Enhancing prime editing efficiency by modified pegRNA with RNA G-quadruplexes

作者：Xiangyang Li, Xin Wang, Wenjun Sun, Shisheng Huang, Mingtian Zhong, Yuan Yao, Quanjiang Ji, Xingxu Huang · 发表于：Journal of Molecular Cell Biology · 年份：2022 · DOI：10.1093/jmcb/mjac022 · 被引用次数：65 · 研究领域：CRISPR and Genetic Engineering、RNA regulation and disease、Advanced biosensing and bioanalysis techniques

Dear Editor, Recent study shows that the prime editing system fusing the Cas9 nickase and reverse transcriptase could perform all types of gene modifications, including base substitutions (transitions and transversions), small insertions, and deletions, without requiring donor DNA or double-strand breaks (DSBs) (Anzalone et al., 2019). Despite the accuracy and versatility, the efficiency of the prime editor (PE) is often insufficient, which limits its broad applications. In principle, the special prime editing guide RNA (pegRNA) of the prime editing system contains an extra reverse transcription (RT) template and a primer binding site (PBS) in the 3′ extension of the scaffold (Anzalone et al., 2019). Compared with original single guide RNA, pegRNA harbors an extra 3′ extension (RT and PBS) of the scaffold and the 3′ non-structured terminal is more prone to being degraded in cells (Houseley and Tollervey, 2009; Supplementary Figure S1A). Previous reports have demonstrated that forming a special motif at the 3′ end of sgRNA can improve the stability of CRISPR/Cas9 and target cleavage efficiency (Nahar et al., 2018). Recently, we demonstrated that incorporating structured RNA motifs to the 3′ terminus of pegRNAs enhances the efficiency of prime editing (Liu et al., 2021). Therefore, we reason that adding a special motif-based rational structure for the 3′ end of pegRNA may improve the stability of pegRNA, and thus improve the PE efficiency. We designed three special types of mod...