Prime editing is an extremely practical but not particularly efficient genome editing method, as its efficiency can be limited by numerous bottlenecks arising during the complex process, and its effective application requires thorough optimization. One factor hindering efficiency is the complementarity of the pegRNA spacer and PBS sequences. Effective editing of every target molecule requires PBSs of varying lengths, which represents a considerable optimization burden; however, longer PBSs (15–17 nucleotides) generally exhibit lower efficiency for the majority of target molecules, likely due to the longer complementary segment associated with the spacer.
We found that by reducing this complementarity, either by introducing mismatches into the spacer or PBS sequences, or by introducing small deletions in a long (20 nt) PBS, or by combining spacer mismatches with PBS deletions, effective prime editing efficiency can be achieved, which sometimes even exceeds the optimal pegRNA efficiency at the given target. However, by introducing a single-nucleotide deletion at the 13th PBS position, we achieved the efficiency of pegRNAs with short PBSs previously characterized at the target site without increasing off-target activity, suggesting that this method could potentially reduce the burden of pegRNA optimization.
We also tested the split prime editor proPE on endogenous targets where the potential edit site was located distally relative to the nick site (>10 positions downstream). We successfully edited the majority of these endogenous and clinically relevant targets in two human cell lines, providing a useful method that could potentially make more than half of known, hard-to-reach human pathogenic SNPs editable.
