Speakers
Amanda Haas
(GE Healthcare Life Sciences, Dharmacon)
Date
15
Mar 2017
Time
10:00:00
Institut Pasteur, Paris, France
Address
Building: Monod (66)
Location
2017-03-15 10:00:00
2017-03-15 00:00:00
Europe/Paris
The power of CRISPR-Cas9 arrayed screen using synthetic crRNA libraries
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 […]
Institut Pasteur, Paris, France
Fabrice Agou
fabrice.agou@pasteur.fr
About
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.
Location
Building: Monod (66)
Address: Institut Pasteur, Paris, France