Abstract
Purpose :
Pathogenic variants in the Crumbs homolog 1 (CRB1) gene are associated with autosomal recessive, early-onset retinal degeneration. The most common pathogenic CRB1 variant c.2843G>A, pCys948Arg occurs in the first nucleotide of exon 9, and is widely classified as a missense mutation. The single nucleotide variant (SNV) c.2842+5G>A is a common CRB1 splice site variant which is positioned in the splice donor site of intron 8. A dual luciferase assay utilising an out-of-frame CRB1 intron-exon hybrid was designed, validated and used to functionally evaluate these two common CRB1 mutations.
Methods :
The out-of-frame CRB1 exon-intron hybrid sequence was inserted into the SGDLuc3.0 backbone between the Renilla and firefly luciferases. Correct splicing, which would result in an in-frame sequence and correlate with firefly luminescence was predicted in-silico. Site-directed mutagenesis introduced the above-mentioned SNVs into the wild-type plasmid. Luciferase assays measuring Renilla and firefly luminescence were performed on transfected HEK293T cells. Splice products were analysed with Sanger sequencing.
Results :
A 7.4-fold increase (SD ± 3.1) in firefly luminesce from baseline Renilla expression was detected in cells transfected with the intron-exon hybrid sequence. Correct splicing of the hCRB1.IE.8.9 hybrid sequence was confirmed with Sanger sequencing. The plasmid containing the SNV c.2842+5G>A resulted in a loss of firefly luciferase luminescence. Sanger sequencing confirmed a read-through of splice donor site. Firefly luminesce fold increase from the plasmid containing the SNV c.2843G>A was significantly reduced (3.31 (SD ± 0.46)) compared to the wild-type plasmid (6.16 (SD ± 1.74)). Sanger sequencing the splice products confirmed a read-through of the splice acceptor site 60% of splice products, while 36% of splice products exhibited correct splicing.
Conclusions :
Due to the location of the most common CRB1 pathogenic variant at the first nucleotide of exon 9, it is plausible that this SNV might affect splicing in addition to causing a missense change. The data generated from the dual luciferase assay is the first to implicate the c.2843G>A SNV as a splice site mutation. A common splice site mutation, c.2842+5G>A, was validated to cause a splice defect in this assay. In future experiments, this dual luciferase assay could be used to screen guide RNA in CRISPR-Cas based therapies.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.