• Successful CRISPR gene editing in primary T cells

    A protocol and webinar detailing use of Edit-R crRNA with electroporation of RNP

    Successful CRISPR gene editing in primary T cells

    Genetic manipulation of primary T cells has the potential for far-reaching implications on human health – everything from novel HIV treatments to personalized cancer therapies. Optimizing the experimental conditions for studies using patient-derived immune cells can be time consuming and subject to variation that are not typical in immortalized cell lines.

    A webinar presented by Dr. Judd Hultquist outlined a successful protocol for electroporation of Cas9 ribonuclear protein (RNP) into primary T cells for targeted gene silencing, including high-throughput screening of hundreds of targets. A few key protocol details include:

    • Cells are most susceptible to editing when fully activated
    • 2:1 ratio of crRNA:tracrRNA to Cas9 protein promotes efficient RNP formation
    • RNPs can be effectively multiplexed at equimolar amounts after generation to knockout more than one gene

    Reagents for robust knockout in primary T cells

    The experiments presented in the webinar made use of the Edit-R two-part guide RNA platform, utilizing a gene-specific, synthetic crRNA combined with Edit-R tracrRNA, then complexed with Cas9 protein to form the ribonuclear complex (RNP). The ability to receive either custom or predesigned crRNAs in a convenient, customizable 96-well plate format saved a lot of time and handling in generating the RNPs prior to electroporation.

    Additional recommendations

    These additional recommendations for consistent and reproducible CRISPR-Cas9 editing in T cells are discussed in the webinar:

    Reagent Recommendation Purpose
    Positive crRNA Controls At least two different target genes Predictable or known effect in your assay
    Non-targeting crRNA Controls At least two distinct designs Control for nonspecific responses to RNP delivery
    crRNA targeting an essential gene (or Lethal Controls) Target a gene known to be needed for cell health Compare viability & cell count to experimental wells
    Experimental crRNAs 3-5 unique guides per gene Targeted knockout; multiple reagents provide redundancy

    Author: Louise Baskin, Senior Product Manager

    Watch the Webinar - High-throughput CRISPR-Cas9 Genome Engineering in Primary T Cells

    Additional Resources

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