• banner:CRISPR Controls

    CRISPRa Controls

    Establish and ensure successful conditions for CRISPR activation experiments

    CRISPR activation controls for gain-of-function experiments

    CRISPR activation (CRISPRa) is based on the CRISPR-Cas9 system which requires two components to be delivered and/or expressed in the cell for efficient transcriptional activation: a guide RNA and a nuclease-deactivated Cas9 (dCas9) fused to transcriptional activators, such as dCas9-VPR. Insufficient amounts of either component will result in inefficient target gene expression.

    To establish optimal experimental conditions and ensure ongoing successful activation, it is recommended to use a species-specific CRISPRa positive control for a characterized gene target. The Edit-R CRISPRa positive controls target human or mouse Titin (TTN) or POU class 5 homeobox 1 (POU5F1) genes.

    Detection of activation

    mRNA and protein levels may change to different degrees and with different timing in CRISPRa experiments; this should be taken into consideration when detecting expression levels.

    qPCR: Detect changes in mRNA compared to baseline expression levels to ascertain fold increase in transcript expression.

    Western blotting: Determine changes in protein level compared to untreated and non-targeting controls

    Edit-R non-targeting negative controls are designed to target no annotated genomic region; therefore, the baseline expression of any given gene should not be altered, and any non-specific effects can be observed.

    Controls should be used for:

    • Optimizing CRISPRa crRNA:tracrRNA transfection conditions
    • Determining the optimal promoter for driving lentiviral dCas9-VPR expression
    • Identifying optimal co-transfection conditions with dCas9-VPR plasmid with either CRISPRa crRNA:tracrRNA or sgRNA plasmid
    • Ensuring experimental consistency and controlling for any possible background effects

    CRISPRa Controls

    • Edit-R CRISPRa Synthetic Positive Controls

      Pooled or individual crRNA controls for assessment of optimal experimental conditions for gene activation

    • Edit-R CRISPRa Non-targeting crRNA Controls

      Non-targeting controls to evaluate baseline cellular responses to CRISPRa components in the absence of gene target-specific crRNA

    • Edit-R CRISPRa Lentiviral sgRNA Positive Controls

      Control sgRNAs targeting well-characterized genes to determine the effectiveness of your experimental conditions for maximum activation

    • Edit-R CRISPRa Lentiviral sgRNA Non-targeting Controls

      Non-targeting controls to evaluate baseline cellular responses to CRISPRa components in the absence of target-specific sgRNA

    • Edit-R dCas9-VPR

      Lentiviral particles, purified plasmid or mRNA that express nuclease-deactivated Cas9 fused to transcriptional activators. When complexed with a guide RNA will trigger an endogenous gene’s expression.

    • Edit-R tracrRNA

      Edit-R trans-activating CRISPR RNA (tracrRNA) is a synthetic, HPLC-purified, long RNA required for use with Edit-R crRNA to form the complex that programs dCas-VPR. It is modified for nuclease resistance.

    Resources for CRISPR Activation