Prime editor (PE) offers significant translational potential due to its diverse editing outcomes. However, its editing efficiency is generally lower than that of wild-type Cas9 and base editor (BE). Previous investigations have suggested that the availability of the primer binding site (PBS) and reverse transcription template (RTT) within the pegRNA limits the installation of desired edits at target sites. In this study, we investigated whether the position and copy number of PBS-RTT sequences are important parameters influencing PE editing efficiencies. We inserted extra copies of the PBS-RTT sequence into the loop 2, tetraloop, or the 3' end of pegRNA, as well as moved the 3' PBS-RTT to the internal stem-loops of sgRNA. Incorporating an extra PBS-RTT copy into loop 2, but not tetraloop, increased PE editing efficiency by 40%-147% across various target sites and cellular contexts. This suggests that both the local concentration and steric hindrance of the PBS-RTT sequence appear to affect its accessibility to the DNA nicking end, ultimately influencing the editing efficiency of PE. By addressing the bottleneck of limited PBS-RTT availability, our strategy may broaden the wide applicability of PE, especially in translational medicine.
CRISPR Gene Editing