Abstract
<title>Abstract</title> <p> Collateral RNA cleavage by CRISPR-Cas13 effectors presents a major obstacle to their application in biological research and therapeutics, yet the molecular determinants of this activity remain poorly understood. Here, we define the molecular basis of collateral activity across Cas13 nucleases and show that target RNA abundance governs potent activation of <italic>Rfx</italic> Cas13d collateral activity. Moderately expressed targets induce limited collateral cleavage, whereas highly abundant targets trigger widespread activation of cellular <italic>Rfx</italic> Cas13d, resulting in global RNA degradation, disruption of proteome homeostasis, and cellular toxicity in human cells and zebrafish embryos. In transgenic zebrafish, collateral activity produces tissue-restricted developmental defects in endothelial and neuronal tissues that mirror the site of target RNA expression, demonstrating that collateral RNA cleavage remains spatially confined. Mechanistically, target abundance determines the proportion of activated <italic>Rfx</italic> Cas13d molecules within a cell, establishing a threshold-dependent switch for collateral RNA degradation. We further identify synthetic guide-target mismatches that substantially reduce collateral activity while preserving on-target silencing, enabling partial uncoupling of these two nuclease activities. Together, our findings reveal fundamental principles governing Cas13 collateral activity and provide a framework for engineering safer and more precise RNA-targeting technologies. </p>