Horizon eBook - 22

Innovations in Cell-Based Screening * CRISPR-Cas9 Knockout Screening

screens, however, have two major limitations:
incomplete knockdown and off-target effects.
RNA-guided Cas9 nuclease is revolutionizing
gene editing in mammalian cells. This key component
of an evolutionally ancient bacterial adaptive immunity
system has been engineered to be active in the nucleus,
where engineered single-guide RNAs (sgRNAs) can
target the nuclease to any 17-20 nucleotide sequence
where it generates a double stranded break that
when repaired by non-homologous end joining
creates small insertions and deletions that inactivate
gene function.
sgRNAs are close in size (70-80 nt) to the shorthairpin RNAs that are used in RNA interference
screens. So can the Cas9/sgRNA system be readily
adapted to the existing shRNA lentiviral screening infrastructure to enable genome-wide knockout
screening? And would this technology overcome
some of the disadvantages of RNA interference?
This year both these questions have been answered
with a definitive "Yes." Four reports have been
published presenting the results of whole genome
screens using lentiviruses to deliver Cas9 and sgRNA

| GENengnews.com

libraries into human or murine cells and next-generation
sequencing to quantify the depletion or accumulation
of the integrated lentiviral DNA sequences specifying
each sgRNA1-4.
All four papers demonstrate the superlative
performance of sgRNA in positive selection screens
(where editing of the sgRNA target confers a
proliferation advantage). For example, Feng Zhang's
group found that the frequency of all the sgRNAs
targeting tumor suppressors whose loss is associated
with resistance to the BRAF inhibitor vemurafenib
were dramatically elevated in a treated population.
Results with shRNAs were far less clear. Similarly,
all 6 sgRNAs vs. enhanced green-fluorescent protein
tested proved able to eliminate GFP expression in
>95% of stably transduced cells, whereas shRNAs
only reduced expression 7-30 fold.
sgRNA screening is set to become an important
tool in oncology for identifying synthetic lethal/
co-dependence targets and can also be applied to
other therapeutic areas where pathway function can
be engineered to regulate survival or expression of
fluorescent markers. n

Cells deleted for tumor suppressors accumulated
under vemurafenib selection


Horizon eBook

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Horizon eBook - 1
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