High-fidelity Cas13 variants for targeted RNA degradation with minimal collateral effects

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. We have designed a dual-fluorescent reporter system that revealed substantial collateral effects of Cas13-mediated RNA knockdown of both exogenous and endogenous genes in mammalian cells. This reporter system was used to screen over 200 Cas13 variants obtained by structure-guided mutagenesis. To study transcriptome-wide off-target effect induced by wild-type Cas13d (RfxCas13d) and its absence in cfCas13d ( named hfCas13d in the publication) variant, we designed a three groups experiment using HEK293 cell lines, including an inactive dead Cas13d control (dCas13d with gRNA targeting endogenous genes), a wild-type Cas13d treatment groups (Cas13d with the corresponding gRNA targeting endogenous genes) and a "collateral cleavage-free" Cas13d treatment groups (cfCas13d with the corresponding gRNA targeting endogenous genes). We chose 2 gRNAs targeting PPIA, and 1 gRNA each targeting RPL4, CA2, or PPARG, respectively. Three replicates were prepared for each group for whole transcriptome sequencing. We finally have 44 RNAseq samples in total, three for each groups except one sample missing in cfCas13d for targeting PPIA with gRNA g1.