In Silico CRISPR-Cas-Mediated Base Editing Strategies for Early-Onset, Severe Cone-Rod Retinal Degeneration in Three Patients, Including the Novel Variant C.2833G>A

Pathogenic variants in the gene lead to severe, childhood-onset retinal degeneration leading to blindness in early adulthood. There are no approved therapies, and traditional adeno-associated viral vector-based gene therapy approaches are challenged by the existence of multiple CRB1 isoforms. Here, we describe three variants, including a novel, previously unreported variant that led to retinal degeneration. We offer a CRISPR-Cas-mediated DNA base editing strategy as a potential future therapeutic approach. This study is a retrospective case series. Clinical and genetic assessments were performed, including deep phenotyping by retinal imaging. In silico analyses were used to predict the pathogenicity of the novel variant and to determine whether the variants are amenable to DNA base editing strategies. Case 1 was a 24-year-old male with cone-rod dystrophy and retinal thickening typical of retinopathy. He had a relatively preserved central outer retinal structure and a best corrected visual acuity (BCVA) of 60 ETDRS letters in both eyes. Genetic testing revealed compound heterozygous variants in exon 9: c.2843G>A, p.(Cys948Tyr) and a novel variant, c.2833G>A, p.(Gly945Arg), which was predicted to likely be pathogenic by an in silico analysis. Cases 2 and 3 were two brothers, aged 20 and 24, who presented with severe cone-rod dystrophy and a significant disruption of the outer nuclear layers. The BCVA was reduced to hand movements in both eyes in Case 2 and to 42 ETDRS letters in both eyes in Case 3. Case 2 was also affected with marked cystoid macular lesions, which are common in CRB1 retinopathy, but responded well to treatment with oral acetazolamide. Genetic testing revealed two c.2234C>T, p.(Thr745Met) variants in both brothers. As G-to-A and C-to-T variants, all three variants are amenable to adenine base editors (ABEs) targeting the forward strand in the Case 1 variants and the reverse strand in Cases 2 and 3. Available PAM sites were detected for KKH-nSaCas9-ABE8e for the c.2843G>A variant, nSaCas9-ABE8e and KKH-nSaCas9-ABE8e for the c.2833G>A variant, and nSpCas9-ABE8e for the c.2234C>T variant. In this case series, we report three pathogenic variants, including a novel c.2833G>A variant associated with early-onset cone-rod dystrophy. We highlight the severity and rapid progression of the disease and offer ABEs as a potential future therapeutic approach for this devastating blinding condition.