Gene therapy trials for inherited retinal dystrophies are ongoing, emphasizing the necessity to ensure that the disease in the selected patients is truly caused by mutations in the gene targeted by the therapeutic approach. This highlights the need for an objective and reliable pathogenicity assessment for individual variants and genotypes of patients. Mutations in CNGA3 cause autosomal recessive achromatopsia (ACHM) and gene therapy trials are ongoing at several centers. CNGA3 and CNGB3 encode the cyclic nucleotide-gated (CNG) cation channel of the cone photoreceptors. We have CNGA3 genotypes of >300 ACHM patients and ~260 different variants - 65% of these are missense variants. We will retrieve data of in silico prediction tools, allele frequencies and segregation analysis and apply the ACMG scoring system for CNGA3 variant evaluation. Using an in vitro aequorin-based bioassay, we will test the functionality of CNG channels with previously uncharacterized, presumably disease-causing missense variants in HEK293 cells allowing us to evaluate, confirm or reject the in silico prediction. We will deliver an objective, functionally confirmed pathogenicity score for CNGA3 variants observed in our patient cohort, the literature and mutation databases, unequivocally identifying patients eligible for future gene therapy trials on ACHM.