工程化环状引导RNA增强基于微型CRISPR/Cas12f的基因激活和腺嘌呤碱基编辑。

Engineered circular guide RNAs enhance miniature CRISPR/Cas12f-based gene activation and adenine base editing.

作者信息

Zhang Xin, Li Mengrao, Chen Kechen, Liu Yuchen, Liu Jiawei, Wang Jiahong, Huang Hongxin, Zhang Yanqun, Huang Tao, Ma Shufeng, Liao Kaitong, Zhou Jiayi, Wang Mei, Lin Ying, Rong Zhili

机构信息

Cancer Research Institute, School of Basic Medical Sciences, State Key Laboratory of Multi-organ Injury Prevention and Treatment, Guangdong Province Key Laboratory of Immune Regulation and Immunotherapy, Southern Medical University, Guangzhou, China.

State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, National Center for Respiratory Medicine, Guangzhou Institute of Respiratory Health, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.

出版信息

Nat Commun. 2025 Mar 28;16(1):3016. doi: 10.1038/s41467-025-58367-4.

DOI:10.1038/s41467-025-58367-4

PMID:40148327

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11950443/

Abstract

CRISPR system has been widely used due to its precision and versatility in gene editing. Un1Cas12f1 from uncultured archaeon (hereafter referred to as Cas12f), known for its compact size (529 aa), exhibits obvious delivery advantage for gene editing in vitro and in vivo. However, its activity remains suboptimal. In this study, we engineer circular guide RNA (cgRNA) for Cas12f and significantly improve the efficiency of gene activation about 1.9-19.2-fold. When combined with a phase separation system, the activation efficiency is further increased about 2.3-3.9-fold. In addition, cgRNA enhances the editing efficiency and narrows the editing window of adenine base editing about 1.2-2.5-fold. Importantly, this optimization strategy also boosts the Cas12f-induced gene activation efficiency in mouse liver. Therefore, we demonstrate that cgRNA is able to enhance Cas12f-based gene activation and adenine base editing, which holds great potential for gene therapy.

摘要

CRISPR系统因其在基因编辑中的精确性和多功能性而被广泛应用。来自未培养古菌的Un1Cas12f1（以下简称Cas12f），以其紧凑的大小（529个氨基酸）而闻名，在体外和体内基因编辑中表现出明显的递送优势。然而，其活性仍不理想。在本研究中，我们为Cas12f设计了环状引导RNA（cgRNA），并将基因激活效率显著提高了约1.9至19.2倍。当与相分离系统结合时，激活效率进一步提高约2.3至3.9倍。此外，cgRNA提高了编辑效率，并将腺嘌呤碱基编辑的编辑窗口缩小了约1.2至2.5倍。重要的是，这种优化策略还提高了Cas12f在小鼠肝脏中诱导的基因激活效率。因此，我们证明cgRNA能够增强基于Cas12f的基因激活和腺嘌呤碱基编辑，这在基因治疗方面具有巨大潜力。