇规则间隔短回文重复序列（CRISPR）的基因编辑技术以其低成本、高效率、易操作、多功能等优点，已广泛应用于多种微生物。本研究基于CRISPR/Cpf1在大肠杆菌中构建了高效、快速的双质粒基因编辑系统pEcCpf1/pcrEG。首先，在八种不同的原型间隔子相邻基序（PAM）区域中验证了基因敲除和整合效率。然后，优化了转化方法，该系统的基因敲除或基因整合效率提高到接近100%，并且大长度片段可以整合到大肠杆菌BL21（DE3）的基因组中。该系统还通过替换质粒pEcCpf1中的同源重组系统进行了优化，产生了pEcCpf1H，它可以使用90 nt（核苷酸）单链引物高效地进行精确的单点突变、终止子插入、短序列插入或基因敲除。此外，可以同时编辑多个基因。接下来，这两个系统在其他大肠杆菌菌株中得到了验证。最后，作为应用，该系统被用于工程化菌株中L-组氨酸的合成途径。摇瓶中L-组氨酸效价达到7.16 g/L，比起始菌株提高了84.1%。因此，本研究为大肠杆菌的代谢工程提供了有效的工具。
Clustered regularly interspaced short palindromic repeat (CRISPR)-based gene-editing technology has been widely used in various microorganisms due to its advantages of low cost, high efficiency, easy operation, and multiple functions. In this study, an efficient and fast double-plasmid gene-editing system pEcCpf1/pcrEG was constructed in Escherichia coli based on CRISPR/Cpf1. First, gene knockout and integration efficiency were verified in eight different kinds of protospacer adjacent motif (PAM) regions. Then, the transformation method was optimized, and the efficiency of gene knockout or gene integration of this system increased to nearly 100%, and the large-length fragments could be integrated into the genome in E. coli BL21 (DE3). The system was also optimized by replacing the homologous recombination system in plasmid pEcCpf1, resulting in pEcCpf1H, which could perform precise single-point mutation, terminator insertion, short-sequence insertion, or gene knockout with high efficiency using a 90 nt (nucleotide) single-stranded primer. Further, multiple genes could be edited simultaneously. Next, these two systems were demonstrated in other E. coli strains. Finally, as an application, the system was used to engineer the synthesis pathway of l-histidine in the engineered strain. The titer of l-histidine in a shake flask reached 7.16 g/L, a value increased by 84.1% compared to the starting strain. Thus, this study provided an effective tool for metabolic engineering of E. coli.