The CRISPR-Cas13 system is an RNA-guided RNA-targeting system, and has been widely used in transcriptome engineering with potentially important clinical applications. However, it is still controversial whether Cas13 exhibits collateral activity in mammalian cells. Here, we found that knocking down gene expression using RfxCas13d in the adult brain neurons caused death of mice, which was not resulted from the loss of target gene function or off-target effects. Mechanistically, we showed that RfxCas13d exhibited collateral activity in mammalian cells, which is positively correlated with the abundance of target RNA. The collateral activity of RfxCas13d could cleave 28s rRNA into two fragments, leading to translation attenuation and activation of the ZAKα-JNK/p38-immediate early gene (IEG) pathway. These results provide new mechanistic insights into the collateral activity of RfxCas13d and warn that the biosafety of CRISPR-Cas13 system needs further evaluation before applying it to clinical treatments.

中文翻译：

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RfxCas13d 对 28s rRNA 的附带切割导致小鼠死亡

CRISPR-Cas13 系统是一种 RNA 引导的 RNA 靶向系统，已广泛用于转录组工程，具有潜在的重要临床应用。然而，Cas13 是否在哺乳动物细胞中表现出附带活性仍然存在争议。在这里，我们发现在成年脑神经元中使用 RfxCas13d 敲低基因表达会导致小鼠死亡，这不是由于靶基因功能丧失或脱靶效应造成的。从机制上讲，我们发现 RfxCas13d 在哺乳动物细胞中表现出侧枝活性，这与靶 RNA 的丰度呈正相关。RfxCas13d 的附带活性可以将 28s rRNA 切割成两个片段，导致 ZAKα-JNK/p38-immediate early gene (IEG) 通路的翻译衰减和激活。