CRISPR/Cas13a系统在大肠杆菌RNA编辑过程中的逃逸现象

Escape phenomenon of CRISPR/Cas13a system during RNA editing in Escherichia coli

【目的】在大肠杆菌(Escherichia coli)MG1655-ΔrecA和Escherichia coli DH10B中构建CRISPR/LshCas13a质粒干扰系统,通过靶向非必需基因lacZ和必需基因polA,分别分析RNA编辑实验中的逃逸现象。【方法】选取来自沙氏纤毛菌(Leptotrichia shahii)中的Cas13a蛋白编码基因LshCas13a,构建可诱导的CRISPR/LshCas13a的RNA编辑系统相关质粒,选取MG1655-ΔrecA和DH10B为研究对象。通过Crisporo算法,设计靶向lacZ和polA的CRISPR RNA(crRNA)序列,考察利用LshCas13a质粒干扰实验靶向lacZ和polA的逃逸现象。再通过对逃逸菌落的数量和序列分析评估LshCas13a系统的逃逸现象,结合PCR和Sanger测序技术探究LshCas13a系统的逃逸事件。选取通过插入序列(insertion sequence,IS)转座破坏LshCas13a系统的LshCas13a基因的逃逸菌落,通过监测OD_(600)进一步考察菌株的生长情况。【结果】利用LshCas13a系统靶向MG1655-ΔrecA和DH10B中的lacZ和polA,发现靶向lacZ时,MG1655-ΔrecA通过LshCas13a基因点突变和IS转座突变方式逃逸;靶向polA时,MG1655-ΔrecA和DH10B通过点突变LshCas13a基因、IS转座和突变crRNA的直接重复(direct repeat,DR)序列等方式逃逸。LshCas13a编码基因的突变促进了菌株生长的恢复。【结论】本研究利用LshCas13a质粒干扰系统研究了E.coli宿主RNA编辑中的多样化逃逸现象,包括了染色体编码的IS介导的LshCas13a转座突变、LshCas13a点突变、crRNA的DR序列突变或重组等情况。本研究为进一步优化CRISPR/LshCas13a基因编辑系统奠定了基础。

[Objective]The plasmid interference system of CRISPR/LshCas13a was constructed in Escherichia coli MG1655-ΔrecA and Escherichia coli DH10B to analyze the escape phenomenon in RNA editing experiments by targeting the non-essential gene lacZ and the essential gene polA.[Methods]An inducible CRISPR/LshCas13a RNA editing systemassociated plasmid was designed with LshCas13a from Leptotrichia shahii.MG1655-ΔrecA and DH10B were selected as the research objects.The Crisporo algorithm was employed to design the CRISPR RNA(crRNA)sequences targeting lacZ and polA,and the LshCas13a plasmid interference experiment was carried out to study the escape phenomena targeting lacZ and polA.The escape phenomenon of the LshCas13a system was evaluated based on the number and sequences of escaped colonies.PCR and Sanger sequencing were conducted to explore the escape events of the LshCas13a system.The escaped colonies carrying the LshCas13a system disrupted by the insertion sequence(IS)were selected,and OD_(600)was measured to evaluate the growth recovery of the strains.[Results]The LshCas13a system was used to target lacZ and polA in MG1655-ΔrecA and DH10B.MG1655-ΔrecA escaped through point mutation of LshCas13a and IS-mediated transposition when lacZ was targeted.When polA was targeted,MG1655-ΔrecA and DH10B escaped by point mutation of LshCas13a,IS-mediated transposition,and mutation of the direct repeat(DR)sequence of crRNA.The mutation of LshCas13a promoted the recovery of strain growth.[Conclusion]The LshCas13a plasmid interference system successfully revealed the diversified escape phenomena during the RNA editing of E.coli,including IS-mediated the transposition of LshCas13a,point mutation of LshCas13a,and DR sequence mutation or recombination of crRNA.The results laid a foundation for optimization of the CRISPR/LshCas13a gene editing system.