MSH2 and MSH6 as size dependent cellular determinants for prime editing in human embryonic stem cells

Potential applications of precise genome editing in human pluripotent stem cells (hPSCs), not only for isogenic disease modeling but also for ex vivo stem cell therapy, have urged the application of diverse genome editing tools in hPSCs. However, unlike differentiated somatic cells, the unique cellular properties of hPSCs (e.g., high susceptibility to DNA damage and active DNA repair) largely determine the overall efficiency of editing tools. Considering high demand of prime editors (PE), mostly due to its broad editing coverage compared to base editors, it is important to characterize the key molecular determinants of PE efficiency in hPSCs. Herein, we showed that MSH2 and MSH6, two main components of the MutS complex of mismatch repair (MMR), are highly expressed in hPSCs and determine PE efficiency in an editing size-dependent manner. Importantly, loss of MSH2, which disrupts both MutS and MutS{beta} complexes, was found to dramatically improve the efficiency of PE from one base to 10 bases, up to 50 folds. In contrast, genetic perturbation of MSH6, which solely abrogates MutS activity, marginally improved the editing efficiency up to 3 base pairs. The size dependent effect of MSH2 or MSH6 on prime editing in hPSCs not only implies MMR is a major determinant of PE efficiency in hPSCs but also highlights the distinct roles of MutS and MutS{beta} in the outcome of genome editing.