Characterizing the Collateral Activity of CRISPR/Cas13 in Mammalian Cells: Implications for RNA Editing and Therapeutic Applications

The CRISPR/Cas13 system has garnered attention as a potential tool for RNA editing. However, the degree of collateral activity among various Cas13 orthologs and their cytotoxic effects in mammalian cells remain contentious, potentially impacting their applications. In this study, we observed differential collateral activities for LwaCas13a and RfxCas13d in 293T and U87 cells by applying both sensitive dual-fluorescence (mRuby/GFP) reporter and quantifiable dual-luciferase (Fluc/Rluc) reporter, with LwaCas13a displaying notable activity contrary to previous reports. However, significant collateral RNA cleavage exerted only a modest impact on cell viability. Furthermore, the collateral activity of LwaCas13a mildly impeded but did not arrest, porcine embryo development. Our findings reveal that distinct collateral RNA cleavage by Cas13 slightly suppresses mammalian cell proliferation and embryo development. This could account for the lack of reported collateral effects in numerous prior studies and offers new insights into the implications of the collateral activity of Cas13 for clinical application. To investigate the collateral activity of Cas13 in mammalian cells, a series of experiments were conducted involving 293T and U87 cells. Cas13a or Cas13d was employed to target the mRuby or Fluc loci, with the inclusion of non-target crRNA as a control. Through RNA-seq analysis of cells treated with various Cas13/crRNA combinations, our objective is to gain a comprehensive understanding of Cas13's collateral activity in mammalian cells and to uncover alterations in gene expression. Comparative analysis of gene expression profiles between experimental groups and the control will be conducted, with a specific focus on genes exhibiting significant differences compared to the control. This study aims to elucidate the regulatory effects of Cas13a and Cas13d on gene expression under different conditions, providing valuable insights into their roles in gene regulation within mammalian cells.