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Low copy CRISPR-Cas13d mitigates collateral RNA cleavage
Abstract:
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.
Insights
CRISPR-Cas13d RNA targeting is precise, but high expression can cause unwanted collateral RNA degradation. Researchers found low expression minimizes this risk, enabling effective transcriptome perturbations.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- CRISPR-Cas13 systems offer precise RNA targeting.
- Collateral RNA degradation is a limitation for therapeutic use and transcriptome studies.
- Rfx Cas13d is a Cas13 variant with potential applications.
Purpose of the Study:
- To evaluate collateral RNA cleavage by Rfx Cas13d under different delivery and expression conditions.
- To assess the impact of Rfx Cas13d expression levels on on-target efficiency and collateral activity.
- To investigate the nuclease activity of a high-fidelity Cas13 variant.
Main Methods:
- Delivery of Rfx Cas13d via plasmid transfection and lentiviral transduction.
- Transcriptome-scale and pooled CRISPR screens in cell lines.
- Analysis of Cas13d expression levels and collateral RNA degradation.
- Characterization of a high-fidelity Cas13 variant.
Main Results:
- Collateral RNA degradation by Rfx Cas13d occurs primarily at high expression levels.
- Low-copy Rfx Cas13d expression enables high on-target knockdown without significant collateral activity.
- Transfection-mediated high expression of Rfx Cas13d leads to collateral RNA degradation.
- A high-fidelity Cas13 variant showed reduced on-target efficiency alongside decreased collateral activity.
Conclusions:
- Controlling Rfx Cas13d expression levels is crucial for minimizing collateral RNA degradation.
- Low-expression systems are suitable for precise transcriptome perturbations.
- High-fidelity Cas13 variants may trade-off on-target efficiency for reduced collateral activity.
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