BAd-CRISPR: Inducible gene knockout in interscapular brown adipose tissue of adult mice The Journal of Biological Chemistry, 2021 Dec 01

"Transfection of adipocyte precursors with sgRNAs predominantly causes frameshift mutations in the target gene.A, U6 promoter-driven sgRNAs were cloned into an AAV expression vector using PmlI and KpnI. The expression vector also contained CMV promoter-driven mCherry and 5′ and 3′ ITRs to facilitate packaging of the cassette into AAV8. B–D, Sanger sequencing traces from Cas9-expressing adipocyte precursors transfected with AAV8-sgRNAs targeted to Adipoq, Atgl, and Plin1. For each target, the primers (purple arrows) were designed upstream and downstream of the sgRNA cut site (yellow arrow). Chromatograms of the reverse strand are shown, and the cut site (black dotted line) and PAM (red underline) are displayed for each sgRNA. The highest frequency indels for each sgRNA and percent contribution as determined by TIDE and the Synthego <t>ICE</t> Analysis tool are shown. Frameshift mutations were calculated by dividing the number of frameshifts over the total number of mutations, as estimated by the Synthego ICE Analysis tool. AAV, adeno-associated virus; Adipoq, adiponectin; ATGL, adipose triglyceride lipase; PAM, protospacer-adjacent motif; PLIN, perilipin; sgRNA, single guide RNA; <t>TIDE,</t> <t>tracking</t> of indels by decomposition. "