Transactivation of cell lines for functional genomic studies

In Australia, ∼50% of individuals with a suspected monogenic disorder do not reach a genetic diagnosis. Many such individuals harbour variants of uncertain significance (VUS), where resolution of their pathogenicity requires additional functional evidence. For VUS predicted to impact splicing, additional evidence can be derived from analysis of the variant genes’ mRNA. The ability to obtain and study the patient’s RNA within the context of their own genetic and cellular context is paramount as it includes critical disease modifiers, and enhances opportunities for precision medicines. For that purpose patient blood and/or skin derived cell lines are used, but their use is limited to the study of genes which are expressed within them. This fundamental limitation excludes their use to study >1400 Mendelian disease genes, whose expression levels are insufficient for the purpose of function investigations in the most frequently used patient cells generated from blood and/or skin. To overcome this limitation we adapted a CRISPR-based gene transactivation technology to induce the expression of otherwise non-expressed Mendelian disease genes in patient derived cell lines. We conduct functional assays on the resulting variant mRNA species to resolve the pathogenicity of VUS predicted to alter splicing.