Combining CRISPR–Cpf1 and Recombineering Facilitates Fast and Efficient Genome Editing in Escherichia coli,ACS Synthetic Biology

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Combining CRISPR–Cpf1 and Recombineering Facilitates Fast and Efficient Genome Editing in Escherichia coli
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2022-04-26 , DOI: 10.1021/acssynbio.2c00041
Xuewen Zhu _{1,

2} , Yaokang Wu _{1,

2} , Xueqin Lv _{1,

2} , Yanfeng Liu _{1,

2} , Guocheng Du _{1,

2} , Jianghua Li _{1,

2} , Long Liu _{1,

2}

Affiliation

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.

中文翻译：

结合 CRISPR–Cpf1 和重组工程有助于在大肠杆菌中进行快速高效的基因组编辑

基于成簇规则间隔短回文重复序列（CRISPR）的基因编辑技术由于具有成本低、效率高、操作简单、功能多等优点，已被广泛应用于各种微生物中。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区相邻基序（PAM）区域中验证了基因敲除和整合效率。然后，对转化方法进行了优化，该系统的基因敲除或基因整合效率提高到近100%，大长度片段可以整合到大肠杆菌的基因组中。BL21 (DE3)。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）单进行精确的单点突变、终止子插入、短序列插入或基因敲除。 -链引物。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证实。最后，作为一个应用，该系统被用于改造工程菌株中l-组氨酸的合成途径。摇瓶中l-组氨酸的效价达到7.16 g/L，与起始菌株相比增加了84.1%。因此，本研究为代谢工程提供了一个有效的工具。大肠杆菌。

更新日期：2022-04-26

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