Two Highly Efficient Prime Editing Systems Based on the Csy4 CRISPR Endonuclease

作者：Yu Lu, Dexin Qiao, Junya Wang, Wei Sun, Zhenghong Cao, Minhui Lu, Yiping Chai, Yuanyuan Jiang, Cuiping Xin, Xiaohan Liu, Siyun Li, Syeda Leeda Gul, Qijun Chen · 发表于：Plant Biotechnology Journal · 年份：2025 · DOI：10.1111/pbi.70337 · 被引用次数：4 · 研究领域：CRISPR and Genetic Engineering、Pluripotent Stem Cells Research、Innovation and Socioeconomic Development

Prime editing is an advanced CRISPR/Cas-derived technology designed to enable precise genetic modifications, including base substitutions, insertions and deletions, at targeted genomic loci (Anzalone et al. 2019). Compared to the unsplit prime editor (PE) system, split PE strategies—such as split inteins, MS2, SunTag, CC-PE and direct split PEs with untethered reverse transcriptase (RT)—offer a more adaptable and efficient approach for size-constrained delivery systems, such as viral delivery, and facilitate the ongoing development of new PEs based on various CRISPR/Cas systems and RTs (Grunewald et al. 2023; Liu et al. 2022; Mu et al. 2024; Wei et al. 2025). Csy4 (also known as Cas6f) is the key enzyme responsible for crRNA production in CRISPR subtype I-F (Sternberg et al. 2012). Csy4 binds with equal affinity to both its substrate pre-crRNA and product crRNA (Sternberg et al. 2012). The Csy4 system has been effectively employed in prime editing for processing pegRNAs (Liu et al. 2021; Ni et al. 2023). In this report, we hypothesised that the Csy4 system could be leveraged to develop a new split PE (sPE) (Figure 1a). Csy4's strong and persistent binding to the Csy4 recognition sequence (Csy4RS), even after cleavage, allows the formation of a functional epegRNA-Csy4RS-Csy4 complex (Figure 1a). By fusing RT to Csy4, this complex (epegRNA-Csy4RS-Csy4-RT) can be captured by the SpCas9KK-H840A nickase to drive prime editing, thereby creating a new sPE (Figure 1a). Based on these...