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

Flight feathers are a defining feature of birds. A recent comparative genomic study identified a 284 bp avian-specific highly conserved element (ASHCE) in non-coding regions that may regulate flight feather development. This element is located within the eighth intron of the Sim1 (SIM bHLH transcription factor 1) gene and is postulated to function as a cis-regulatory element. To investigate its role in flight feather formation, we used CRISPR/Cas9 to generate genome-edited (GE) primordial germ cell (PGC) lines with ASHCE deletions, producing GE chickens by mating germline chimeric males with wild-type (WT) hens. The GE chickens exhibited 257–260 bp deletions, including approximately half of the Sim1-ASHCE sequence. 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. Despite this, GE chickens displayed normal flight and tail feather development. In situ hybridization revealed Sim1 expression in the posterior mesenchyme of flight feather buds in E8 WT embryos, but not in the feather buds themselves. These results suggest that the 257–260 bp deletions, including approximately half of the Sim1-ASHCE, are not required for the formation of flight feathers. However, the deleted region exhibits enhancer activity for Sim1. Further research is necessary to fully remove the ASHCE and clarify the role of Sim1 in feather development.