Development of CRISPR-Cas guideRNA screening platform for allele-specific reduction of mutant Huntingtin protein

Project Summary RNA guided DNA endonucleases derived from bacterial CRISPR systems promise to rapidly advance genome editing based therapeuticsHoweverthis approach is significantly limited by the ability to target only a mutated allele in the human genome in a safe and specific mannerHuntingtonandapos s diseaseHDis caused by such a dominant genetic defect and there is clear evidence that mutant allele disruption can positively impact disease progressionWe have previously published a safe and efficient method to deliver CRISPR Casto neurons in vivo and the key limitation we now face to bringing a CRISPR based therapeutic to clinical trials is the lack of therapeutically relevant guides that target the mutant HuntingtinHTTgeneTo this endwe aim to identify CRISPR guide RNAs that will reduce the burden of mutant HTT protein on the cellTo accomplish this we propose a screen that can rapidly readout the effect a genetic edit has on both copies of the HTT gene in highthroughput and on a single cell basisWe will apply this screen to test thousands of guides for activity and allele specificityIf successfulthese experiments will advance a core technology for genome editing and identify individual products that will be rigorously characterized in preclinical work for a HD therapeutic product during an application oriented Phase II Narrative Programmable nuclease based genome editing promises to create a new class of genetic therapiesHere we propose to build and utilize a screening system to identify the possible therapeutic targets for the HuntingtinHTTgenewhich when mutatedis the underlying cause of genetically dominant Huntingtonandapos s disease