ProPE expands the prime editing window and enhances gene editing efficiency where prime editing is inefficient

Prime editing (PE) is a promising gene editing method that exploits a reverse transcriptase fused to a Cas9, whose single guide RNA (sgRNA) is extended with a reverse transcriptase template containing the desired DNA modifications. Its efficiency and specificity are inconsistent, requiring extensive optimization. To address this, we propose prime editing with prolonged editing window (proPE), which uses a second non-cleaving sgRNA to target the reverse transcriptase template near the edit site. ProPE requires less optimization than PE and extends PE’s potential for allele-specific modifications. By overcoming five limitations of PE, proPE significantly increases overall editing efficiency 6.2-fold up to 29.3% for low-performing edits (<5% with PE) and broadens its applicability to modifications beyond the typical PE range, encompassing a major portion of human pathogenic single nucleotide polymorphisms. With these enhanced properties, proPE holds considerable promise for improved gene editing, including disease modelling and therapeutic intervention. Prime editing is a CRISPR methodology whose efficiency declines with distance from the target sequence. Here the authors demonstrate prime editing with prolonged editing window, proPE, which extends the editing distance, enabling the use of prime editing for therapeutic interventions.