CRISPR/RfxCas13d 系统在人类细胞中的非靶向活性。

Department of Medicine and Department of Systems Biology, Columbia University Irving Medical Center, New York, NY, USA.

Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, USA.

Commun Biol. 2023 Mar 28;6(1):334. doi: 10.1038/s42003-023-04708-2.

CRISPR/Cas13 systems are increasingly used for programmable targeting of RNAs. While Cas13 nucleases are capable of degrading both target RNAs and bystander RNAs in vitro and in bacteria, initial studies fail to detect collateral degradation of non-target RNAs in eukaryotic cells. Here we show that RfxCas13d, also known as CasRx, a widely used Cas13 system, can cause collateral transcriptome destruction when targeting abundant reporter RNA and endogenous RNAs, resulting in proliferation defect in target cells. While these results call for caution of using RfxCas13d for targeted RNA knockdown, we demonstrated that the collateral activity can be harnessed for selective depletion of a specific cell population defined by a marker RNA in an in vitro setting.

CRISPR/Cas13 系统越来越多地被用于可编程靶向 RNA。虽然 Cas13 核酸酶能够在体外和细菌中降解靶 RNA 和旁观者 RNA，但最初的研究未能检测到真核细胞中非靶 RNA 的 collateral 降解。在这里，我们展示了 RfxCas13d，也称为 CasRx，一种广泛使用的 Cas13 系统，在靶向丰富的报告 RNA 和内源性 RNA 时，可以导致 collateral 转录组破坏，从而导致靶细胞增殖缺陷。虽然这些结果呼吁谨慎使用 RfxCas13d 进行靶向 RNA 敲低，但我们证明 collateral 活性可用于在体外环境中选择性耗尽特定标记 RNA 定义的特定细胞群体。