Highly efficient prime editing by introducing same-sense mutations in pegRNA or stabilizing its structure

Prime editors can mediate all twelve types of base substitutions and small insertions or deletions in living cells but its efficiency remains low. Here the authors introduce same-sense mutations into pegRNAs to increase base-editing efficiency and the pegRNA secondary structure was altered to increase indel-editing efficiency. Prime editor (PE), which is developed by combining Cas9 nickase and an engineered reverse transcriptase, can mediate all twelve types of base substitutions and small insertions or deletions in living cells but its efficiency remains low. Here, we develop spegRNA by introducing same-sense mutations at proper positions in the reverse-transcription template of pegRNA to increase PE's base-editing efficiency up-to 4,976-fold (on-average 353-fold). We also develop apegRNA by altering the pegRNA secondary structure to increase PE's indel-editing efficiency up-to 10.6-fold (on-average 2.77-fold). The spegRNA and apegRNA can be combined to further enhance editing efficiency. When spegRNA and apegRNA are used in PE3 and PE5 systems, the efficiencies of sPE3, aPE3, sPE5 and aPE5 systems are all enhanced significantly. The strategies developed in this study realize highly efficient prime editing at certain previously uneditable sites.

Automated summary

In this study, the authors optimized the gene editing system to increase the efficiency of Prime editor by altering the secondary structure of the guide RNA and introducing mutations. The developed strategies achieved highly efficient Prime editing at previously uneditable sites.