Disruption of Pik3r1 promotes muscle hyperplasia and lipolysis in grass carp (Ctenopharyngodon idella).

Grass carp (Ctenopharyngodon idella) is a major aquaculture species, yet production efficiency is often constrained by growth performance and metabolic disorders such as hepatic lipid deposition. Conventional selective breeding faces challenges in further improving these complex traits, necessitating precision genetic interventions. In this study, we combined comparative transcriptomics with CRISPR/Cas9 mediated gene disruption to investigate the regulatory role of Pik3r1. Comparative RNA-seq of fast- and slow-growing individuals together with protein-protein interaction network analysis identified Pik3r1 as a candidate negative regulator of growth. To validate its function, we generated Pik3r1 mosaic F0 mutants by targeting the SH3 domain of Pik3r1. Disruption of Pik3r1 conferred a profound growth advantage, increasing body weight by 51.8% and body length by 13.5% compared to wild-type controls. Histological analyses revealed that enhanced muscle accretion was driven by both hypertrophy with a 22.50% increase in myofiber cross-sectional area and hyperplasia characterized by an 18.90% rise in fiber density, coupled with a 67.7% reduction in hepatic lipid accumulation. At the molecular level, Pik3r1 disruption relieved the inhibitory effect on the PI3K/AKT/mTOR cascade, with upregulation of PI3K, AKT, mTOR, S6K1, Myod1, and Mybpc1 and downregulation of 4ebp1 and Pik3ca in muscle, accompanied by upregulation of PPARα and CPT1a and downregulation of FASN and ACC1 in the liver. Furthermore, a clear genotype phenotype correlation was observed: fish carrying confirmed premature termination codons exhibited substantially greater growth enhancement (77.6% increase in body weight) than those with in-frame mutations. Collectively, our results support Pik3r1 as a key regulator linking somatic growth and lipid metabolism in grass carp, and highlight its potential as a target for molecular breeding aimed at improving yield and carcass quality.