Optimizing CRISPR/Cas9-Mediated Bax Gene Knock-Out in Sympathetic Neurons (2024)

Undergraduate: Gretchen Davis

Faculty Advisor: Mohanish Deshmukh
Department: School of Medicine (SOM), UNC Neuroscience Center

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The CRISPR-Cas9 editing system utilizes a Cas9 nuclease and target specific single guide (sgRNA) to produce precise genomic knockouts or deletions. However, this system still requires optimization in certain cell types. Specifically, establishing an effective CRISPR-Cas9 system in sympathetic neurons remains a challenge. Utilizing three experimental approaches, we tested the most efficient knockout approach utilizing the apoptotic regulator gene Bax as the target gene. Experimental approach one consisted of electroporating Bax sgRNA into neurons isolated from a Cas9 floxed mouse line. This mouse line includes inducible Cre-dependent Cas9/GFP expression; therefore, Cre lentivirus was added to cultures to induce Cas9 and GFP expression. The second experimental approach consisted of transducing a single Bax sgRNA-Cas9 construct in wild-type neurons. Lastly, the third experimental approach involved transducing two lentiviruses into neurons from Cas9 floxed mice, one consisting of Cre recombinase and one with Bax sgRNA. The efficacy of these approaches was assessed by measuring neuronal resistance to apoptosis upon growth factor deprivation. As the Bax gene is necessary for apoptosis to occur, neuronal survival indicated successful Bax knockout. Ultimately, the approach consisting of transducing two lentiviruses (Cre and sgRNA) yielded the highest knockout efficacy.