Megabase Deletion of the Human EYS Locus Using CRISPR/Cas9.

Mutations in the Eyes Shut Homolog (EYS) gene are associated with autosomal recessive retinitis pigmentosa (arRP). To date, four retinal isoforms of EYS have been identified. However, the precise retinal function of EYS is not fully understood, but it has apparent roles in retinal morphogenesis, architecture, and ciliary transport. Clustered-regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated (Cas) nuclease-mediated approaches are powerful tools for genome engineering in mammalian cells. The use of paired CRISPR/Cas9-induced double-strand breaks (DSBs) using dual single guide RNAs (sgRNA) can lead to precise genomic deletions. In this study, we developed a dual sgRNA strategy to facilitate CRISPR/Cas9-mediated deletion of 1,988,210 bp of the EYS locus, removing the four currently identified human retinal EYS isoforms. This approach can be used to produce EYSdel induced pluripotent stem cell (iPSC) lines to explore the function of EYS in human iPSC-derived retinal organoids.