eBook: Optimizing Gene-Editing Workflows - 19

Applying a selection pressure is optional
A selective pressure, such as a drug, may be applied
to arrayed screens in order to test what genes, when
ablated, affect one or more cellular phenotypes
when under certain contexts.
Analysis of arrayed screening results
Edited cells are assessed using an appropriate assay.
Because each target is separated across wells, one
can easily connect the phenotype to each genotype.
When to use a pooled or arrayed
screen?
Figure 2. Steps involved in performing an arrayed
CRISPR screen
Constructing an arrayed library
Arrayed gRNA libraries can be produced in a variety
of formats: plasmid, virus, and RNA oligonucleotides
(synthetic sgRNA).
Constructing an arrayed library to cells
The gRNA is introduced to cells (one target per well)
through one of a variety of methods. Cas9 is either
co-transfected in plasmid format or in complexed ribonucleoprotein
(RNP). Alternatively, Cas9-expressing
cells may be used.
The decision on what screening format to use is
based on a number of considerations, including the
assay, cell model, and labor, among others. Below,
we outline some of the advantages and disadvantages
of each. A more comprehensive comparison of
the benefits and drawbacks of each screen format is
summarized in Table 1.
Assay compatibility is essential
As described above, pooled screens are restricted to
binary assays, while arrayed screens are compatible
with binary and multiparametric assays. Given these
differences in versatility, it is important
about what phenotype(s) would be most informative
for answering your research question. For instance,
if identifying genes that sensitize/desensitize
a disease cell type to a given drug is the goal, then
a pooled screen may be adequate. However, if the
objective is to identify gene disruptions that cause
changes in multiple morphological features or complex
phenotypes, then an arrayed screen would be
more appropriate.
to think
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