Marker-free coselection for successive rounds of prime editing in human cells

Prime editing enables the introduction of precise point mutations, small insertions, or short deletions without requiring donor DNA templates. However, editing efficiency remains a key challenge in a broad range of cell types. We designed a robust coselection strategy to perform successive rounds of genetic changes in human cells using CRISPR-Cas nucleases and prime editors. Through coediting, the recovery of cells modified at a target gene of interest is coupled to selection for dominant alleles of the ubiquitous and essential sodium/potassium pump (Na+/K+ ATPase). The method improves prime editing efficiency 1.5-10-fold in K562 and HeLa cells, reaching up to 83% of desired alleles in cell populations, for previously optimized prime editing guide RNAs (pegRNAs). Using pegRNAs designed de novo, we readily engineered homozygous cell lines with cancer-associated or drug resistant MTOR mutations displaying altered mTORC1 signaling. Our approach streamlines the production of cell lines with multiple genetic modifications to create cellular models for biological research and lays the foundation for the development of cell-type specific coselection strategies.