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"The power of CRISPR-Cas9 arrayed screen using synthetic crRNA libraries"
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|14h00||Café d’accueil des participants|
|14h30||Introduction – GE Healthcare Life Science|
|14h45||The power of CRISPR-Cas9 arrayed screen using synthetic crRNA libraries
Amanda HAAS, PhD, GE Healthcare Dharmacon, Lafayette CO, USA
CRISPR-Cas9 is a powerful tool for loss-of-function analysis, and to date has been adopted for large-scale functional screening primarily in the form of pooled sgRNA libraries. However, pooled screening is limited to enrichment or depletion phenotypic assays, thereby reducing the breadth and depth of biological questions that can be applied. An arrayed platform, which supports one-gene-per-well knockout, greatly expands the types of phenotypic assays to morphological and other high-content readouts, including multiparametric analysis. The experimental considerations for optimization and successful execution of an arrayed CRISPR-Cas9 screen using a two-part (dual) synthetic guide RNA system will be briefly described. We will present results from a multiparametric HCA screen using an arrayed crRNA library of cell cycle-related genes in a G1S cell cycle phase reporter cell line. This cell line expresses EGFP fused to a subcellular localization domain of Helicase B, which translocates between the nucleus and cytoplasm based on its phosphorylation state, driven by the cell cycle phase. Finally, we will review two recent publications using arrayed synthetic crRNAs and the utility of this approach.
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