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Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced...
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CRISPR Gene Editing Tool for MicroRNA Cluster Network Analysis
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Low copy CRISPR-Cas13d mitigates collateral RNA cleavage.

Sydney K Hart, Hans-Hermann Wessels, Alejandro Méndez-Mancilla

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    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.

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    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.