CRISPR targeting of SNPs associated with age-related macular degeneration in ARPE-19 cells: a potential model for manipulating the complement system

Abstract

This study explores a novel CRISPR targeting approach to manipulate the complement cascade in AMD using the reliable ARPE19 cell line as an in vitro model. Our results demonstrate efficient DNA editing with CRISPR in ARPE-19 cells, with notable allele specificity in the editing of various AMD-related SNPs in the complement system. The key factor influencing allele specificity is the distance of the target SNP from the PAM site.

Age-related Macular degeneration (AMD) is a major cause of vision loss and is linked to several predisposing single nucleotide polymorphisms (SNPs). CRISPR-mediated genome editing offers the potential to target negatively associated SNPs in an allele-specific manner, necessitating the need for a relevant cell model. The ARPE-19 cell line, with its stable monolayer growth and retinal pigment epithelium (RPE) characteristics, serves as an ideal model for AMD studies. Chronic inflammation and complement system dysregulation are implicated in AMD pathogenesis. Most genetic variations associated with AMD are in complement genes, suggesting their regulatory role. In this study, we conducted targeted PCRs to identify AMD-related SNPs in ARPE-19 cells and used CRISPR constructs to assess allele-specific activity. Guide RNA sequences were cloned into an EF-1-driven SpCas9 vector and packaged into lentivirus. Targeting efficiencies were evaluated with TIDE analysis, and allele-specificity was measured with NGS analysis 30 days post-transduction. Our results showed varying targeting efficiencies depending on guide RNA efficacy. For example, TIDE analysis of CFH SNPs rs1061170 and rs1410996 revealed efficiencies of 35.5% and 33.8%, respectively. CFB SNP rs4541862 showed efficiencies from 3% to 36.7%, and rs641153 ranged from 3.4% to 23.8%. Additionally, allele-specific targeting of AMD-related SNPs rs1061170, rs1410996, rs4541862, and rs641153 ranged from 48% to 52% in heterozygous differentiated ARPE-19 cells. These findings demonstrate the potential to manipulate the complement system in an AMD model by targeting disease-associated SNPs in an allele-specific manner, offering a promising therapeutic approach.

© 2025. The Author(s).

Summary

This study tested whether the CRISPR tool, which is used to change DNA, could change gene variants linked to age-related macular degeneration in lab-grown eye cells.

The researchers found that some variants could be targeted more accurately than others. CRISPR worked best when the change was close to a nearby DNA guide point. The study suggests these cells could help test future gene-editing treatments, although other tools may work better for some variants.

Keywords: age-related macular degeneration, CRISPR, eye cells, immune system genes, targeted gene editing, small DNA changes, CFH, CFB, retinal pigment cells, gene editing, DNA guide point