UC San Diego

Autism Spectrum Disorder is one of the most genetically heterogenous neurodevelopmental disorders known making it difficult to characterize and understand its genetic basis. Efforts to screen and curate Autism genes have revealed an overrepresentation of Chromatin Regulator genes, suggesting a significant role in the etiology of this condition. This paper seeks to establish a novel means of studying chromatin regulator genes involved in neurodevelopmental disorders by utilizing the dTAG inducible protein depletion system which would allow for specific, rapid, and reversible protein depletion. In this paper we construct the CRISPR plasmid vectors for the tagging of the Chromatin Regulator SETD5 with the dTAG cassette for inducible protein depletion. One Cas9 vector targeting the SETD5 Stop codon and two HDR vectors were produced, each with a different selection marker to allow for ease in producing homozygous clones in the future. Two confirmed heterozygous HEK293T cell lines were confirmed to contain the dTAG cassette through sequencing. Partial degradation of the SETD5 protein upon induction was demonstrated as expected with heterozygotes and blotting for the HA-tag further confirmed this result. Overall these plasmid vector constructs provide a means of SETD5 tagging with an effective inducible protein system for further investigation into the effects of haploinsufficiency of SETD5. This framework is ideal for use in human embryonic stem cells which can recapitulate the neurodevelopmental stages during which haploinsufficiency has an effect. Therefore, it also lays a framework for the investigation of other genes implicated in neurodevelopment disorders.