Generation of Cacng2a Promotor-less Mutant Line in Zebrafish Using CRISPR/Cas9 Genome Editing Technology

George "AJ" Holmes*, Samantha Hershman, and Sadie A Bergeron

Department of Biology, West Virginia University, Morgantown, WV 26506

Presentation Category: Biological & Biochemical Sciences (Poster Presentation #91)

Student’s Major: Biology and Psychology

Eye-tracking dysfunction (ETD) is a common endophenotype of neurodevelopmental disorders like schizophrenia and autism. Several genes have been associated with the development of ETD, including calcium voltage-gated channel auxiliary subunit gamma 2 (CACNG2) which functions as a transmembrane AMPA receptor regulatory protein that facilitates cell-cell communication in the central nervous system. To determine the function of CACNG2 and its influence on visual neural circuit development and behavior CRISPR/Cas9 gene editing technology was used to knockout its zebrafish ortholog cacng2a. sgRNAs were designed using CHOPCHOP (https://chopchop.cbu.uib.no) to target ~500 basepairs upstream and downstream of the transcription start site. Transgenic zebrafish embryos that will develop to have GFP expression in neural circuits connecting the eyes to the brain were microinjected at the one-cell stage with Cas9 mRNA, sgRNA, and GFP mRNA. Injected embryos were screened for ubiquitous GFP expression at 1-day post fertilization to verify mRNA integrity, and a sample of injected, GFP-positive embryos as well as uninjected controls were lysed to extract genomic DNA. Fluorescent polymerase chain reaction amplified genomic fragments containing the sgRNA target site were used for fragment analysis to determine the efficacy of Cas9 cutting activity. Additional GFP-positive larvae were raised to adulthood and bred with wild type zebrafish to generate lines of putative carriers of cacng2a promoter mutations which will be screened. I expect to generate and perform initial genotypic and phenotypic characterization of zebrafish lacking cacng2a expression which could be used for future analysis of visual neural circuit development and function.

Funding: West Virginia University Department of Biology, West Virginia University Honors EXCEL

Program/mechanism supporting research/creative efforts: Other, WVU Honors EXCEL