Developing a CRISPR-mediated safe harbor strategy to correct patient-derived induced pluripotent stem cells

Abstract

Purpose : CRISPR-Cas9 genome editing has revolutionized basic and translational vision research. The ability to select for corrected disease-causing variants provides an effective strategy in disease modelling and development of cell therapy for inherited retinal diseases. However, in some cases the placement of the selection cassette interferes with expression from the target locus. We hypothesized that delivering CRISPR-Cas9 reagents, a donor template targeting the locus of interest, and a donor template carrying the selection cassette targeting a separate locus would address selection-based transcriptional interference at the intended locus. The purpose of this study was to develop a co-targeting strategy employing the AAVS1 safe harbor site as a selection locus.

Methods : We created a plasmid carrying a S. pyogenes Cas9 expression cassette and single guide RNA expression cassettes targeting both the locus of interest and the AAVS1 site. The CRISPR-Cas9 expression plasmid, a donor template carrying homologous sequence to the locus of interest, and a donor template carrying a puromycin selection cassette flanked by AAVS1 homology sequence were delivered to human cells via cationic lipid transfection and cultured under puromycin selection for 10-14 days. Genomic DNA was isolated from puromycin resistant clones and screened for modification at the locus of interest via PCR.

Results : We cloned and screened single guide RNAs targeting five loci: CRX, GRK1, VSX2, USH2A, and CEP290. We achieved successful co-targeting at all five loci as revealed by amplification of donor-target junctions and RFLP analysis. Clonal analysis at three loci – CRX, GRK1, and VSX2 – indicated co-targeting in 5%, 20%, and 35% of puromycin-resistant clones, respectively.

Conclusions : This work may accelerate the development of CRISPR-Cas9 therapies for inherited retinal dystrophies.

This is a 2021 ARVO Annual Meeting abstract.