Despite the immense progress the identification of disease-associated genetic variants, our understanding of the functional and pathogenic significance of most mutations is still limited. Current technologies screen variants on a large scale, but they lack quantitative measurements and validation, rendering them impractical as universally applicable tools. In Nature Genetics, Niu et al. report CRISPR-Select, a multi-parametric functional CRISPR screen that quantitatively links distinct variants of uncertain significance (VUS) to (pathogenic) phenotypes.
CRISPR-Select uses CRISPR–Cas9 to introduce a VUS or a synonymous silent control mutation into a cell population. The method then calculates the absolute frequencies of the variant cells as a ratio to wild-type cells over time, across space or in relation to a given cellular marker. Using CRISPR-SelectTIME, the authors look at the sensitivity to different drug therapies over time of cancer cells with known or uncharacterized gene mutations, in cell lines in vitro and in xenograft models in vivo. While this assay is limited to a single coding variant, high-throughput assays in a multi-well format allow multiplexed investigation of several different mutations simultaneously. CRISPR-SelectSPACE allows the analysis of variants involved in cell migration and invasion, whereas CRISPR-SelectSTATE can be used to determine the significance of a particular VUS to any biological state or biochemical process of a cell assessable by fluorescence-activated cell sorting markers. In this way, CRISPR-Select extends current technologies of proliferation assays to variant effects on cell motility and physiological or metabolic states.
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