• Strategies to detect and validate your CRISPR gene edit

    Once you have carried out your gene editing experiment, how will you monitor the result?

    You’ve chosen a CRISPR strategy to introduce a gene edit into your target cells. How will you verify and characterize the edit?

    Depending on your experimental purpose and the nature of the gene edit, a variety of assays may be used, yielding various amounts and types of information.

    This article summarizes the most commonly applied assays (Table 1), together with examples that apply them (see below, application examples A-J).

    Application examples

    Summary

    Common assays to detect and validate your CRISPR gene edit

    For most purposes, validation and characterization of edits on both the molecular and phenotypic level, will be required to assess their biological relevance.

    For genomic screening purposes (application examples B, H, I, J), a phenotypic assay may be a good starting point to rapidly screen gene edits that show a desired phenotype.

    DNA mismatch assays, TIDE, RFLP, or phenotypic assays are often applied as starting points to assess the success of the CRISPR experiment and screen positive clones with desired knockout or knockin mutations. Usually this first step, will be followed by more in-depth characterization of the nature of the edit on the sequence level, as well as on the functional level.

    Hence, in a typical gene editing experiment, a multitude of assays will be applied successively (See application examples above).

    References

    1. R. D. Mashal, J. Koontz, J. Sklar, Detection of mutations by cleavage of DNA heteroduplexes with bacteriophage resolvases. Nat Genet 9, 177-183 (1995).
    2. L. Vouillot, A. Thelie, N. Pollet, Comparison of T7EI and surveyor mismatch cleavage assays to detect mutations triggered by engineered nucleases. G3 (Bethesda) 5, 407-415 (2015).
    3. E. K. Brinkman, T. Chen, M. Amendola, B. van Steensel, Easy quantitative assessment of genome editing by sequence trace decomposition. NAR 42, Issue 22, Pages e168 (2014).

    Author: Kathrin Kerschgens Ph.D. | Project Manager Cell Line Engineering

    Additional Resources

  • On-demand Webinar: Getting started with CRISPR-Cas9