Effects of targeted deletion of a 284 bp avian-specific highly conserved element within the Sim1 gene on flight feather development in chickens.

Flight feathers represent a hallmark innovation of avian evolution. Recent comparative genomic analyses identified a 284 bp avian-specific highly conserved element (ASHCE) located within the eighth intron of the SIM bHLH transcription factor 1 ( Sim1) gene, postulated to act as a cis-regulatory element governing flight feather morphogenesis. To investigate its functional significance, genome-edited (GE) primordial germ cell (PGC) lines carrying targeted ASHCE deletions were generated using CRISPR/Cas9-mediated editing, with germline chimeric males subsequently mated with wild-type (WT) hens to obtain GE progeny. The resulting GE chickens harbored 257-260 bp deletions, excising approximately half of the Sim1-ASHCE sequence. Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) analysis showed an average 0.32-fold reduction in Sim1 expression in the forelimbs of GE embryos at day 8 (E8) compared to WT counterparts. Despite this, GE chickens developed structurally normal flight and tail feathers. In situ hybridization localized Sim1 expression to the posterior mesenchyme surrounding flight feather buds in E8 WT embryos, but not within the buds themselves. These results suggest that partial deletion of Sim1-ASHCE, despite diminishing Sim1 expression, does not disrupt flight feather formation. The excised region appears to possess enhancer activity toward Sim1 but is dispensable for flight feather development. Complete ablation of the ASHCE will be necessary to fully resolve the regulatory role of Sim1 in avian feather morphogenesis.