Precise RNA targeting with CRISPR-Cas13d

The possibility of collateral RNA degradation poses a concern for transcriptome perturbations and therapeutic applications using CRISPR-Cas13. We show that collateral activity only occurs with high RfxCas13d expression. Using low-copy RfxCas13d in transcriptome-scale and combinatorial pooled screens, we achieve high on-target knockdown without extensive collateral activity. Further, analysis of a high-fidelity Cas13 variant suggests that its reduced collateral activity may be due to overall diminished nuclease capability. We engineered three human cell lines, HAP1, HEK293FT, and A375, to express the nuclear-localized RfxCas13d effector under doxycycline-inducible control. We also engineered HAP1 cells to express nuclear-localized High fidelity Cas13d under doxycycline-inducible control. Next, we transduced each cell line with one of 3 lentiviral gRNA libraries (Screen 1: RfxCas13d-expressing HAP1 and HEK293FT cells; Screen 2: RfxCas13d expressing A375 cells; or Screen 3: HfCas13d-expressing HAP1 cells) at a low multiplicity-of-infection to ensure each cell received only a single perturbation and induced Cas13 expression via doxycycline addition. We harvested genomic DNA from these cells at day 0 or 14 days post-Cas13 induction and computed changes in gRNA abundance via amplicon sequencing.