Homo sapiens Raw sequence reads. Homo sapiens

Prime editing (PE) has its advantages for small insertion, deletion or point mutations without double-stranded DNA breaks. The 3-extension of pegRNAs could negatively affect its stability or folding and comprise the PE activity. Here we engineered pegRNA by additional RNA stem loop aptamers at 3-end, or splitting pegRNA into sgRNA and prime RNA containing PBS and RTT. We tethered RNA aptamer binding proteins to Cas9 nickase either directly or in an inducible dimerization manner. We show that additional RNA stem loop increases the PE efficiency. Split pegRNA Prime Editing maintains the PE activity and makes the PE to be chemically inducible

先导编辑（Prime editing, PE）在无需引入双链DNA断裂的前提下，可高效介导小型插入、缺失及点突变的精准编辑。先导编辑向导RNA（prime editing guide RNA, pegRNA）的3'端延伸可能会对其自身稳定性与RNA折叠产生负面影响，进而削弱PE的编辑活性。本研究通过两种策略对pegRNA进行工程化改造：其一，在其3'端引入额外的RNA茎环适配体；其二，将pegRNA拆分为单导RNA（single guide RNA, sgRNA）与包含引物结合位点（primer binding site, PBS）及逆转录模板区（reverse transcriptase template, RTT）的先导RNA。我们将RNA适配体结合蛋白与Cas9切口酶（Cas9 nickase）进行直接偶联，或采用诱导型二聚化的方式实现二者的靶向结合。实验结果证实，在pegRNA 3'端添加RNA茎环适配体可显著提升PE的编辑效率。拆分型pegRNA先导编辑不仅能够保留PE的固有编辑活性，还可实现先导编辑的化学诱导调控。