Project description:We found that several variants with 2-4 mutations on Higher Eukaryotes and Prokaryotes Nucleotide-binding (HEPN) domains showed undiminished on-target activity but markedly reduced collateral effects. Furthermore, transcriptome-wide off-target effects and cell growth arrest induced by wild-type Cas13d were found to be absent for a Cas13d variant. Thus, high-fidelity Cas13 variants with minimal collateral effects are now available for the targeted degradation of RNAs in basic research and therapeutic applications.
Project description: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.