Horizon eBook - 18

Innovations in Cell-Based Screening * Finding a Signal Amid the Noise

Figure 1. Pooled approaches aim to express one lentivirus and therefore
one sgRNA per cell.


| GENengnews.com

rely on disrupted genetic and epigenetic networks and signals from surrounding cells rather than on a single mutation), making identification of potential
targets difficult. Genome-wide screens aid the identification of targets that are
important in these networks and do not require a priori knowledge of the
interactions between the networks. Many screens have been carried out using
shRNA or siRNA, but off-target effects have proved to be a substantial
limitation. Moreover, if the intention of a screen is to identify true synthetic
lethal targets, then a robust loss of the target is warranted rather than its
transient or partial knockdown. So, can cell-line gene editing technologies
help in determining a distinct signal within the noise of a genetically heterogeneous population, such as one might find in a population of tumor cells?
Several recent high-profile publications have demonstrated the feasibility
of whole-genome knockout screens using Cas9-sgRNA.7-9 The effectiveness
of this approach for target identification and drug discovery is promising, but
is yet to be fully tested and may well depend on what one is hoping to find.
For example, it is fairly straightforward to use a pooled sgRNA approach
(Figure 1) in positive selection screens, where one is looking for genes that
when lost enable survival in the presence of a drug or a specific mutation. In
these cases, the sgRNAs targeting relevant genes will increase in abundance
as the screen progresses, making them easy to identify using next-generation
sequencing (NGS) at the end of the screen. Conversely, negative, drop-out
or synthetic lethal screens where loss of a gene might contribute to increased
sensitivity to a drug or genetic mutation rely on finding sgRNAs that have


Horizon eBook

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