Low copy CRISPR-Cas13d mitigates collateral RNA cleavage.
Sydney K HartHans-Hermann WesselsAlejandro Méndez-MancillaSimon MüllerGediminas DrabaviciusOlivia ChoiNeville E SanjanaPublished in: bioRxiv : the preprint server for biology (2024)
While 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 when Rfx Cas13d is delivered via plasmid transfection or lentiviral transduction and find that collateral activity only occurs with high levels of Rfx Cas13d expression. Using transcriptome-scale and combinatorial CRISPR pooled screens in cell lines with low-copy Rfx Cas13d, we find high on-target knockdown, without extensive collateral activity regardless of the expression level of the target gene. In contrast, transfection of Rfx Cas13d, 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.
Keyphrases
- crispr cas
- genome editing
- poor prognosis
- genome wide
- dna binding
- gene expression
- nucleic acid
- escherichia coli
- magnetic resonance
- binding protein
- rna seq
- single cell
- randomized controlled trial
- computed tomography
- magnetic resonance imaging
- dna methylation
- copy number
- electronic health record
- drug delivery
- transcription factor
- radiation therapy
- contrast enhanced
- open label
- study protocol