Abstract
Purpose :
Several deep-intronic variants (DIVs) in ABCA4 have been classified as causative for Stargardt disease. By strengthening a cryptic splice site, ABCA4 DIV c.5197-557G>T determines the retention of an intronic sequence in the mature mRNA, resulting in a premature termination codon. For the first time, we investigated CRISPR/Cas9 genome editing for the potential to correct a splice defect caused by a DIV in ABCA4 - specifically c.5197-557G>T.
Methods :
Three CRISPR/Cas9 approaches, implementing Streptococcus pyogenes Cas9 (SpCas9) or SpCas9-nickase were designed: dual gRNA/SpCas9, utilizing two paired gRNAs designed to target regions flanking the c.5197-557G>T DIV, dual gRNA/SpCas9-nickase rescue based on the use of two paired gRNAs coupled to SpCas9-nickase, and (single) gRNA/SpCas9 rescue employing single gRNAs. Minigene splicing assay in HEK293T of the DIV c.5197-557G>T was used for preliminary testing the rescue potential of sixteen strategies. Subsequently, eight most-promising ones were validated in patient-derived heterozygous ABCA4 c.5197-557G>T cone photoreceptor precursor cells (CPCs).
Results :
Splicing rescue in the minigene splicing assay ranged from 10±3% to 100±0%. In CPCs, two gRNA combinations each for dual gRNAs/SpCas9, as well as for dual gRNAs/SpCas9-nickase, and four single gRNAs for gRNA/SpCas9 were validated. Due to the partial degradation of the aberrant transcript via nonsense-mediated mRNA decay, CPCs show 28±2% aberrant transcript. The selected strategies reduced the fraction of aberrant transcript up to 5±2%, corresponding to a splicing rescue of 83±5% achieved by a (single) gRNA/SpCas9 strategy. Upon CRISPR/Cas9 treatment, a significant fold increase up to 1.8±0.2 in ABCA4 transcript levels was also detected. Interestingly, when using two paired gRNAs coupled to SpCas9 to target the DIV in CPCs, the level of editing at the ABCA4 target locus quantified 35 days compared to 7 days after treatment revealed a drop in the percentage of edited cells of around 50%, hinting at potential genotoxicity of this design.
Conclusions :
In conclusion, we proved CRISPR/Cas9 effective in addressing a splice defects in ABCA4. This study paves the way to the investigation of CRISPR/Cas9 genome editing as a therapeutic tool to correct the several DIVs in the ABCA4 gene, providing initial evidence of possible permanent splicing correction for Stargardt disease.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.