CRISPR‐Cas9 Gene Editing Yields a Novel Rat Model with Genetic Regulation of Obesity
Authors: Clark Karen C , Pettus Janette M , Grobe Justin L , Kwitek Anne E 
Published: November 2023

Metabolic Syndrome (MetS) is the coexistence of obesity, hypertension, dyslipidemia, and hyperinsulinemia that collectively increases risk for heart disease, diabetes, and stroke. More than 20% of US adults suffer from MetS, and due to the sizeable public health burden, numerous studies have undertaken to determine the genetics underlying the disease. Gene discovery studies in MetS have met with modest success, due to complex genetic underpinnings, uncertain genetic background effects, and highly variable environmental stressors. For this reason, we employ the genetically and environmentally tractable Lyon Hypertensive (LH) and Lyon Normotensive (LN) rats—well‐characterized rat models of Metabolic Syndrome sensitivity and resistance, respectively.Using a genome wide approach, we identified a candidate gene, RGD1562963 (RGD), that has genetic effects on components of MetS. We have generated CRISPR‐Cas9 mediated frameshift mutations in exon 2 of RGD in an LH derived strain and are studying the effects of the mutations on the obesity phenotype in the LH derived rat.We have determined that the mutant RGD allele acts in an additive manner to significantly decrease body weight in adult animals. At 18 weeks of age, heterozygous (RGD+/−) and homozygous mutant (RGD−/−) animals are 60–80 grams leaner, respectively, compared to wild‐type (RGD+/+) sex‐matched controls, representing a 13–17% reduction in body weight (RGD+/+: 450.4g±4.025, n=11; RGD+/−: 392.3g±1.732, n=3; RGD−/−370.3g±12.45, n=3; p < 0.01 ANOVA vs RGD+/+ control).We speculate this difference may be due to an increase in resting aerobic metabolic rate (RMR) observed in RGD mutants compared to wild‐type (RGD+/− :1.05 kcal/hr ± 0.007, n=3; RGD+/+ 0.85 kcal/hr ± 0.007, n=2, p < 0.01). Interestingly, this difference only appears after the animals have reached adulthood and completed the pubertal transition, which occurs around 10 weeks of age. As juveniles, all animals have high RMR, irrespective of genotype. As animals reach sexual maturity, a significant RMR reduction occurs in the wild‐type controls (30%) compared to only 10% in the RGD mutants, resulting in a higher metabolic rate overall. The decrease in RMR may drive the later divergence in body weight, so that a significant decrease in body weight is observed in adult mutants compared to wild‐type controls.RGD1562963 is uncharacterized, and has no annotated functional domains. Our studies suggest RGD1562963 is an obesity susceptibility gene. Furthermore, inhibition of this gene at the whole body level is tolerated, and induces increased resting metabolic rate and prevention of obesity, making it a possible therapeutic target. Future studies of RGD function will yield insight into the mechanism underlying the different phenotypes observed in animals with an RGD mutation, and provide information on the complex regulation of metabolism in humans.Support or Funding InformationNIH T32 GM008629, R01 HL089895, R21 DK089417, R24 HL114474; AHA 14GRNT20410043; Fraternal Order of Eagles Diabetes Research Center