Second Generation CRISPR Repressors Enable Highly Efficient Regulation of Sophisticated Genetic Circuits in Mammalian Cells

The CRISPR-associated endonuclease Cas9 is a versatile tool for targeted genome editing and transcriptional regulation. It’s nuclease-deactivated form, dCas9 can be coupled with sequence-specific sgRNA and can be converted into a modular and multiplexable transcriptional repressor. However, due to its limited ability to achieve complete silencing of target genes, there has been only a handful of applications reported so far. In this work, we report an improved dCas9 based repressor, which was engineered by fusing a rationally designed bipartite KRAB super repressor to its C-terminal domain. In addition, we demonstrate the superiority of this repressor over a library of previous tools not only in silencing coding and non-coding genes but also in the context of generation of sophisticated synthetic genetic circuits. When employed in a two-layer cascade circuit, the repressor surpasses other tools in efficiently transferring information over a sophisticated circuit topology, thereby rescuing full functionality of the cascaded circuit. These systems can be taken as valuable toolbox to engineer robust mammalian synthetic circuits for future gene and cellular therapies.