Transcriptional response to RfxCas13d with mCherry targeting crRNA in HEK293T

Here, we systematically investigate the collateral activity of several Cas13 variants in human cells in vitro and zebrafish embryos in vivo. Among these nucleases, RfxCas13d displays robust collateral activity whose extent scales continuously with target RNA abundance.

Targeting moderately expressed RNAs activates only a limited fraction of RfxCas13d molecules, producing limited collateral cleavage that predominantly affects abundant, accessible co-expressed transcripts while the broader endogenous transcriptome and proteome, and cell fitness, remain largely intact. In contrast, highly abundant RNA targets drive simultaneous activation of a large fraction of cellular RfxCas13d, leading to widespread collateral cleavage of cellular RNAs, disruption of proteome homeostasis, and consequent cell toxicity and developmental defects in zebrafish embryos.