An upgraded nuclease prime editor platform enables high-efficiency singled or multiplexed knock-in/knockout of genes in mouse and sheep zygotes

作者：Weijia Mao, Pei Wang, Lei Zhou, Dongxu Li, Xiangyang Li, Xin Lou, Xingxu Huang, Feng Wang, Yanli Zhang, Jianghuai Liu, Yongjie Wan · 发表于：Protein & Cell · 年份：2025 · DOI：10.1093/procel/pwaf006 · 被引用次数：8 · 研究领域：CRISPR and Genetic Engineering、Animal Genetics and Reproduction、RNA and protein synthesis mechanisms

Dear Editor, The emergence of CRISPR/Cas9-based genome editing technology has energized many applied biotechnology fields, including livestock breeding (Zhao et al., 2019). Given that the most naturally occurring beneficial variants in farm animals are regulatory alleles (Georges et al., 2019), practical livestock editing should mostly involve knocking-in of the natural variants, or of designed elements to regulate key genes. However, earlier CRISPR/Cas9-dependent strategies for installation of knock-in alleles showed limitations of low efficiencies and purities (Anzalone et al., 2020). An important breakthrough toward precise genome editing was marked by the development of prime editor (PE), which presented a highly innovative design for the installation of small-sized edits (Anzalone et al., 2019). The canonical PE protein features a fusion of Cas9 nickase (nCas9, H840A) moiety and a reverse transcriptase (RTase) domain. A prime editing guide RNA (pegRNA) is comprised of a conventional sgRNA module (for initially directing a nick at the target site) and an additional 3′ sequence extension (for subsequently programming synthesis of edits at the nick by reverse transcription (RT)). The resultant intermediate 3′-flap structure may be subsequently resolved by the cellular repair pathways for the eventual installation of edits (Anzalone et al., 2019). Empowered by such a double-strand DNA break (DSB)-independent and RT-dependent mode of action, PE has exhibited high editing vers...