大肠杆菌必需基因的CRISPR-Cas9敲除策略及高效质粒置换方法

CRISPR/Cas9 knockout strategy for essential genes in Escherichia coli and efficient plasmid replacement methods

由于必需基因的功能必须性使其难以从基因组上被删除,而此前报道的方法存在效率低、非无痕敲除等缺点。基于此,该文建立了一种基于CRISPR/Cas9的基因组必需基因高效无痕敲除方法。以大肠杆菌metK基因(编码S-腺苷甲硫氨酸合酶)为例,首先将metK基因克隆至质粒pTarget-metK,并采用定点突变方式将metK基因进行同义突变移除对应的PAM序列,构建获得质粒pHL3。进一步构建含有与pHL3相同metK的回补质粒,pCas、pHL3及回补质粒依次转化即可完成基因组必需基因的高效无痕敲除。该方法对必需基因metK编辑时编辑效率可达100%。为了研究必需基因的酶学性质改变对宿主代谢的影响,探索了基于质粒不相容及Flp/FRT系统对回补质粒的置换效率,发现基于Flp/FRT系统可实现回补质粒高效置换。生长测试表明携带metK野生型或是PAM位点突变的metK编辑菌株与野生型菌株在LB培养基和M9培养基中生长无明显差异,而对L-甲硫氨酸亲和力下降MetK突变体则表现明显的生长延滞。

Deleting essential genes from the genome is challenging due to their functional importance.Existing editing methods have drawbacks such as low efficiency and non-marker knockout.In this study,we present an efficient and traceless CRISPR/Cas9-based knockout method for essential genes in the Escherichia coli genome using metK disruption as an example.Firstly,we constructed plasmid pHL3 by cloning the metK gene into plasmid pTarget-metK.Through site-specific mutation,we made the metK gene synonymous,thereby removing the corresponding PAM sequence.Subsequently,we created a complement plasmid containing the same metK sequence as pHL3.The deletion of essential genes in the genome,in a highly efficient and marker manner,can be achieved by sequentially transforming pCas,pHL3,and the complement plasmid.The efficiency of metK deletion by this method can reach 100%.To investigate the impact of changes in the properties of proteins encoded by essential genes on host metabolism,we explored the replacement efficiency of the complement plasmid based on plasmid incompatibility and the Flp/FRT system.The Flp/FRT system proved capable of efficiently replacing the complement plasmid.Comparing the growth of genomic metK-deleted strains with wild-type metK or PAM site mutation to that of wild-type strains,no significant difference in growth between LB medium and M9 medium was observed.However,as expected,MetK mutants with reduced affinity for L-methionine exhibited a significant decrease in cell growth.