• A multiparametric, high-content, arrayed CRISPR screen for cell cycle regulators

    Synthetic CRISPR RNA & IN Cell Analyzer are ideal for high-throughput, complex phenotype screening

    Synthetic CRISPR RNA & IN Cell Analyzer are ideal for high-throughput, complex phenotype screening

    Have you heard the news? Synthetic crRNA libraries are gaining in popularity for arrayed functional genomics screens, even for the whole human genome! A smaller gene family libraries, the Human Edit-R Cell Cycle Regulation library of over 600 crRNAs, was used in a high-content analysis screen examining cell cycle phenotypes. To perform the screening, the IN Cell Analyzer 2200 high content analysis system, with sensitive and flexible wide-field LIVE cell imaging acquired the images, and IN Cell Investigator Software quantified cells in different cell cycle phases. A peer-reviewed publication in the Journal of Biotechnology shows how synthetic CRISPR RNAs are ideally suited for arrayed screening in a wide variety of phenotypic readouts, including high-content microscopy assays.

    Check out these highlights from the publication!

    • The cell cycle reporter expresses a GFP-fusion protein that translocates between the nucleus and the cytoplasm depending on the phase of the cell cycle
    • Optimization with positive and negative crRNA controls demonstrated a robust, high-content assay
    • Multiple crRNA sequences per gene showed a phenotype, which led to very reliable hit-calling
    • Due to the robustness of the assay, many genes were identified with high-confidence even when moderate phenotypes were observed
    • RNAi, mismatch detection assays, expression analysis, and other strategies for hit follow-up and validation were employed to confirm genes’ role in cell cycle regulation

    High-content imaging was performed by the IN Cell Analyzer 2200 on live cells:

    • Seven experimental parameters were measured based on intensity and localization of GFP reporter and nuclear staining
    • The IN Cell Investigator Software used the seven parameters to classify cells into different categories (G1 phase; S or G2 phase; mitosis; condensed chromatin indicative of either aberrant mitosis or early apoptosis; cells with multinuclear DNA component).

    These types of one-well-per-gene data nicely highlight the power of arrayed CRISPR-Cas9 knockout screening in a wide variety of phenotypic readouts, including multiparametric high-content microscopy assays.

    Are you curious to learn more? Take a closer look at the publication here!

    High-content analysis screening for cell cycle regulators using arrayed synthetic crRNA libraries »

    Additional Resources

    • Edit-R crRNA Libraries

      Plated pre-defined collections of popular human and mouse gene families for arrayed knockout screening

    • Cherry-pick libraries

      Customize and order plates of predesigned crRNA for knockout studies for your targets of interest

    • IN Cell Analyzer 2200

      Learn how the IN Cell Analyzer platform can assist in your high-throughput CRISPR screens