Silencing Genes with dCas9: A Story of Fine-Tuned Control, High-Throughput Screens and Surprising Toxicity

The catalytically dead variant of Cas9 known as dCas9 can be guided by small RNAs to block transcription of target genes, in a strategy also known as CRISPRi. The level of complementarity between the guide RNA and the target controls the rate at which RNA polymerase 'kicks out' dCas9 from the target and completes transcription. We used this mechanism to precisely and robustly reduce gene expression by defined relative amounts. Silencing genes with dCas9 can also be performed in high-throughput screens to decipher gene functions and genetic interactions. We recently used a genome-wide library of guide RNAs to direct dCas9 to block gene transcriptions in E. coli. The results can be used to investigate gene essentiality and provide insights into the effect of dCas9 binding position and orientation, as well as its polar effects on gene expression. Surprisingly, we revealed that guide RNAs sharing specific 5 nucleotides seed sequences can produce strong fitness defects and even kill E. coli regardless of the other 15nt of guide sequence. This effect which occurs at high dCas9 concentrations can be alleviated by tuning the expression of dCas9 while maintaining strong on-target repression. Altogether our work demonstrates the usefulness of CRISPRi in bacteria while providing important design rules.