Abstract
Purpose :
Age-Related Macular Degeneration (AMD) is a leading cause of blindness in the developed world. Despite extensive research, there remains incomplete understanding of disease development and progression. Single nucleotide polymorphisms (SNPs) are one of several mechanisms that have been associated with a predisposition to develop AMD. SNPs have previously been shown to be editable with CRISPR machinery, causing endonuclease mediated double-strand DNA breaks specified by guide RNA (gRNA) targeting. In this instance, CRISPR gene editing can target SNPs in an allele-specific manner. To investigate CRISPR editing of AMD-related SNPs, a retinal pigment epithelium (RPE) cell line, ARPE19, which possesses several AMD-related SNPs, was used. ARPE19 is a spontaneously arising RPE cell line derived from the healthy eye of a 19-year-old male. These cells grow in a stable monolayer, express RPE-specific markers CRALBP and RPE-65 and exhibit a typical RPE morphology including tight junction formation, representing a suitable model to validate CRISPR targeting of AMD-related SNPs.
Methods :
SNPs in the ARPE19 cell line were identified via genotyping. U6 promoter driven gRNAs targeting AMD-related SNPs and VEGF were cloned into a gRNA cloning plasmid using BbsI cut sites. gRNAs were transfected into ARPE19 and HEK293T cells in combination with a CMV promoter driven SpCas9 plasmid to achieve a dual transfection system. To assess editing efficiency via tracking of indels by decomposition (TIDE) analysis, target SNPs were PCR amplified using primers designed to flank the target SNP with ~200bp on the 5’ end and ~500bp on the 3’ end.
Results :
TIDE analysis of PCR products from ARPE19 cells with dual plasmid transfection of SpCas9 and gRNAs against the following AMD-related SNPs [rs1410996, rs380390, rs2230199, rs147859257, rs641153 CFB SNP, rs4541862] and VEGF targeting gRNA, showed 4-5% targeting efficiency, compared to 30% in HEK293T cells.
Conclusions :
The dual plasmid targeting approach using SpCas9 and U6-driven gRNA plasmids efficiently delivers the CRISPR system to HEK293T cells. Low editing efficiency in ARPE19 cells may be due to inadequate time for CRISPR mechanisms to elucidate effect, as ARPE19 cells begin to degrade after 24 hours, or due to the more stable differentiation of these cells which may involve epigenetic changes to targeted genes.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.