npj Systems Biology and Applications (Sep 2022)

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

  • Michael A. Crone,
  • James T. MacDonald,
  • Paul S. Freemont,
  • Velia Siciliano

DOI
https://doi.org/10.1038/s41540-022-00241-w
Journal volume & issue
Vol. 8, no. 1
pp. 1 – 7

Abstract

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Abstract 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.