Background <p>Prime editing is a versatile and precise genome-editing technique that enables targeted modifications with unprecedented accuracy. Unlike other CRISPR-based methods, prime editing does not induce double-stranded DNA breaks, thereby minimizing genomic instability and off-target effects. This technology allows for precise insertions, deletions, and substitutions, surpassing the limitations of conventional genome-editing approaches. However, a major challenge remains—low editing efficiency in human pluripotent stem cells (hPSCs), which restricts its broader application in stem cell research and regenerative medicine.</p> Methods <p>This protocol describes Prime-Induced Nucleotide Engineering using a Transient Reporter for Editing Enrichment (PINE-TREE), a fluorescence-based system designed to enhance prime editing efficiency in hPSCs. The step-by-step methodology details plasmid construction, transfection, and clonal isolation strategies, facilitating real-time enrichment of prime-edited cells. Additionally, the protocol includes comprehensive methods for evaluating off-target effects and confirming the maintenance of pluripotency, ensuring precision and reliability in edited cell lines.</p> Discussion <p>PINE-TREE significantly improves the efficiency of prime editing in hPSCs, addressing key limitations associated with low editing rates. By enabling real-time fluorescence-based enrichment, this system enhances the isolation of successfully edited cells, reducing the need for labor-intensive selection processes. Moreover, its adaptability across multiple cell types and compatibility with various prime editing strategies make it a valuable tool for researchers in genome engineering, disease modeling, and regenerative medicine.</p> Clinical trial number <p>Not applicable.</p>