Bacterial resistance to CRISPR-Cas antimicrobials

We systematically investigated mutations that arise from CRISPR-Cas9 induced death. We designed a genetic circuit using Cas9 from Streptococcus pyogenes (SpCas9) to study the different parameters that affect the efficiency of the system and elucidated the spontaneous genetic alterations that confer resistance to this novel type of antimicrobial agent. Our findings highlight that killing efficiency is not correlated with the type of target or number of cutting sites, however we observed that multiple target sites are valuable in order to prevent genomic mutations that can result in resistance. Furthermore, we observed that in most cases SpCas9 is the preferred target for mutations inactivating the activity of the antimicrobial by large-scale bacterial genome rearrangements involving mobile genetic elements. The work project presents a guide to design strategies that reduce resistance and improve the activity of CRISPR-Cas antimicrobials.

本研究系统探究了CRISPR-Cas9诱导致死过程中产生的突变。我们采用来自化脓链球菌的Cas9（Streptococcus pyogenes Cas9, SpCas9）构建遗传回路，以研究影响该系统效率的各类参数，并阐明赋予该新型抗菌剂抗性的自发遗传变异。研究结果表明，杀伤效率与靶标类型或切割位点数量并无关联，但我们发现，设置多个靶标位点可有效预防可引发抗性的基因组突变。此外，我们观察到，在多数情况下，通过涉及移动遗传元件的大规模细菌基因组重排使该抗菌剂失活的突变，其优先靶标为SpCas9。本研究可为设计降低抗性、提升CRISPR-Cas类抗菌剂活性的策略提供参考指南。