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

Abstract

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.

Impact of CRISPR interference on strain development in biotechnology

“…Moreover, inherent problems such as ambiguous interpretation of CRISPRi results when targeting a bidirectional promoter or upstream versus downstream genes in operons exist [135,136] and require caution and further experimentation for full comprehension. Several studies reported dCas9 protein off-target effect, besides toxicity in bacterial cells [137][138][139][140][141][142]. In E. coli, it was observed that a high level of dCas9 expression led to abnormal cell morphology [137].…”

“…Several studies reported dCas9 protein off-target effect, besides toxicity in bacterial cells [137][138][139][140][141][142]. In E. coli, it was observed that a high level of dCas9 expression led to abnormal cell morphology [137]. This may negatively affect laboratory routine work on molecular cloning when plasmid-borne dcas9 is expressed in E. coli cloning host.…”

Strategies for Developing CRISPR‐Based Gene Editing Methods in Bacteria

“…Jiang et al found that expression of SpCas9 is strongly toxic to Corynebacterium glutamicum even in the absence of any sgRNA, resulting in a transformation efficiency reaching to zero. Another study also found that overexpression of SpCas9 in E. coli significantly altered the cell morphology and growth rates . As overdosed SpCas9 may spontaneously interrogate the genomic DNA searching for PAM sites in a high frequency, these transient SpCas9‐binding events may affect some essential cellular biological processes, thus, manifesting as toxicity to host bacterial cells.…”

“…Another study also found that overexpression of SpCas9 in E. coli significantly altered the cell morphology and growth rates. [58] As overdosed SpCas9 may spontaneously interrogate the genomic DNA searching for PAM sites in a high frequency, these transient SpCas9-binding events may affect some essential cellular biological processes, thus, manifesting as toxicity to host bacterial cells. To alleviate the toxicity of SpCas9, its nickase form has been demonstrated to be practical in some studies.…”

Modeling predicts that CRISPR-based activators, unlike CRISPR-based repressors, scale well with increasing gRNA competition and dCas9 bottlenecking

“…Any expression of one gRNA in a network of CRISPRtfs sequesters shared dCas9 away from other gRNAs, decreasing their effectiveness as outlined in detail in [24]. The dCas9 bottleneck can be loosened by producing more dCas9, but this strategy is limited by the toxicity of dCas9 when expressed at high concentration, especially in prokaryotes [25,26].…”