Prime editing enables the installation of virtually any combination of point mutations, small insertions or small deletions in the DNA of living cells. A prime editing guide RNA (pegRNA) directs the prime editor protein to the targeted locus and also encodes the desired edit. Here we show that degradation of the 3′ region of the pegRNA that contains the reverse transcriptase template and the primer binding site can poison the activity of prime editing systems, impeding editing efficiency. We incorporated structured RNA motifs to the 3′ terminus of pegRNAs that enhance their stability and prevent degradation of the 3′ extension. The resulting engineered pegRNAs (epegRNAs) improve prime editing efficiency 3–4-fold in HeLa, U2OS and K562 cells and in primary human fibroblasts without increasing off-target editing activity. We optimized the choice of 3′ structural motif and developed pegLIT, a computational tool to identify non-interfering nucleotide linkers between pegRNAs and 3′ motifs. Finally, we showed that epegRNAs enhance the efficiency of the installation or correction of disease-relevant mutations.

Prime 编辑可以在活细胞 DNA 中安装点突变、小插入或小删除的几乎任何组合。prime editing guide RNA (pegRNA) 将 prime editor 蛋白引导至目标位点，并对所需的编辑进行编码。在这里，我们表明包含逆转录酶模板和引物结合位点的 pegRNA 3' 区域的降解会毒害 prime 编辑系统的活性，从而降低编辑效率。我们将结构化 RNA 基序整合到 pegRNA 的 3' 末端，以增强其稳定性并防止 3' 延伸的降解。由此产生的工程化 pegRNA (epegRNA) 可将 HeLa、U2OS 和 K562 细胞以及原代人成纤维细胞中的主要编辑效率提高 3–4 倍，而不会增加脱靶编辑活动。我们优化了 3' 结构基序的选择并开发了 pegLIT，这是一种计算工具，用于识别 pegRNA 和 3' 基序之间的非干扰核苷酸接头。最后，我们发现 epegRNA 提高了安装或校正疾病相关突变的效率。