Cas9 is an RNA-guided DNA endonuclease that is being used for sequence-specific DNA recognition, genome engineering, targeted gene activation/repression and genome imaging. Two commonly used variants of Cas9 are SpCas9 and SaCas9, which naturally occur in S pyogenes and S aureus, respectively. A pressing concern in the therapeutic genome editing using Cas9 is undesired off-target editing and chromosomal translocations that are associated with high activity levels of Cas9. Much interest also exists for rapid termination of Cas9 activity upon completion of on-target editing. Additionally, rapid, dosable, reversible, temporal, and orthogonal control of Cas9-based technologies (e.g., transcriptional activation and repression) will significantly expand the application of these technologies in biomedical research. We propose to develop small-molecule inhibitors of SpCas9 and SaCas9 that will allow rapid, dosable, temporal, and orthogonal control of Cas9 activities in HIV-associated immune cells as a model. To this end, we propose to deploy state-of-art technologies from organic chemistry and chemical informatics, biophysics, structural biology, and genome engineering.
Cas9 is an RNA-guided DNA endonuclease associated with CRISPR adaptive bacterial immunity system that has revealed an enormous opportunity for gene therapy. Two commonly used variants of Cas9 are SpCas9 and SaCas9. The goal of the proposal is to develop small- molecule inhibitors of SpCas9 and SaCas9 that will allow rapid, dosable, temporal, and orthogonal control of Cas9 activities in mammalian cells.
|Maji, Basudeb; Moore, Christopher L; Zetsche, Bernd et al. (2017) Multidimensional chemical control of CRISPR-Cas9. Nat Chem Biol 13:9-11|