Transcriptional response to RfxCas13d with mCherry targeting crRNA in HEK293T

ABSTRACT: Collateral RNA cleavage by CRISPR-Cas13 effectors presents a critical obstacle to their application in biological research and therapeutics, yet the molecular determinants of this activity remain poorly understood. 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. Notably, transgenic zebrafish with target RNA expression restricted to endothelial or neuronal lineages exhibit collateral activity confined to the target-expressing cells, producing tissue-specific developmental abnormalities and motility deficits. These findings reveal that RfxCas13d collateral activity is governed by target RNA abundance, becoming extensive above a threshold level of target expression rather than acting in an all-or-none manner. This work underscores the need to consider target abundance when deploying RfxCas13d, and highlights PspCas13b as an alternative with low collateral activity for RNA silencing.

ORGANISM(S): Homo sapiens

PROVIDER: GSE337551 | GEO | 2026/07/09

REPOSITORIES: GEO