High-Level dCas9 Expression Induces Abnormal Cell Morphology in Escherichia coli

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• CRISPR/Cas9 technology has functional advances in genome editing.
• Endonuclease-deficient Cas9 (dCas9) is a versatile tool for exploring gene functions via RNA-guided transcriptional modulation.
• The impact of intracellular dCas9 expression on bacterial morphology remains underexplored.

Purpose of the Study:

• To investigate the effects of dCas9 expression on Escherichia coli morphology.
• To characterize the molecular mechanisms underlying dCas9-induced phenotypic changes.
• To assess the implications of dCas9 on bacterial cell division and membrane structure.

Main Methods:

• Observation of morphological changes in E. coli expressing dCas9.
• RNA sequencing (RNA-Seq) to analyze gene expression profiles.
• Chromatin immunoprecipitation sequencing (ChIP-Seq) to identify dCas9 binding sites.

Main Results:

• dCas9 expression led to severe growth rate reduction (approx. 50%) and abnormal filamentous morphology in E. coli.
• RNA-Seq revealed significant differential transcription of 574 upregulated genes (cell division-associated) and 221 downregulated genes (membrane proteins).
• ChIP-Seq identified dCas9 binding upstream of 37 genes, including fimA, without single-guide RNA.

Conclusions:

• dCas9 significantly impacts bacterial cell division and inner/outer membrane structure in E. coli.
• Unexpected morphological changes can occur, necessitating regulation of intracellular dCas9 levels.
• Precise control of dCas9 expression is vital for reliable gene function studies using transcriptional modulation.