Horizon eBook - 19

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

a substantially reduced abundance at the end of a pooled screen. For this
readout to work effectively using NGS, we must assume that a majority of
the sgRNAs targeted to a particular essential gene will cut effectively in the
cells in which they are expressed, leading to genetic disruption. However, if
only two out of five sgRNAs effectively result in gene disruption in only 30%
of the cells in which they are expressed, and the other three guides result in
minimal genetic disruption, loss of this 30% of cells and their sgRNAs could
be easily missed. At Horizon, we are investigating whether building smaller
sgRNA libraries against specific subsets of the genome will enable synthetic
lethal targets to be more readily identified.
The identification of new potential therapeutic targets from an initial
Cas9-sgRNA screen is arguably the easy part. Target validation comes next,
with new challenges. Many approaches can be taken here, and if several
hundred potential hits have been identified then re-screening with new
sgRNAs to potential hits using a pooled approach should help to narrow
down the field. For tens rather than hundreds of hits, standard shRNA or
siRNA approaches could be used. However, the correlation between targets
identified using RNAi techniques and those identified using Cas9-sgRNAs
seems to be poor.8 The recently described use of CRISPRi to induce target
repression, or CRISPRa to activate gene expression, both requiring modified
versions of Cas9, appear on paper to be another useful method that could be
readily applied to target validation.10 Rapid validation of a small number of
hits could also be achieved using vectors that express fluorescent proteins, by

| GENengnews.com

Figure 2. Using fluorescence to help validate new drug targets.

looking for changes in the ratio of red to green fluorescence (Figure 2).11
The use of sgRNA-Cas9 genome engineering is evolving rapidly12-15
and although technically challenging, these techniques should broaden the
range of available approaches and reduce the time taken to identify new
drug targets and to validate them. n


Horizon eBook

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