MutSa and MutSb as size-dependent cellular determinants for prime editing in human embryonic stem cells

Precise genome editing in human pluripotent stem cells (hPSCs) has potential applications in isogenic disease modeling and ex vivo stem cell therapy, necessitating diverse genome editing tools. However, unlike differentiated somatic cells, hPSCs have unique cellular properties that maintain genome integrity, which largely determine the overall efficiency of an editing tool. Consid-ering the high demand for prime editors (PEs), it is imperative to characterize the key molecular determinants of PE outcomes in hPSCs. Through homozygous knockout (KO) of MMR pathway key proteins MSH2, MSH3, and MSH6, we reveal that MutSa and MutSss determine PE efficiency in an editing size-dependent manner. Notably, MSH2 perturbation disrupted both MutSa and MutSss complexes, dramatically escalating PE efficiency from base mispair to 10 bases, up to 50 folds. Similarly, impaired MutSa by MSH6 KO improved editing efficiency from single to three base pairs, while defective MutSss by MSH3 KO heightened efficiency from three to 10 base pairs. Thus, the size-dependent effect of MutSa and MutSss on prime editing implies that MMR is a vital PE efficiency determinant in hPSCs and highlights the distinct roles of MutSa and MutSss in its outcome.