gDesigner: computational design of synthetic gRNAs for Cas12a-based transcriptional repression in mammalian cells

Synthetic networks require complex intertwined genetic regulation often relying on transcriptional activation or repression of target genes. CRISPRi-based transcription factors facilitate the programmable modulation of endogenous or synthetic promoter activity and the process can be optimised by using software to select appropriate gRNAs and limit non-specific gene modulation. Here, we develop a computational software pipeline, gDesigner, that enables the automated selection of orthogonal gRNAs with minimized off-target effects and promoter crosstalk. We next engineered a Lachnospiraceae bacterium Cas12a (dLbCas12a)-based repression system that downregulates target gene expression by means of steric hindrance of the cognate promoter. Finally, we generated a library of orthogonal synthetic dCas12a-repressed promoters and experimentally demonstrated it in HEK293FT, U2OS and H1299 cells lines. Our system expands the toolkit of mammalian synthetic promoters with a new complementary and orthogonal CRISPRi-based system, ultimately enabling the design of synthetic promoter libraries for multiplex gene perturbation that facilitate the understanding of complex cellular phenotypes.

Automated summary

In this study, a computational software pipeline called gDesigner was developed to automate the selection of orthogonal gRNAs and minimize off-target effects and promoter crosstalk. A repression system based on dLbCas12a was engineered, and a library of orthogonal synthetic dCas12a-repressed promoters was created and experimentally validated in various cell lines. This system expands the toolkit of mammalian synthetic promoters and enables the design of synthetic promoter libraries for multiplex gene perturbation, facilitating the understanding of complex cellular phenotypes.