Altered primer binding site and spacer sequences that weaken their intramolecular complementarity can enhance prime editing efficiency

For efficient prime editing, the length and sequence of the primer binding site (PBS) are criticaland its intramolecular complementarity with the prime editing guide RNA (pegRNA) spacer is amajor drawback. We investigate these effects by analysing literature data and by testing over 300modified pegRNAs with weakened PBS-spacer interactions. It has been suggested that theeffective PBS length for plasmid delivered pegRNAs is considerably longer than what anefficient priming would necessarily require, likely because of exonuclease digestion of the PBS.However, analysing literature data of over 3000 pegRNAs reveals no significant shift in theoptimum PBS length for engineered pegRNAs compared to conventional pegRNAs. Byintroducing mismatches in the spacer or PBS sequence to disrupt complementarity, we find toimprove editing efficiency up to seven-fold, but this effect is less pronounced with an optimalPBS length. Combination of spacer mismatches and PBS deletions led to further editingimprovements over the best matching PBS, although finding the best pairing requires extensiveoptimization. Here, using SPELL (Streamlined Prime Editing with fixed-Length PBS Leverage),which employs only a deletion in a 17-20 nucleotide-long PBS, we achieve near- optimal editingefficiency in most cases without requiring prior pegRNA optimization.