Low copy CRISPR-Cas13d mitigates collateral RNA cleavage

Abstract

AbstractWhile CRISPR-Cas13 systems excel in accurately targeting RNA, the potential for collateral RNA degradation poses a concern for therapeutic applications and limits broader adoption for transcriptome perturbations. We evaluate the extent to which collateral RNA cleavage occurs whenRfxCas13d is delivered via plasmid transfection or lentiviral transduction and find that collateral activity only occurs with high levels ofRfxCas13d expression. Using transcriptome-scale and combinatorial CRISPR pooled screens in cell lines with low-copyRfxCas13d, we find high on-target knockdown, without extensive collateral activity regardless of the expression level of the target gene. In contrast, transfection ofRfxCas13d, which yields higher nuclease expression, results in collateral RNA degradation. Further, our analysis of a high-fidelity Cas13 variant uncovers a marked decrease in on-target efficiency, suggesting that its reduced collateral activity may be due to an overall diminished nuclease capability.