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Megabase Deletion of the Human EYS Locus Using CRISPR/Cas9
Bruna Lopes da Costa1,2, Anders Steen Knudsen1,2, C Henrique Alves3,4,5,6
1Department of Biomedical Engineering, Columbia University, New York, NY, USA.
Advances in Experimental Medicine and Biology
|February 10, 2025
Summary
Researchers precisely deleted the Eyes Shut Homolog (EYS) gene locus using CRISPR/Cas9 technology. This created EYS-deleted induced pluripotent stem cell lines to study EYS gene function in retinal organoids.
Area of Science:
- Genetics
- Molecular Biology
- Ophthalmology
Background:
- Mutations in the Eyes Shut Homolog (EYS) gene are linked to autosomal recessive retinitis pigmentosa (arRP).
- The exact function of the four identified retinal EYS isoforms in the retina remains unclear, despite known roles in retinal development and ciliary transport.
Purpose of the Study:
- To develop a method for precise genomic deletion of the EYS locus.
- To create EYS-deleted induced pluripotent stem cell (iPSC) lines for functional studies.
Main Methods:
- Utilized a dual single guide RNA (sgRNA) strategy with CRISPR/Cas9.
- Induced paired double-strand breaks (DSBs) to achieve a precise deletion of 1,988,210 bp within the EYS gene locus.
- Generated EYS-deleted (EYSdel) iPSC lines.
Main Results:
- Successfully deleted the entire EYS locus, encompassing all four known human retinal EYS isoforms.
- Established EYSdel iPSC lines suitable for further research.
Conclusions:
- The developed CRISPR/Cas9 dual sgRNA strategy enables precise and large-scale genomic deletions.
- EYSdel iPSC lines provide a valuable tool for investigating the function of EYS in human retinal organoids and understanding arRP pathogenesis.
