34 128
essential host response to pathogens [68], which would otherwise be technically challenging
with other genome-editing tools.
Cas9-mediated loss-of-function screens have also performed to knock out pairs of genes in
combination [69]. A library of 23 409 barcoded dual sgRNA combinations was created and a
pooled screen was performed to identify gene pairs in human cells that inhibit ovarian cancer cell
growth in the presence of small-molecule drugs. While further work is needed to characterize the
ef
fi
cacy and accuracy of multiplex genetic screening, this work highlights the potential of more
sophisticated functional screening studies using CRISPR.
Beyond Cas9-based complete loss-of-function screens, the invention of CRISPRi and CRISPRa
further enables both partial loss-of-function and gain-of-function genetic screens [47,48].
Growth-based screens using CRISPRi/a have been used to identify essential genes, tumor
suppressor genes, and potential mechanisms that confer cytotoxicity induced by a cholera-
diphtheria toxin [47]. Using a library comprising approximately 70 000 guides targeting the
human RefSeq coding isoforms, a CRISPRa-based screen identi
fi
ed genes that, upon activa-
tion, conferred resistance to a BRAF inhibitor [48].
In addition to the use of pooled screens, multi-well plates have been used in combination with
the partial repression feature of CRISPRi to study the function of the full set of essential genes in
the Gram-positive bacterium
Bacillus subtilis
[45]. Given that knocking out essential genes
results in lethality that prevents further assay of the phenotype, partial knockdown of essential
genes becomes a powerful approach. A mutant
B. subtilis
library was created to include gene
partial knockdowns (approximately threefold) of all essential genes using CRISPRi, which was
tested for the growth phenotype under 35 unique compounds. Using this chemical genomic
approach, a comprehensive interconnecting essential gene network was identi
fi
ed, as well as
targeted genes that interact with uncharacterized antibiotics. Inducible knockdown of essential
genes also allowed for systematic characterization of cell morphology and terminal death
phenotypes.
An important question is how these screens compare with each other and with other existing
approaches. Several works compared different screens based on CRISPR, CRISPRi, and RNAi.
One work performed comparative screens of 46 essential and 47 nonessential genes, and
concluded that the CRISPR/Cas9 nuclease system outperformed the shRNA- and CRISPRi/
dCas9-based gene regulation systems for the sets of essential and nonessential genes [70].
From the CRISPR screening data, the authors observed less variation across the data, and
detected more functional constructs with fewer off-target effects. Another study concluded that
CRISPR could identify more essential gene targets compared with RNAi [71]. Since similar
precision was observed between the two approaches, it was suggested that combining data
from both screens would improve the predictive accuracy. The systematic comparison of
different approaches suggests that a comparative screening approach will be more powerful
for studying complex cell biology phenotypes.
In addition, new methods to generate CRISPR libraries may help reduce the overall cost
associated with this technique and extend its uses to screen a larger chromosomal region
(e.g., the tiling along a whole chromosome). While most CRISPR libraries are generated via
chemical synthesis of large pools of oligos, a new method, termed CRISPR EATING (Everything
Available Turned Into New Guides), can inexpensively generate large quantities of sgRNAs for
whole-genome targeting [72]. In this approach, PAM-proximal sequences are extracted by
digesting input DNA with restriction enzymes that target immediately 5
0
to an NGG or NAG (the
PAM sequences for
S. pyogenes
Cas9, N = any nucleic acid). In this study, one library was
generated and used to label the whole 3.4-mb region on
Xenopus laevis
chromosome 4 in the
882
Trends in Cell Biology, November 2016, Vol. 26, No. 11