Substantial collateral effects induced by CasRx in mammalian cells and mice

CRISPR-Cas13 systems have recently been employed to silence RNAs in yeast, plants, mammalian cell lines and animals, however, collateral effects induced by Cas13 may impede their research and therapeutic applications. Here, we found Cas13a and CasRx-mediated RNA knockdown induced substantial collateral effects on both exogenous and endogenous genes in mammalian cells. Moreover, transgenic mice carrying CasRx, with or without targeting guide RNA, exhibited high lethality. Therefore, our results present limitations of CasRx, requiring a solution to address its fidelity before clinical applications. To study the collateral effects of casRx system targeting endogenous genes in mammalian cells, we designed a three groups experiment using HEK293T cell lines, including a control (casRx with exogenous targets) and two treatment groups (casRx with endogenous targets). Three replicates were prepared for each group for whole transcriptome sequencing. One sample in the control group was discarded due to quality issue. We finally have 8 RNAseq samples in total, two for the control group, labeled with NT, and three for each endogenous-target groups, labeled with casRx+RPL4 g1 and casRx + RPL4 g2, respectively.