Fig. 1

Experimental flow chart. The flow chart describes the workflow, exclusion criteria, and the final number of larvae included in the statistical analysis in all experiments. (a) Transgenic (Tg[−1.2insH2b:mCherry; 2.8fabp10a:GFP]) larvae (AB) were fed on either a control amount (n = 350), 3x as much (n = 366) from day 5 to day 9 post fertilization. (b) Zebrafish orthologues of 12 established human obesity genes were targeted using a multiplexed CRISPR/Cas9 approach. Two sets of CRISPR/Cas9 founders for eight (arid5b, mc4r, lepr, negr1, pcsk1, pomca, pomcb, sec16b) and seven (bdnf, irs1, irs2a, irs2b, sh2b1, sim1a, sim1) orthologues were generated using fertilized eggs from parents with fluorescently labelled liver and pancreatic beta cell nuclei. Founders were raised and in-crossed, and F₁ larvae were overfed and phenotypically characterized at 10 days post-fertilization (dpf) for 14 traits, using semi-automated fluorescence microscopy and biochemistry. (c) Fertilized eggs from parents with fluorescently labelled liver and pancreatic beta cell nuclei were microinjected at the single cell stage with a duplex gRNA targeting kita (controls) or additionally with a duplex gRNA to generate affected larvae for the candidate gene of interest. Larvae were overfed from day 5 to day 9. Larvae were imaged on day 10 or 11 and/or collected for biochemistry. (d) A duplex gRNA targeting the kita gene was injected in fertilized eggs from AB parents to generate controls; while affected larvae were generated by micro-injecting up to three duplex gRNAs targeting early exons or domains of each orthologue of the human gene of interest, alongside the duplex gRNA targeting kita. Larvae were overfed from day 5 to day 7. At day 8, larvae were fed with fluorescently labeled food and imaged to quantify food intake.