Along with functional advances in the use of CRISPR/Cas9 for genome editing, endonuclease-deficient Cas9 (dCas9) has provided a versatile molecular tool for exploring gene functions. In principle, differences in cell phenotypes that result from the RNA-guided modulation of transcription levels by dCas9 are critical for inferring with gene function; however, the effect of intracellular dCas9 expression on bacterial morphology has not been systematically elucidated. Here, we observed unexpected morphological changes in Escherichia coli mediated by dCas9, which were then characterized using RNA sequencing (RNA-Seq) and chromatin immunoprecipitation sequencing (ChIP-Seq). Growth rates were severely decreased, to approximately 50% of those of wild type cells, depending on the expression levels of dCas9. Cell shape was changed to abnormal filamentous morphology, indicating that dCas9 affects bacterial cell division. RNA-Seq revealed that 574 genes were differentially transcribed in the presence of high expression levels of dCas9. Genes associated with cell division were upregulated, which was consistent with the observed atypical morphologies. In contrast, 221 genes were downregulated, and these mostly encoded proteins located in the cell membrane. Further, ChIP-Seq results showed that dCas9 directly binds upstream of 37 genes without single-guide RNA, including fimA, which encodes bacterial fimbriae. These results support the fact that dCas9 has critical effects on cell division as well as inner and outer membrane structure. Thus, to precisely understand gene functions using dCas9-driven transcriptional modulation, the regulation of intracellular levels of dCas9 is pivotal to avoid unexpected morphological changes in E. coli.

中文翻译：

---

高水平的dCas9表达诱导大肠杆菌细胞形态异常。

随着在使用CRISPR / Cas9进行基因组编辑方面的功能进步，核酸内切酶缺陷型Cas9（dCas9）为探索基因功能提供了一种多功能的分子工具。原则上，由dCas9的RNA指导的转录水平调节导致的细胞表型差异对于推断基因功能至关重要。但是，尚未系统阐明细胞内dCas9表达对细菌形态的影响。在这里，我们观察到了大肠杆菌中意想不到的形态变化由dCas9介导，然后使用RNA测序（RNA-Seq）和染色质免疫沉淀测序（ChIP-Seq）进行表征。取决于dCas9的表达水平，其生长速度严重降低，约为野生型细胞的50％。细胞形状变为异常的丝状形态，表明dCas9影响细菌细胞分裂。RNA-Seq显示，在dCas9高表达水平下，有574个基因被差异转录。与细胞分裂相关的基因被上调，这与观察到的非典型形态一致。相反，有221个基因被下调，而这些主要编码的蛋白质位于细胞膜中。此外，ChIP-Seq结果显示，dCas9直接结合37个基因的上游，而没有单向导RNA，包括fimA，它编码细菌菌毛。这些结果支持了dCas9对细胞分裂以及内部和外部膜结构具有关键作用的事实。因此，为了使用dCas9驱动的转录调节精确地了解基因功能，调节细胞内dCas9的水平至关重要，以避免大肠杆菌中发生意想不到的形态变化。