CRISPRi-based screening for identifying regulators of NRXN1-specific Alternative Splicing

Presenter

Rachel Mary Hobson

Campus

UMass Amherst

Sponsor

ChangHui Pak, Department of Biochemistry and Molecular Biology, UMass Amherst

Schedule

Session 5, 3:30 PM - 4:15 PM [Schedule by Time][Poster Grid for Time/Location]

Location

Poster Board A49, Campus Center Auditorium, Row 3 (A41-A60) [Poster Location Map]

Abstract

Neurexin-1 (NRXN1) is a presynaptic cell adhesion protein critical during synaptic development and maintenance. NRXN1 has extensive alternative splicing from six canonical splice sites (SS1-6), giving rise to over one thousand possible splice variants. Copy number variations in the NRXN1 gene are implicated in autism, schizophrenia, and intellectual disabilities. Recent work from our lab demonstrated that heterozygous NRXN1 deletions in human glutamatergic induced neurons induce misregulation of alternative splicing of key synaptic genes, including NRXN1 itself and impair synaptic transmission, specifically reducing excitatory neurotransmitter release probability. Therefore, it is critical to test whether NRXN1 alternative splicing misregulation is driving this functional deficit. To this end, we are creating a CRISPRi-based system coupled with NRXN1-specific splicing reporters to identify novel proteins responsible for NRXN1 splicing regulation. Through molecular cloning, three single guide RNAs (sgRNA) targeted to specific splicing regulators, are inserted into a lentiviral backbone to create an infectable sgRNA construct. Confirmed constructs, via Sanger sequencing, will be tested in human glioblastoma cells (H4i) stably expressing dCas9-KRAB. Once knockdown is confirmed by qRT-PCR, we will co-introduce sgRNAs and fluorescence reporters specific to NRXN1 alternative splicing to test whether KD of certain genes impact NRXN1 splicing patterns. Manipulation of both known and unknown regulators of splicing machinery will reveal components of NRXN1 splicing regulation. Ultimately, this experimental system will be applied to human glutamatergic induced neuronal system (i3N). Results will provide foundational knowledge on the molecular basis of NRXN1 alternative splicing-mediated contributions to neuropsychiatric disorders.

Keywords

Alternative Splicing, CRISPRi, Synaptic development

Research Area

Neuroscience and Cognitive Science

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