ProPE, a prime editing method with prolonged editing window for enhanced gene editing

Prime editing (PE) is a promising gene editing method that exploits a reverse transcriptase (RT) fused to a Cas9 (CRISPR-associated protein 9), whose single guideRNA (sgRNA) is extended with an RT template, containing the desired DNA modifications. Though its efficiency and specificity have been inconsistent, requiring extensive optimization. To address this, we propose proPE (prime editing with prolonged editing window), 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 the potential of PE for allele-specific modifications. By overcoming five limitations of PE, proPE significantly increases overall editing efficiency by 6.2 fold for low-performing edits (<5% with PE) up to 29.3%, and broadens its applicability to modifications beyond the typical editing range of PE, encompassing a major portion of human pathogenic SNPs. With these enhanced properties, proPE holds considerable promise for improved gene editing including disease modelling and therapeutic intervention.