Successful CRISPR gene editing in primary T cells

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

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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

Edit-R predesigned crRNA

  • Guaranteed to edit your target! Algorithm-optimized crRNA for genome-wide coverage of human, mouse, or rat genes. Modifications for nuclease resistance improve DNA-free editing. Simply search for your gene!

Edit-R Cas9 Nuclease protein NLS

  • Purified Cas9 protein ready to use for DNA-free nuclease expression.

Edit-R Cas9 nuclease protein electroporation - Protocol

  • Electroporation protocol for use with Edit-R Cas9 protein NLS and synthetic guide RNA.

CRISPR-Cas9 guide RNAs and the innate immune response

  • Comparing in vitro transcribed and chemically synthesized guide RNA for induction of innate immune response genes.