LwaCas13a indiscriminately targets the human transcriptome [version 3; peer review: 1 approved, 2 approved with reservations, 1 not approved]

Background Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 13a (Cas13a) has been described as a superior tool to short-interfering RNAs (siRNAs) for specific gene silencing. Cas13 targets RNAs through Watson-Crick binding of the target CRISPR RNA (crRNA) and activation of nuclease activity. In bacteria, once Leptotrichia wadei Cas13a (LwCas13a) has cut its specific target, the trans-collateral activity of the protein degrades any single-stranded RNA present in the cell independent of its sequence or homology to the crRNA. This transcollateral activity has been reported to be absent in mammalian cells. Therefore, in this study, we aimed to downregulate mRNAs expression in mammalian cells (HaCaT and HEK293T) using LwaCas13a. Methods We developed a doxycycline-inducible system to express LwaCas13a in HEK293T cells. The off-target activity of LwaCas13a in HEK293T cells was analyzed using RNA-seq. Results In this study, we observed that activation of LwaCas13a in HEK293T cells led to non-specific targeting of RNAs, which caused cell toxicity and death. Conclusion This study provides evidence of the off-target activity of LwaCas13a in HEK293T cells, making it an unsuitable tool for the specific downregulation of RNAs.