利用CRISPR/Cas9和λ-Red级联技术构建产肠毒素大肠杆菌LT敲除菌株

doi:10.16431/j.cnki.1671-7236.2020.03.003

摘要/Abstract

摘要： 本研究旨在利用CRISPR/Cas9和λ-Red级联的技术对产肠毒素大肠杆菌（enterotoxigenic Escherichia coli，ETEC）K88的热不稳定性肠毒素（heat-labile toxin，LT）基因进行无痕敲除并获得K88 LT^-缺陷菌株。通过序列比对获取LT两端同源序列，并构建包含LT边界、氯霉素筛选标记、sgRNA和LT同源臂的供体片段；将供体片段转化至ETEC K88，同时分别利用λ-Red同源重组系统和CRISPR/Cas9基因编辑系统，对LT基因进行敲除；通过PCR验证获得了K88 LT^-缺陷菌株，并通过试验测定了敲除菌株的溶血能力和生长曲线。结果显示，λ-Red同源重组系统可成功地将LT基因替换为相应的供体片段，CRISPR/Cas9基因编辑系统可高效地对筛选标记进行删除，最终通过λ-Red和CRISPR/Cas9结合的基因编辑系统可成功对ETEC K88的LT基因进行无痕敲除。体外试验结果表明，K88 LT^-缺陷菌株的溶血能力丧失，并且生长速度比野生型菌株减缓，LT可能和ETEC K88的致病能力和生长性能有关。表明λ-Red和CRISPR/Cas9级联的基因敲除方法可用于LT毒素基因及其他一些大肠杆菌基因的敲除。K88 LT^-缺陷菌株的构建为下一步研究LT毒素的致病机制奠定基础。

关键词: 产肠毒素大肠杆菌(ETEC); CRISPR/Cas9; λ-Red同源重组系统; 热不稳定性肠毒素

Abstract: The aim of this study was to use the CRISPR/Cas9 and λ-Red cascade technology to perform a seamless knockout the heat-labile toxin (LT) gene of enterotoxigenic Escherichia coli (ETEC) K88 and obtain a K88 LT deficient strain.The homologous sequences of boundary sequences at both ends were obtained by sequence alignment,and a donor fragment was constructed containing the LT boundary sequences,the chloromycetin selection marker,sgRNA sequences and the LT homology arm.The donor fragment was transformed into ETEC K88,and LT gene was knocked out using λ-Red homologous recombination system and CRISPR/Cas9 gene editing system,respectively.The K88 LT-deficient strain was obtained by PCR,and the hemolysis ability and growth curve of the knockout strain were determined.The results showed that LT gene was successfully replaced by the corresponding donor fragment using λ-Red homologous recombination system,and the chloromycetin selection marker was efficiently deleted using CRISPR/Cas9 gene editing system.The gene editing system successfully performed a seamless knockout of LT gene of ETEC K88.In vitro experiment results showed that the K88 LT deficient strain had no capacity to hemolysis and grew slowly than the wild-type strain.It indicated that LT affected the pathogenicity and growth performance of the strain.Therefore,the results suggested that λ-Red and CRISPR/Cas9 cascade knockout methods could be used for knockout of LT toxin genes,and could also be used for other E.coli knockouts.The construction of K88 LT-strain laid the foundation for further study on the pathogenesis of LT toxin.

Key words: enterotoxigenic Escherichia coli(ETEC); CRISPR/Cas9; λ-Red recombination system; heat-labile enterotoxin

中图分类号:

S852.61⁺2

檀克勤, 马现永, 崔艺燕, 田志梅, 邓盾. 利用CRISPR/Cas9和λ-Red级联技术构建产肠毒素大肠杆菌LT敲除菌株[J]. 中国畜牧兽医, 2020, 47(3): 666-675.

TAN Keqin, MA Xianyong, CUI Yiyan, TIAN Zhimei, DENG Dun. Construction of Enterotoxigenic E.coli LT Knockout Strain Using CRISPR/Cas9 and λ-Red Cascaded Technology[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(3): 666-675.

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