Tong Huawei, Huang Jia, Xiao Qingquan, He Bingbing, Dong Xue, Liu Yuanhua, Yang Xiali, Han Dingyi, Wang Zikang, Wang Xuchen, Ying Wenqin, Zhang Runze, Wei Yu, Xu Chunlong, Zhou Yingsi, Li Yanfei, Cai Minqing, Wang Qifang, Xue Mingxing, Li Guoling, Fang Kailun, Zhang Hainan, Yang Hui
HuiGene Therapeutics Co., Ltd., Shanghai, China.
Institute of Neuroscience, State Key Laboratory of Neuroscience, Key Laboratory of Primate Neurobiology, Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai, China.
Nat Biotechnol. 2023 Jan;41(1):108-119. doi: 10.1038/s41587-022-01419-7. Epub 2022 Aug 11.
CRISPR-Cas13 systems have recently been used for targeted RNA degradation in various organisms. However, collateral degradation of bystander RNAs has limited their in vivo applications. Here, we design a dual-fluorescence reporter system for detecting collateral effects and screening Cas13 variants in mammalian cells. Among over 200 engineered variants, several Cas13 variants including Cas13d and Cas13X exhibit efficient on-target activity but markedly reduced collateral activity. Furthermore, transcriptome-wide off-targets and cell growth arrest induced by Cas13 are absent for these variants. High-fidelity Cas13 variants show similar RNA knockdown activity to wild-type Cas13 but no detectable collateral damage in transgenic mice or adeno-associated-virus-mediated somatic cell targeting. Thus, high-fidelity Cas13 variants with minimal collateral effects are now available for targeted degradation of RNAs in basic research and therapeutic applications.
CRISPR-Cas13系统最近已被用于多种生物体中的靶向RNA降解。然而，旁系RNA的附带降解限制了它们在体内的应用。在这里，我们设计了一种双荧光报告系统，用于检测附带效应并在哺乳动物细胞中筛选Cas13变体。在200多种工程变体中，包括Cas13d和Cas13X在内的几种Cas13变体表现出高效的靶向活性，但附带活性明显降低。此外，这些变体不存在由Cas13诱导的全转录组脱靶效应和细胞生长停滞。高保真Cas13变体在转基因小鼠或腺相关病毒介导的体细胞靶向中显示出与野生型Cas13相似的RNA敲低活性，但没有可检测到的附带损伤。因此，具有最小附带效应的高保真Cas13变体现在可用于基础研究和治疗应用中的RNA靶向降解。
