McCullough, M.; Poti, A.; Munteanu, M.; Supek, F.

Mismatch repair deficiency (MMRd) is a central driver of cancer genome evolution, yet it is commonly reduced to a binary MSI-high versus microsatellite-stable call. Here we combine multiplex CRISPR/Cas12a editing, long-term mutation accumulation experiments in human cells, and whole-genome sequencing to map the mutational consequences of single and pairwise perturbations across canonical and accessory MMR genes, together with selected base excision repair and direct-repair factors. Across 72 isogenic lineages representing 58 genotypes, we recover reproducible substitution, indel and short-tandem-repeat mutational states that distinguish MSH2, MSH6, MLH1, PMS2, MSH3 and MLH3 deficiencies. A dedicated STR spectrum reveals genome-wide evidence for MSH3-driven EMAST and enables design of compact loci panels for mechanism-aware MSI classification. Experimental spectra generalize across independent cell systems and to cancer whole genome sequences, where Elastic-net classifiers accurately identify causal MMR genes in Lynch tumors and uncover common occurrences of intermediate MMRd states. Pairwise perturbations further reveal repair crosstalk reflected in mutational spectra, including enhancement of MUTYH-associated oxidative mutagenesis by MMR loss. Together, these findings recast mismatch repair deficiency as a multidimensional genomic phenotype and provide a framework for interpreting DNA repair deficiencies by bridging mutational phenotypes from experimental systems and human cancers.