Collateral activity of the CRISPR/RfxCas13d system in human cells

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. The collateral activity of the CRISPR/RfxCas13d system in mammalian cells is explored using spike-in RNA-seq and reporter assays.

Automated summary

CRISPR/Cas13 systems are increasingly used for programmable targeting of RNAs. Initial studies fail to detect collateral degradation of non-target RNAs in eukaryotic cells, while this study shows that the widely used Cas13 system RfxCas13d can cause collateral transcriptome destruction in target cells. However, this collateral activity can be harnessed for selective depletion of specific cell population in vitro.