利用CRISPR/Cas9核酸酶靶向细菌必需基因的特异性抗菌剂研究

Specific Antimicrobials Targeting Bacterial Essential Genes Using CRISPR/Cas9 Nucleases

CRISPR/Cas9核酸酶是一种高效和精确的基因组DNA编辑工具。目前,CRISPR/Cas9被开发成一种新型抗菌剂,通过靶向破坏目标基因,例如抗生素耐药基因来诱导细菌死亡。本研究利用CRISPR携带多靶点优势,同时靶向大肠杆菌必需基因gyrA、gyrB和folA,以达到杀菌作用。本设计针对3个内源基因的CRISPR系列载体,分别含有1个、2个或3个靶点。当IPTG诱导Cas9表达后,CRISPR RNA (crRNA)和反式作用CRISPR RNA (trans-activating CRISPR RNA, tracrRNA)复合物募集Cas9切割靶基因,导致细菌死亡。随着crRNA中靶点数量的增加,杀菌效率显著提高。当含有3个靶点的crRNA作用于细菌基因组时,杀菌效率达到99.35%。此外,在存活的大肠杆菌中,crRNA表达质粒的靶序列和直接重复序列发现碱基缺失,而内源靶基因和Cas9基因均未发生突变。最后,该CRISPR系统还应用于其他大肠杆菌实验室菌株和产肠毒素K88菌株,并表现出高效的杀菌作用。我们设计了一种基于CRISPR/Cas9核酸酶的新型抗菌剂,为抗菌剂的研发提供新策略。

The CRISPR/Cas9 nuclease exhibits highly efficient and precise modification of genomic DNA in various species.Currently,CRISPR/Cas9 was created as a novel antimicrobial to induce cell deaths by targeting genes such as antibiotic resistance genes.In this study,we designed an alternative CRISPR vector system harboring multiple spacers for targeting bacterial essential genes to induce cell deaths in E.coli.For E.coli endogenous genes,such as gyrA,gyrB and folA,a series of CRISPR vectors were designed harboring one,two and three spacers.In the presence of IPTG,Cas9 was expressed and CRISPR RNA(crRNA)/trans-activating CRISPR RNA(tracrRNA)recruited Cas9 to cut target genes,resulting in cell death.When more spacers were constructed in the crRNA array,the antibacterial efficiencies were significantly increased.For crRNA harboring three different spacers,the efficiency of cell death was up to 99.35%.In addition,no mutations were observed in the target genes or the Cas9 gene in surviving E.coli cells.However,various deletions of spacers and direct repeats were examined in the crRNA plasmids isolated from survival colonies.Furthermore,this CRISPR system demonstrated bactericidal effect in other E.coli laboratory strains and enterotoxigenic K88.Collectively,we designed a simple and feasible CRISPR/Cas9 system as a novel antimicrobial agent for killing bacteria,which also provides an alternative strategy for antibiotics research.