A Singular System with Precise Dosing and Spatiotemporal Control of CRISPR‐Cas9
Abstract
Several genome engineering applications of CRISPR‐Cas9, an RNA‐guided DNA endonuclease, require precision control of Cas9 activity over dosage, timing, and targeted site in an organism. While some control of Cas9 activity over dose and time have been achieved using small molecules, and spatial control using light, no singular system with control over all the three attributes exists. Furthermore, the reported small‐molecule systems lack wide dynamic range, have background activity in the absence of the small‐molecule controller, and are not biologically inert, while the optogenetic systems require prolonged exposure to high‐intensity light. We previously reported a small‐molecule‐controlled Cas9 system with some dosage and temporal control. By photocaging this Cas9 activator to render it biologically inert and photoactivatable, and employing next‐generation protein engineering approaches, we have built a system with a wide dynamic range, low background, and fast photoactivation using a low‐intensity light while rendering the small‐molecule activator biologically inert. We anticipate these precision controls will propel the development of practical applications of Cas9.
Document Details
- Document Type
- Pub Defense Publication
- Publication Date
- Apr 02, 2019
- Source ID
- 10.1002/ange.201900788
Entities
People
- Amit Choudhary
- Basudeb Maji
- Benjamin K. Law
- Debasish Manna
- Kurt J Cox
- Qingxuan Zhou
- Ralph Mazitschek
- Soumyashree A. Gangopadhyay
Organizations
- Brigham and Women's Hospital
- Broad Institute
- Burroughs Wellcome Fund
- Defense Advanced Research Projects Agency
- Harvard Medical School
- Massachusetts General Hospital
- National Institutes of Health