摘要
目前Ⅱ型CRISPR/Cas9工具被广泛应用于基因组编辑中,利用CRISPR/Cas9基因组编辑技术对大肠杆菌(Escherichia coli)基因组进行编辑,可应用于代谢工程及细菌耐药性等研究领域中。利用单缺口核酸酶Cas9 nickase(Cas9n)工具对宿主细胞基因进行修饰,可降低由于双链断裂对宿主带来的毒性。但目前利用CRISPR/Cas9n对大肠杆菌基因组编辑的研究较少,不利于缺口酶Cas9 D10A和Cas9 H840A在大肠杆菌基因组编辑的应用。本研究在前人报道的细菌双质粒Cas9基因编辑系统(pCas/pTarget)基础上,通过分子突变的方法构建缺口酶Cas9突变体质粒pCas-D10A和pCas-H840A,以及携带修复模板或无修复模板的靶向大肠杆菌sseA基因的质粒pTargetT-△sseA(993 bp)和pTargetT-△sseA,系统研究了两种缺口酶编辑大肠杆菌内源性基因sseA的有效性和效率。在稳定表达pCas质粒的MG1655菌株中转入携带修复模板的pTargetT-△sseA(993 bp)质粒,通过菌落PCR及DNA测序方法验证基因编辑的有效性和效率,同时用醋酸铅试纸生化方法对敲除sseA基因的菌株进行功能验证。同时,将不含修复模板的pTargetT-△sseA质粒转入含有pCas的MG1655菌株中,通过平板菌落计数检测编辑效率。实验结果表明,在有修复模板和无修复模板两种系统中,Cas9 D10A和Cas9 H840A均可以编辑大肠杆菌内源性基因sseA,两者编辑效率基本一致,但都低于Cas9-WT的编辑效率。相对于野生型Cas9、Cas9 D10A或Cas9 H840A编辑大肠杆菌基因组时具有更小的细菌毒性。本研究为后续发展大肠杆菌的基因组编辑技术提供了一定的研究基础和新的研究思路。
At present,typeⅡCRISPR/Cas9 tools are widely used in genome editing.The use of CRISPR/Cas9 genome editing technology to edit the Escherichia coli genome can be used in the research of metabolic engineering and bacterial drug resistance.Using Cas9 nickase(Cas9 n)tool to modify host cell genes could reduce the toxicity of double-strand breaks to the host.However,currently there are few studies on the Escherichia coli genome editing using CRISPR/Cas9 n,which hinders the application of Cas9 D10 A or Cas9 H840 A nickase for Escherichia coli genome editing.In this study,on the basis of the previously reported dual-plasmid Cas9 gene editing system(pCas/pTarget)in bacteria,we systematically studied the effectiveness and efficiency of Cas9 D10 A or Cas9 H840 A nickase on the editing of the endogenous sseA gene in Escherichia coli MG1655 strain.The pCas-D10 A and pCas-H840 A plasmids were constructed by molecular mutation methods.Next,the MG1655 strain stably expressing the pCas plasmid was transformed with a plasmid carrying the repair template pTargetT-△sseA(993 bp),and the efficiency of gene editing were determined by colony PCR and DNA sequencing methods.The function of the knocked out of sseA gene in MG1655 strain was verified by the biochemical method of lead acetate test stripe.Meanwhile,the pTargetT-△sseA plasmid lack of repair template was transformed into the MG1655 strain expressing of pCas,and the editing efficiency was resolved by counting the number of survived colony in the plate.Regardless of the presence or absence of the repair template,both Cas9 D10 A and Cas9 H840 A nickase could edit the endogenous gene sseA in Escherichia coli.The editing efficiency of Cas9 D10 A or Cas9 H840 A was found to be similar,but both of them are lower than that of Cas9-WT.Compared with the wild-type Cas9,Cas9 D10 A or Cas9 H840 A has much less bacterial toxicity when editing the Escherichia coli genome.This research provides new research basis and ideas for the development of genome editing technology in Escherichia coli.
作者
韦佳妤
吴方
Wei Jiayu;Wu Fang(Key Laboratory of Ministry of Education for Systems Biomedicine,Shanghai Center for Systems Biomedicine,Shanghai Jiao Tong University,Shanghai,200240)
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2022年第8期1677-1691,共15页
Genomics and Applied Biology
基金
国家自然科学基金面上项目(31870763)资助
