Abstract Gene editing in human induced pluripotent stem (iPS) cells with programmable nucleases facilitates reliable disease models, but methods using double-strand break repair often produce random on-target by-products. Prime editing (PE) combines Cas9 nickase with reverse transcriptase (RT) and a prime editing guide RNA (pegRNA) encoding a repair template to reduce by-products. We implemented a GMP-compatible protocol for transfecting Cas9- or PE-2A-mCherry plasmids to track and fractionate human iPS cells based on PE expression level. We compared the editing outcomes of Cas9- and PE-based methods in a GFP-to-BFP conversion assay, at the HEK3 benchmark locus, and at the APOE Alzheimer’s risk locus, revealing superior precision of PE at high expression levels. Moreover, sorting cells for PE expression level influenced allelic editing outcomes at the target loci. We expect that our findings will aid in the creation of gene-edited human iPS cells with intentional heterozygous and homozygous genotypes. Highlights Delivered large plasmids to human iPS cells under GMP-compliant conditions Developed a flow cytometry-based approach to enrich for PE in human iPS cells Demonstrated few on-target indels in cells regardless of PE expression Sorted iPS cells based on PE expression level to influence mono- or bi-allelic editing

Enrichment of allelic editing outcomes by Prime Editing in iPS cells