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Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library


Identification of genes influencing a phenotype of interest is frequently achieved through genetic screening by RNA interference (RNAi) or knockouts. However, RNAi may only achieve partial depletion of gene activity, and knockout-based screens are difficult in diploid mammalian cells. Here we took advantage of the efficiency and high throughput of genome editing based on type II, clustered, regularly interspaced, short palindromic repeats (CRISPR)–CRISPR-associated (Cas) systems to introduce genome-wide targeted mutations in mouse embryonic stem cells (ESCs). We designed 87,897 guide RNAs (gRNAs) targeting 19,150 mouse protein-coding genes and used a lentiviral vector to express these gRNAs in ESCs that constitutively express Cas9. Screening the resulting ESC mutant libraries for resistance to either Clostridium septicum alpha-toxin or 6-thioguanine identified 27 known and 4 previously unknown genes implicated in these phenotypes. Our results demonstrate the potential for efficient loss-of-function screening using the CRISPR-Cas9 system.

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Figure 1: Stable expression of Cas9 and gRNA from single-copy transgenes can induce site-specific DSBs.
Figure 2: Lentiviral delivery of gRNA expression cassettes.
Figure 3: Analyses of 52 gRNAs targeting 26 genes involved in the GPI-anchor biosynthesis pathway.
Figure 4: Generation of a mouse genome-wide lentiviral gRNA library.
Figure 5: Genetic screens using the genome-wide gRNA library and genetic validation assays of the novel candidate genes.

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We thank A. Bradley for comments on the manuscript, B. Ng and W. Cheng for the flow cytometry analyses, and the Sanger Institute DNA pipeline for the sequence analyses. We also thank J. Takeda and T. Kinoshita for providing us alpha-toxin and the cDNA expression vectors, respectively. Y.L. is supported by the Wellcome Trust PhD program. M.D.C.V.-H. is supported by the Cancer Research UK and Wellcome Trust PhD program. This work was supported by Wellcome Trust (WT077187). The mouse CRISPR library is available through Addgene. The plasmid DNAs are available at the Wellcome Trust Sanger Institute Archives (

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Authors and Affiliations



K.Y. conceived the research and wrote the manuscript with comments from all authors. H.K.-Y., E.-P.T. and K.Y. performed the experiments. Y.L. and M.D.C.V.-H. performed the bioinformatics analyses.

Corresponding author

Correspondence to Kosuke Yusa.

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Competing interests

K.Y. and Y.L. filed a patent application based on the results reported in this paper.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–19 and Supplementary Tables 3–7 (PDF 2520 kb)

Supplementary Table 1

Off-target cleavage analyses of the gRNA targeting Site 2 of the Piga gene (with no bulge) (XLSX 101 kb)

Supplementary Table 2

Off-target cleavage analyses of the gRNA targeting Site 2 of the Piga gene (with bulges) (XLSX 157 kb)

Supplementary Table 8

List of shRNAs and oligo seqeunces (XLSX 10 kb)

Supplementary Table 9

List of genes, gRNA target sequences and oligonucleotide sequences used in this study. (XLSX 18 kb)

Supplementary Data 1

(XLSX 35502 kb)

Supplementary Data 2

(XLSX 6296 kb)

Supplementary Data 3

A full list of potential off-target sites (with NGG PAM) of the gRNA targeting site 2 of the Piga gene. (XLSX 338 kb)

Supplementary Data 4

A full list of potential off-target sites (with NAG PAM) of the gRNA targeting Site 2 of the Piga gene. (XLSX 455 kb)

Supplementary Data 5

A full list of potential off-target sites (with bulges) of the gRNA targeting site 2 of the Piga gene. (XLSX 76 kb)

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Koike-Yusa, H., Li, Y., Tan, EP. et al. Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library. Nat Biotechnol 32, 267–273 (2014).

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