Nonspecific toxicities of streptococcus pyogenes and Staphylococcus aureus dCas9 in Chlamydia trachomatis.
Nonspecific toxicities of streptococcus pyogenes and Staphylococcus aureus dCas9 in Chlamydia trachomatis.
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DOI：
10.1093/femspd/ftaa005
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发表时间：
2019-12
影响因子：
3.3
通讯作者：
W. Wurihan;Yehong Huang;Alec M. Weber;Xiang Wu;Huizhou Fan
中科院分区：
文献类型：
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作者：
W. Wurihan;Yehong Huang;Alec M. Weber;Xiang Wu;Huizhou Fan
衣原体是人类和动物常见的重要病原体。尽管遗传学最近取得了进展，但科学家们仍在寻找有效的工具来敲除或敲低染色体基因的表达。我们尝试采用基于dCas 9的CRISPR干扰（CRISPRi）技术，使用无水四环素（ATC）诱导的表达系统有条件地敲低沙眼衣原体中的基因表达。令人惊讶的是，常用的化脓性链球菌dCas 9在C.沙眼衣原体在不存在任何指导RNA（gRNA）的情况下引起强抑制。金黄色葡萄球菌（Staphylococcus aureus）dCas 9在仅存在空gRNA支架的情况下也显示出强毒性。S.在低至0.2 nM ATC的情况下容易观察到化脓性链球菌dCas 9。生长抑制S.金黄色葡萄球菌dCas 9在1.0 nM ATC开始是明显的。与此相反，C.沙眼衣原体的生长不受10 nM ATC诱导的甲硫氨酸-tRNA连接酶过表达的影响。我们的结论是S.化脓链球菌和S.目前形式的金黄色葡萄球菌dCas 9蛋白对衣原体研究的效用有限，并提出了克服该问题的策略。
Chlamydiae are common, important pathogens for humans and animals alike. Despite recent advancement in genetics, scientists are still searching for efficient tools to knock out or knock down the expression of chromosomal genes. We attempted to adopt a dCas9-based CRISPR interference (CRISPRi) technology to conditionally knock down gene expression in Chlamydia trachomatis using an anhydrotetracycline (ATC)-inducible expression system. Surprisingly, expression of the commonly used Streptococcus pyogenes dCas9 in C. trachomatis causes strong inhibition in the absence of any guide RNA (gRNA). Staphylococcus aureus dCas9 also shows strong toxicity in the presence of only an empty gRNA scaffold. Toxicity of the S. pyogenes dCas9 is readily observed with as little as 0.2 nM ATC. Growth inhibition by S. aureus dCas9 is evident starting at 1.0 nM ATC. In contrast, C. trachomatis growth was not affected by methionine-tRNA ligase overexpression induced with 10 nM ATC. We conclude that S. pyogenes and S. aureus dCas9 proteins in their current forms have limited utility for chlamydial research and suggest strategies to overcome this problem.