Rapid, in vitro phenotyping of monogenic neurological disorders using CRISPRi and high-throughput all-optical electrophysiology.

Project SummaryAutism spectrum disordersASDare estimated to affect more thanof children underyears of age in the USThese neurodevelopmental disorders are characterized by impaired social communicationrestrictive interests and repetitive or stereotyped behaviorsThere are frequent co morbidities in ASDincluding epilepsy and intellectual disabilityASD can be divided into two main categories based on differing causal factorsSyndromic ASD are caused by highly penetrantsingle gene mutations or genetic copy number variants that impact clinically relevant geneswhile idiopathic ASD result from more complex combinations of genetic and environmental factorsDespite decades of effortno drugs are available for specific treatment of ASDwhich remain a major area of unmet medical needDrug discovery for ASD has been limited by a lack of knowledge of underlying disease biologyRecent progress in elucidating the genetic basis of ASD is providing a foundation for new therapeutic discovery approachesEvidence for synaptic dysfunction in genetic models of syndromic ASD and the identification of mutations in genes encoding synaptic proteins in idiopathic autism suggest that alterations in neurotransmission represent a core pathogenic mechanismMoreoverthere is evidence for shared disease mechanisms among ASDincluding syndromic and idiopathic formsThese lines of evidence support therapeutic discovery strategies aimed at identifying and targeting shared neurophysiological disease mechanisms in ASDThe overall objective of this proposal is to combine our Optopatch all optical electrophysiology platform with a newly developed method to modulate gene expressionCRISPR interferenceand to apply this system to parallel generation and neurophysiological characterization of multiple ASD genetic loss of function modelsOptopatch is the only optogenetic system with compatiblegenetically encoded actuator and voltage sensor componentsenabling light mediated stimulation and precise recording of electrical activity in neurons with unprecedented throughputCRISPRi achieves gene knockdown by targeting a transcriptional repressor hybrid proteinKRAB dCasto a specified gene target via a complementary guide RNAEfficient knockdown of multiple genes is achieved simply by changing the guide RNAWe will first optimize the CRISPRi Optopatch system and then apply it to characterizing effects on neuronal activity and synaptic transmission in neuronal knockdown models forASD genes defined by lossof function mutationsThrough this large scale phenotyping effortwe propose to identify core disease mechanisms in ASDIf successfulthe program will be positioned for Phase II research aimed at generating commercially valuablevalidated phenotypic screening assays for ASD drug discoveryValidated assays will provide an attractive basis for discovery partnerships with pharmaceutical companies to discover and develop novelmechanism based treatments for ASDUltimatelythis program stands to benefit the large population of ASD patients and their families and caregivers by enabling discovery of new treatments