Abstract
Targeted genome editing technologies are powerful tools for studying biology and disease, and have a broad range of research applications1,2,3,4,5,6,7. In contrast to the rapid development of toolkits to manipulate individual genes, large-scale screening methods based on the complete loss of gene expression are only now beginning to be developed8,9. Here we report the development of a focused CRISPR/Cas-based (clustered regularly interspaced short palindromic repeats/CRISPR-associated) lentiviral library in human cells and a method of gene identification based on functional screening and high-throughput sequencing analysis. Using knockout library screens, we successfully identified the host genes essential for the intoxication of cells by anthrax and diphtheria toxins, which were confirmed by functional validation. The broad application of this powerful genetic screening strategy will not only facilitate the rapid identification of genes important for bacterial toxicity but will also enable the discovery of genes that participate in other biological processes.
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Acknowledgements
We thank J. Xi for providing the Cas9-encoding construct, F. Tang for technical advice regarding the high-throughput sequencing analysis, Z. Jiang for aid with the cell lines, Y. Yang for assistance in primer design, Y. Wang for data analysis, and D. Trono for aid with the lentivirus packaging system. We thank Peking University High-throughput Sequencing Center operated by BIOPIC. This work was supported by funds from the National Basic Research Program of China (2010CB911800), the National Science Foundation of China (NSFC31170126, NSFC31070115), and the Peking-Tsinghua Centre for Life Sciences.
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Contributions
W.W., Y.Z., S.Z. and C.C. conceived the methodology and designed the experiments. Y.Z., S.Z., C.C., P.Y. and C.L. performed the experiments. W.W., Y.Z., S.Z., C.C., P.Y. and Y.H. analysed the data. W.W., C.C., Y.Z. and S.Z. wrote the manuscript with help from the other authors.
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The authors declare no competing financial interests.
Extended data figures and tables
Extended Data Figure 1 Relative expression level of Cas9 by real-time PCR.
Two pairs of primers (a, 5′-GGGGGACAGTCTTCACGAGC-3′ and 5′-CACGTACATGTCCCTGCCGT-3′; b, 5′-GCACGTGGCCCAAATTCTCG-3′ and 5′-AGCGGTGGCCTTGCCTATTT-3′) were designed to test Cas9 expression level. cDNA of HEK293T serves as the negative control. β-Actin was used as the internal control and the Cas9 expression of HEK293TOC was set as the calibrator. The data are the mean ± s.d. (n = 3).
Extended Data Figure 2 Images of cells after three rounds of toxin treatment: diphtheria toxin (7.5 ng ml−1) for 60 h or PA/LFnDTA (70 ng ml−1/ 50 ng ml−1) for 48 h.
The fluorescence signals were superimposed onto white light images. Scale bar: 100 μm.
Extended Data Figure 3 PCR amplification of the sgRNA region from the sgRNA library for deep-sequencing analysis, as indicated by electrophoresis.
The genomic DNAs isolated from the indicated cells were used as templates for PCR amplification.
Extended Data Figure 4 Scatter plots of log2 normalized read counts between two sgRNA libraries.
a, Scatter plots of log2 normalized read counts between two of the three replicated sgRNA libraries. b, Scatter plots of log2 normalized read counts between two sgRNA libraries with or without prolonged culturing (14 days), and three replicates of the original library were used for the comparison.
Extended Data Figure 5 Effect of ANTXR1 deficiency on PA/LFnDTA- and diphtheria-toxin-triggered cytotoxicity in HeLa cells.
a, Partial sequences of ANTXR1 gene in genome containing TALENs binding regions (underlined) and the mutated alleles from 10 randomly selected TALENs clones (in HeLa cells). The tall letter indicates insertion, and the dashes indicate deletions in this and other figures. b, DNA sequencing analysis of mutated alleles in the human ANTXR1 locus. Partial coding sequences of targeted genes in the genome containing the CRISPR/Cas9 sgRNA3ANTXR1 binding region (underlined) and sequence analysis of the mutated alleles from two independent clones are shown. Primers used for the PCR reactions are listed in Supplementary Table 5, and the shaded nucleotides represent the PAM sequences that guide the Cas9 for DNA recognition and cleavage in this and other figures. c, d, Susceptibility assay of two HeLa ANTXR1−/− clones on PA/LFnDTA- and diphtheria-toxin-triggered cytotoxicity. The cells were treated with different doses of toxins, and the XTT cell viability assay and LDH lethality assay were performed 48 h after toxin treatment. Data are presented as the mean ± s.d. (n = 3).
Extended Data Figure 6 Effect of HBEGF deficiency on diphtheria-toxin-triggered cytotoxicity in HeLa cells.
a, DNA sequencing analysis of mutated alleles in the human HBEGF locus. Partial coding sequences of targeted genes in the genome containing the CRISPR/Cas9 sgRNA3HBEGF binding region (underlined) and sequencing analysis of the mutated alleles from two independent clones. b, Susceptibility assay of HeLa HBEGF−/− clone 2 on diphtheria-toxin-triggered cytotoxicity. The cells were treated with different doses of toxins, and the XTT cell viability assay and LDH lethality assay were performed 48 h after toxin treatment. Data are presented as the mean ± s.d. (n = 3).
Extended Data Figure 7 Light microscopic images of HeLaOC-SC cells transfected with indicated sgRNA constructs.
Four days after transfection, cells were incubated with PA (70 ng ml−1) plus LFnDTA (50 ng ml−1) for 48 h. The representative images for every condition (six images for each) are shown. Scale bar, 200 μm.
Extended Data Figure 8 Effects of RAB2A sgRNAs on diphtheria-toxin-triggered cytotoxicity in HeLa cells.
HeLaOC-SC cells were transfected with indicated sgRNA constructs. Four days after transfection, the LDH lethality assay (a) and the XTT cell viability assay (b) were performed 48 h after toxin treatment. HBEGF_sg3, labelled in red, was used as the positive control. The data are the mean ± s.d. (n = 3); *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant; Dunnett’s multiple comparison test, one-way ANOVA.
Extended Data Figure 9 Effect of PECR deficiency on PA/LFnDTA- and diphtheria-toxin-triggered cytotoxicity in HeLa cells.
a, b, DNA sequencing analysis of mutated alleles in the human PECR locus. Partial coding sequences of targeted genes in the genome containing the CRISPR/Cas9 sgRNA1PECR (a) and sgRNA2PECR (b) binding region (underlined) and sequencing analysis of the mutated alleles from two independent clones are shown. c, Susceptibility assay of HeLa PECR−/−_sg2 clone on PA/LFnDTA- and diphtheria-toxin-triggered cytotoxicity. The cells were treated with different doses of toxin, and the LDH lethality assay was performed 48 h after toxin treatment. Data are presented as the mean ± s.d. (n = 3). d, Effect of PECR-targeting shRNA smart pool on HeLa cells to PA/LFnDTA toxicity. Three different shRNAs (TRCN0000046538: 5′-CCGGGCAGTGGAATACGGATCAATTCTCGAGAATTGATCCGTATTCCACTGCTTTTTG-3′, TRCN0000046539: 5′-CCGGCCTCACCAAATCTTTAGCTTTCTCGAGAAAGCTAAAGATTTGGTGAGGTTTTTG-3′, and TRCN0000046541: 5′-CCGGCCCGCTAAACGAATTGGTGTTCTCGAGAACACCAATTCGTTTAGCGGGTTTTTG-3′) from MISSION shRNA library (Sigma, Inc.) were chosen, and the MISSION shRNA control vector (SHC002) was used, which contains a sequence that engages with RISC without targeting any known mammalian gene. After transient transfection, 1.5 μg ml−1 puromycin was added to obtain the stable shRNA expression cells. The cells were treated with PA (40 ng ml−1) plus LFnDTA (50 ng ml−1), and the XTT cell viability assay was performed 48 h after toxin treatment. Data are presented as the mean ± s.d., n = 3, **P < 0.01; t-test.
Extended Data Figure 10 Effects of ANTXR1 and ANTXR2 gene expression on the susceptibility of HeLa and HEK293T cells to PA/LFnDTA.
a, Effects of ANTXR1 and ANTXR2 gene knockout on the susceptibility of the indicated cells to the treatment of PA and LFnDTA. Details of the TALENs constructs used for gene targeting have been previously described16. Cell clones transfected with TALENs were randomly picked and cultured with PA (100 ng ml−1) and LFnDTA (50 ng ml−1) for 3 days to determine their sensitivity to toxins. b, Level of ANTXR1 and ANTXR2 transcripts in HeLa and HEK293T cells was measured by RT–PCR, and β-actin and GAPDH were included as internal controls. To ensure accuracy, two pairs of primers were used to measure ANTXR1 and ANTXR2 transcript levels, respectively. The primers used in PCR reactions were: for ANTXR1, primer pair 1 5′-GGAAACGGCTTCCGACATGC-3′and 5′-TGTTGTGGGGCCTGATGCAA-3′, primer pair 2 5′-GCCCGGATTGCGGACAGTAA-3′ and 5′-GCTGACCTGGAGTGCAGCTT-3′; for ANTXR2, primer pair 1 5′-TTGGACGGTCTGGTGCCATC-3′ and 5′-TCCTCCCCCACACAGACACT-3′, and primer pair 2 5′-ATGTGGTGGTTTTGGCCCCT-3′ and 5′-GTGTGGGTTTGGGTCGAGGT-3′; for β-actin, 5′-AGAGCTACGAGCTGCCTGAC-3′ and 5′-AGCACTGTGTTGGCGTACAG-3′; for GAPDH, 5′-ACCACAGTCCATGCCATCAC-3′ and 5′-TCCACCACCCTGTTGCTGTA-3′.
Supplementary information
Supplementary Information
This file contains Supplementary Figure 1 and Supplementary Tables 1, 5 and 6. (PDF 2349 kb)
Supplementary Table 2
Oligos for the sgRNA library construction. (XLSX 92 kb)
Supplementary Table 3
Screening results of the sgRNA Library. (XLSX 92 kb)
Supplementary Table 4
Statistical analysis of the deep-sequencing data. (XLSX 175 kb)
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Zhou, Y., Zhu, S., Cai, C. et al. High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells. Nature 509, 487–491 (2014). https://doi.org/10.1038/nature13166
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DOI: https://doi.org/10.1038/nature13166
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