transcriptome-wide collateral effects of Cas13d variants

The intrinsic collateral cleavage activity of Cas13d,which refers to the gRNA-independent degradation of bystander RNA, significantly limits its therapeutic potential. To extensively evaluate the collateral effects of hpCas13d genome-wide, we conducted RNA-seq analysis of the transcriptome in HEK293T cells after the transfections with dCas13d, wtCas13d, hpCas13d, and hfCas13d . Compared to dCas13d, wtCas13d with a PPIA gRNA (reported to induce the significant collateral cleavage) resulted in significant downregulations of thousands genes. In sharp contrast, hpCas13d and hfCas13d showed much less off-target genes, respectively. These findings demonstrate that hpCas13d exhibits reduced collateral activity, rendering it suitable for in vivo applications. To assess the transcriptome-wide off-target effect, we sorted 1x10^6 GFP-positive HEK293T cells using the BD FACS Aria III, 48 hours after transfection with Cas13d variants. Total RNA was then extracted and RNA sequencing was performed.