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 系统最近已用于多种生物体中的靶向 RNA 降解。然而，旁观者 RNA 的附带降解限制了它们的体内应用。在这里，我们设计了一种双荧光报告系统，用于检测哺乳动物细胞中的附带效应并筛选 Cas13 变异。在 200 多个工程变体中，包括 Cas13d 和 Cas13X 在内的几种 Cas13 变体表现出高效的靶向活性，但附带活性显着降低。此外，这些变体不存在 Cas13 诱导的全转录组脱靶和细胞生长停滞。高保真 Cas13 变体表现出与野生型 Cas13 相似的 RNA 敲低活性，但在转基因小鼠或腺相关病毒介导的体细胞靶向中没有检测到附带损伤。因此，具有最小附带影响的高保真 Cas13 变体现在可用于基础研究和治疗应用中的 RNA 靶向降解。