Cas9 gRNA engineering for genome editing, activation and repression

Abstract

We demonstrate that by altering the length of Cas9-associated guide RNA(gRNA) we were able to control Cas9 nuclease activity and simultaneously perform genome editing and transcriptional regulation with a single Cas9 protein. We exploited these principles to engineer mammalian synthetic circuits with combined transcriptional regulation and kill functions governed by a single multifunctional Cas9 protein.

Genome Editing Reveals Glioblastoma Addiction to MicroRNA-10b

“…Although the application of human lentiviral gene therapy is hampered by the risk of carcinogenesis by random proviral integration into the genome of normal somatic cells, future studies should determine whether this risk is acceptable for local GBM treatment, given the lack of efficacious drugs and poor life expectancy of patients with the disease. The identification of Staphylococcus aureus (SaCas9) and other smaller Cas9 enzymes that can be packaged into adeno-associated viral vectors, which are highly stable and effective in vivo, [29][30][31] …”

Genome-Editing Technologies: Principles and Applications

“…Similarly, dCas9 repressor proteins targeted to distal regulatory elements have been found to facilitate chromatin remodeling and gene repression via epigenomic modification . Finally, by simply reducing the length of the gRNA, catalytically active variants of Cas9 can stimulate transcription without inducing DNA breaks (Dahlman et al 2015;Kiani et al 2015), enabling orthogonal gene knockout and activation with the same Cas9 variant in a single cell.…”

CRISPR-Cas type II-based Synthetic Biology applications in eukaryotic cells

“…Similar results were presented in. 61 Here, Cas9 (from S. thermophilus and S. aureus) was fused to the VPR activation domain and proved to be able to both activate and repress promoters when interacting with short gRNAs sharing a 14 -nucleotide-complementary sequence with their target promoters.…”