Functionally characterizing obesity-susceptibility genes using CRISPR/Cas9, in vivo imaging and deep learning

Hundreds of loci have been robustly associated with obesity-related traits, but functional characterization of candidate genes remains a bottleneck. Aiming to systematically characterize candidate genes for a role in accumulation of lipids in adipocytes and other cardiometabolic traits, we developed a pipeline using CRISPR/Cas9, non-invasive, semi-automated fluorescence imaging and deep learning-based image analysis in live zebrafish larvae. Results from a dietary intervention show that 5 days of overfeeding is sufficient to increase the odds of lipid accumulation in adipocytes by 10 days post-fertilization (dpf, n = 275). However, subsequent experiments show that across 12 to 16 established obesity genes, 10 dpf is too early to detect an effect of CRISPR/Cas9-induced mutations on lipid accumulation in adipocytes (n = 1014), and effects on food intake at 8 dpf (n = 1127) are inconsistent with earlier results from mammals. Despite this, we observe effects of CRISPR/Cas9-induced mutations on ectopic accumulation of lipids in the vasculature (sh2b1 and sim1b) and liver (bdnf); as well as on body size (pcsk1, pomca, irs1); whole-body LDLc and/or total cholesterol content (irs2b and sh2b1); and pancreatic beta cell traits and/or glucose content (pcsk1, pomca, and sim1a). Taken together, our results illustrate that CRISPR/Cas9- and image-based experiments in zebrafish larvae can highlight direct effects of obesity genes on cardiometabolic traits, unconfounded by their - not yet apparent - effect on excess adiposity.