Massively parallel functional testing of MSH2 missense variants conferring Lynch Syndrome risk

We performed a massively parallel screen in human HAP1 cells to identify loss-of-function missense variants in the key DNA mismatch repair factor MSH2. Resulting variant loss-of-function (LOF) scores are strongly concordant with previous functional evidence and available variant classification. Single amino-acid saturation mutagenesis was used to generate libraries comprising every possible missense, synonymous and nonsense variant within each of 21 tiles across MSH2 cDNA, which were tagged with degenerate barcodes, and pacakged into lentivirus and used to transduce HAP1 MSH2 KO cells were transduced at low multiplicity (<0.1). Genomic DNA was harvested at baseline ('P0') and after two rounds of functional selection by 6-TG ('DB6') or mock treatemnt ('DB'). Integrated MSH2 constructs were sequenced within each mutant tile to identify and quantify loss-of-function alelles enriched by 6-TG selection.