eBook: Optimizing Gene-Editing Workflows - 16

Pooled vs. Arrayed
CRISPR Screens
Considerations before choosing a screening format.
Amanda Mah, Ph.D.
The drug discovery process begins with identifying
genes or targets that play a role in the specific disease
of interest. This critical step of target identification is
done through a process called screening, a method
that assesses a large number of genes at one time to
identify the gene(s) responsible for a particular outcome
or phenotype. CRISPR has made screening
and target identification much more precise and
reliable compared to previous methods. There are
two choices of CRISPR screening approaches.
In this article, we discuss the two types of CRISPR
screens, pooled and arrayed, explain what they are,
how they are different, and when to use each one.
What is a pooled screen?
Pooled screens involve introducing a " pool " or mixture
of sgRNA into a single population of cells as a
whole. Because the edits occur across all targets in
a single tube of cells, it is difficult to link the phenotype
of each individual cell with the underlying
genetic perturbation. Pooled screens are thus only
compatible with binary assays that physically separate
edited cells exhibiting a phenotype of interest
from those that do not.
Pooled screens commonly involve packaging sgRNA-containing
plasmids into lentiviral particles
(one per vector) and then combining each virus together
equally. This pooled lentiviral library is then
transduced into host cells. The stable expression of
guide (along with Cas9) facilities the knockouts of
targeted genes.
Steps of a pooled screen
Performing a pooled screen can be divided into four
main stages. The schematic in Figure 1 highlights
the main steps within each stage. Further details are
provided in the descriptions of each stage below.
Constructing a pooled library
Plasmids encoding sgRNAs are sold as E. coli glycerol
stocks. The plasmids are PCR-amplified and validated
via NGS to ensure that equal representation
is maintained. The plasmids are then packaged into
lentiviral particles (one guide per vector) containing
a selectable marker (e.g., antibiotic resistance gene).
Typically, more than one sgRNA is designed per
gene to increase confidence in genotype to phenotype
correlations. Libraries can also be purchased as
pre-packaged viral particles.
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https://www.synthego.com/workflows/drug-discovery https://www.synthego.com/learn/crispr https://www.synthego.com/applications/screening-target-identification https://www.synthego.com/applications/screening-target-identification https://www.synthego.com/guide/crispr-methods/crispr-screen https://www.synthego.com/guide/crispr-methods/crispr-screen

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