A multiparametric highcontent arrayed CRISPR screen for cell cycle regulators

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

For high-throughput, complex phenotype screening use Synthetic CRISPR RNA

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 library, the Human Edit-R Cell Cycle Regulation of over 600 crRNAs, was used in a high-content analysis screen examining cell cycle phenotypes. A peer-reviewed publication in the Journal of Biotechnology illustrates 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 on live cells:

Seven experimental parameters were measured based on intensity and localization of GFP reporter and nuclear staining.

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