Human induced pluripotent stem (iPS) cell lines harboring mutations in disease-related genes serve as invaluable in vitro models for unraveling disease mechanisms and accelerating drug discovery efforts. Introducing mutations into iPS cells using traditional gene editing approaches based on the CRISPR-Cas9 endonuclease often encounters challenges such as unintended insertions/deletions (indels) and off-target effects. To address these limitations, we present a streamlined protocol for introducing highly accurate gene mutations into human iPS cells using prime editing, a "search-and-replace" genome-editing technology that combines unwanted indel-minimized CRISPR-Cas9 nickase with reverse transcriptase. This protocol encompasses the design of prime editing guide RNAs (pegRNAs) required for binding and replacement at target loci, construction of prime editor and pegRNA expression vectors, gene transfer into iPS cells, and cell line selection. This protocol allows for the efficient establishment of disease-associated gene variants within 6-8 weeks while preserving critical genomic context. Key features • Dramatic improvement in efficiency of In-Fusion cloning using inserts assembled from the three pegRNA components (spacer, spCas9 scaffold, and 3' extension) via overlap extension PCR. • Cost-effective and time-saving selection of pegRNAs for prime editing via bulk Sanger sequencing. • Straightforward gene transfection using polymer-based reagents, which requires no specialized equipment or techniques and offers high reproducibility and broad applicability across different cell lines. • Precise genome editing based on pegRNA/prime editing minimizes off-target effects, enabling a wide range of applications in the study of disease-associated genetic variants. Graphical overview Key steps of generation of human induced pluripotent stem (iPS) cell lines harboring disease-relevant single nucleotide variants (SNVs) using a prime editing system.
