Table 3.

Description of key published studies to date using pooled CRISPR-based screening libraries.

Study reference	Cas9 system	Library size, genes targeted	Cell type(s)	Selection	Key findings with implications for screening
Wang et al., 2014	WT Cas9 stably expressed.	73,151 sgRNAs 7,114 genes 10 sgRNA/gene	Human KBM7 (CML) HL60 (PML)	6-thioguanine Etoposide Proliferation	CRISPR-based KO screening is effective in both haploid and diploid cells. Off-target effects are minimal.
Shalem et al., 2014	WT Cas9 encoded in same lentivirus as sgRNA	64,751 sgRNAs 18,080 genes 3-4 sgRNA/gene	Human A375 (melanoma) HUES62 (stem cell line)	Proliferation Vemurafenib	CRISPR-based KO screening produces strong phenotypes that may be undetectable using RNAi. Hits have a high validation rate.
Koike-Yusa et al., 2014	WT Cas9 stably expressed	87,897 sgRNAs 19,150 genes 2-5 sgRNA/gene	Mouse JM8 (ES cell line)	Alpha toxin 6-thioguanine	CRISPR is powerful enough to be used for recessive screens. Off target effects are low, subject to effective sgRNA design. Lentiviral-delivered sgRNA guide sequences are most effective if the first nucleotide is a G.
Zhou et al., 2014	WT Cas9 stably expressed, OCT4 stably expressed to boost U6 promoter	869 sgRNA 291 genes ~3 sgRNA/gene	Human HeLa (adenocarcinoma)	Diptheria toxin Chimaeric anthrax	CRISPR has advantages over RNAi for knowledge-based screening with small focused libraries. It is important to select a clonal Cas9 cell line with a high modification efficiency.
Bassett et al., 2015	WT Cas9 encoded in same plasmid as sgRNA	40,279 sgRNA 13,501 genes ~3 sgRNA/gene	Drosophila S2R+ cells	Proliferation	First Drosophila whole genome sgRNA library. Plasmid transfection can be used in pooled approaches if DNA is diluted and cells are selected.
Gilbert et al., 2014	CRISPRi: dCas9-KRAB stably expressed CRISPRa: sunCas9 stably expressed	206,421 sgRNAs 15,977 genes ~10 sgRNA/gene 198,810 sgRNAs 15,977 genes ~10 sgRNA/gene	Human K562 (CML)	Proliferation Chimaeric cholera toxin-diptheria toxin catalytic A subunit	Genome-wide activation and repression-based screens are robust and give complementary results. Off-target effects are extremely low due to mismatch intolerance. Perturbation of non-coding elements is achievable.
Konermann et al., 2015	SAM (synergistic activation mediator) with protein components stably expressed	70,290 sgRNAs 23,430 genes 3 sgRNA/gene	Human A375 (melanoma)	Vemurafenib	The crystal structure of Cas9 can inform effective engineering strategies for gene activation. Activation-based screening is an alternative to cDNA overexpression, and hits have a high validation rate.

CML, chronic myeloid leukemia; CRISPRa, CRISPR activation; CRISPRi, CRISPR interference; PML, promyelocytic leukemia.