Toward tunable dynamic repression using CRISPRi

CRISPR interference (CRISPRi) is widely utilized for regulation of target gene expression by repressing transcription. Simple design rules for the single guide RNA (sgRNA) and multiplexity won this method immense popularity. However, quantitative control of the expression levels at varying degrees in a dynamic manner using CRISPRi has been regarded difficult. To deal with this limitation, Fontana et al. modulated the expression levels of the components of CRISPRi, the deactivated Cas9 (dCas9), and the sgRNAs, using various constitutive or inducible promoters (Fontana et al., Biotechnol. J. 2018, 13, 1800069). They found that the expression level of sgRNA is the key to controlling CRISPRi. Modulation of sgRNA expression levels enabled quantitative tuning of the CRISPRi-regulated gene expression level. This approach is expected to be easily applied to diverse applications owing to its simplicity compared to the conventional approaches that modified target sequence or changed the expression level of dCas9.