Genome editing using a novel CRISPR protein

Project Summary RNA guidedDNA targeting endonucleases derived from bacterial CRISPR systems promise to rapidly advance genome editing based therapeuticsThis approachhoweveris significantly limited by the paucity of available programmable endonucleasesA protein known as CasX was recently discovered by the Doudna group at UC Berkeley and shown to function in a fashion conceptually similar to the unrelated CRISPR proteins Casand CpfThe genome editing activity of CasX has not been tested in human cellsHerein Phase I feasibility studieswe propose to verify and quantify the activity of CasX for genome editing applicationsInitial work and analysis of CasX suggests that its activity may be lower than that of Casor CpfTo this endwe have developed a high throughput screening platform for identifying proteins with higher activityThis platform will be used to engineer more active variants of CasX and the most promising variants will be screened for improved activity in human cellsIf successfulthese experiments will advance a core technology for genome editing that will be rigorously characterized and optimized in an application oriented Phase II Narrative Programmable nuclease based genome editing promises to create a new class of genetic therapiesHere we optimize the unique yet minimally characterized protein CasX for genome editing in human cells