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A Dual-reporter system for real-time monitoring and high-throughput CRISPR/Cas9 library screening of the hepatitis C virus.

Ren Q, Li C, Yuan P, Cai C, Zhang L, Luo GG, Wei W - Sci Rep (2015)

Bottom Line: The hepatitis C virus (HCV) is one of the leading causes of chronic hepatitis, liver cirrhosis and hepatocellular carcinomas and infects approximately 170 million people worldwide.Using the NIrD system and a focused CRISPR/Cas9 library, we identified CLDN1, OCLN and CD81 as essential genes for both the cell-free entry and the cell-to-cell transmission of HCV.The combination of this ultra-sensitive reporter system and the CRISPR knockout screening provides a powerful and high-throughput strategy for the identification of critical host components for HCV infections.

View Article: PubMed Central - PubMed

Affiliation: Biodynamic Optical Imaging Center (BIOPIC), Peking-Tsinghua Center for Life Sciences, State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing 100871, China.

ABSTRACT
The hepatitis C virus (HCV) is one of the leading causes of chronic hepatitis, liver cirrhosis and hepatocellular carcinomas and infects approximately 170 million people worldwide. Although several reporter systems have been developed, many shortcomings limit their use in the assessment of HCV infections. Here, we report a real-time live-cell reporter, termed the NIrD (NS3-4A Inducible rtTA-mediated Dual-reporter) system, which provides an on-off switch specifically in response to an HCV infection. Using the NIrD system and a focused CRISPR/Cas9 library, we identified CLDN1, OCLN and CD81 as essential genes for both the cell-free entry and the cell-to-cell transmission of HCV. The combination of this ultra-sensitive reporter system and the CRISPR knockout screening provides a powerful and high-throughput strategy for the identification of critical host components for HCV infections.

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Screen of host genes essential for an HCV infection.(a) Primary HCVcc screening data. The count of every sgRNA is the number of reads that match the sgRNA target sequence. (b) sgRNA ranking was based on the fold change of normalised counts of every sgRNA in the HCV treated and untreated populations. (c) The FDR (false discovery rate) of every gene in the library was calculated by MAGeCK32 based on the counts and kinds of sgRNAs in the three replicates. (d) Effects of the gene knockout of CLND1, OCLN and CD81 on cell-free entry of HCVcc. All cells indicated carry the NIrD system. Light and fluorescence images were taken 72 h post-HCVcc infection in the presence of Dox (2 μg/ml). Scale bar, 100 μm. (e) Effects of the gene knockout of CLND1, OCLN and CD81 on cell-to-cell transmission of HCVcc. Huh7.5(NΙrD) cells with the indicated background (WT, CLDN1−/−, CLDN1−/−/CLDN1, OCLN−/−, OCLN−/−/OCLN, CD81−/− or CD81−/−/CD81) were co-cultured with HCVcc pre-infected (24 h prior) Huh7.5 cells. HCVcc carries the EGFP gene in its genome28, resulting in a punctuated green fluorescence pattern in the cells. OCLN−/− and CD81−/− knockout cells expressed diffused green fluorescence because they were derived from cells expressing EGFP. The light and fluorescence (green and red) images were taken 72 h following the co-culturing of the cells. Scale bar, 100 μm.
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f4: Screen of host genes essential for an HCV infection.(a) Primary HCVcc screening data. The count of every sgRNA is the number of reads that match the sgRNA target sequence. (b) sgRNA ranking was based on the fold change of normalised counts of every sgRNA in the HCV treated and untreated populations. (c) The FDR (false discovery rate) of every gene in the library was calculated by MAGeCK32 based on the counts and kinds of sgRNAs in the three replicates. (d) Effects of the gene knockout of CLND1, OCLN and CD81 on cell-free entry of HCVcc. All cells indicated carry the NIrD system. Light and fluorescence images were taken 72 h post-HCVcc infection in the presence of Dox (2 μg/ml). Scale bar, 100 μm. (e) Effects of the gene knockout of CLND1, OCLN and CD81 on cell-to-cell transmission of HCVcc. Huh7.5(NΙrD) cells with the indicated background (WT, CLDN1−/−, CLDN1−/−/CLDN1, OCLN−/−, OCLN−/−/OCLN, CD81−/− or CD81−/−/CD81) were co-cultured with HCVcc pre-infected (24 h prior) Huh7.5 cells. HCVcc carries the EGFP gene in its genome28, resulting in a punctuated green fluorescence pattern in the cells. OCLN−/− and CD81−/− knockout cells expressed diffused green fluorescence because they were derived from cells expressing EGFP. The light and fluorescence (green and red) images were taken 72 h following the co-culturing of the cells. Scale bar, 100 μm.

Mentions: High-throughput sequencing analysis revealed a total of 912 sgRNA sequences (99.3% of the 918 designed) from the original library (Supplementary Table 2). We compared the abundance of each sgRNA between the final enriched Huh7.5(NIrD)OC cells that no longer responded to HCV inoculation and the untreated populations and calculated a score for each sgRNA or gene using different algorithms (Fig. 4a–c). Three genes, CLDN1, OCLN and CD81, were confirmed to encode receptor proteins essential for HCV infections, based on false discovery rate (FDR) calculations32. It remains to be determined whether the other genes in the library are minimally involved in HCV infections or whether the number of designed sgRNAs targeting these genes were simply too small to produce data with statistical significance.


A Dual-reporter system for real-time monitoring and high-throughput CRISPR/Cas9 library screening of the hepatitis C virus.

Ren Q, Li C, Yuan P, Cai C, Zhang L, Luo GG, Wei W - Sci Rep (2015)

Screen of host genes essential for an HCV infection.(a) Primary HCVcc screening data. The count of every sgRNA is the number of reads that match the sgRNA target sequence. (b) sgRNA ranking was based on the fold change of normalised counts of every sgRNA in the HCV treated and untreated populations. (c) The FDR (false discovery rate) of every gene in the library was calculated by MAGeCK32 based on the counts and kinds of sgRNAs in the three replicates. (d) Effects of the gene knockout of CLND1, OCLN and CD81 on cell-free entry of HCVcc. All cells indicated carry the NIrD system. Light and fluorescence images were taken 72 h post-HCVcc infection in the presence of Dox (2 μg/ml). Scale bar, 100 μm. (e) Effects of the gene knockout of CLND1, OCLN and CD81 on cell-to-cell transmission of HCVcc. Huh7.5(NΙrD) cells with the indicated background (WT, CLDN1−/−, CLDN1−/−/CLDN1, OCLN−/−, OCLN−/−/OCLN, CD81−/− or CD81−/−/CD81) were co-cultured with HCVcc pre-infected (24 h prior) Huh7.5 cells. HCVcc carries the EGFP gene in its genome28, resulting in a punctuated green fluorescence pattern in the cells. OCLN−/− and CD81−/− knockout cells expressed diffused green fluorescence because they were derived from cells expressing EGFP. The light and fluorescence (green and red) images were taken 72 h following the co-culturing of the cells. Scale bar, 100 μm.
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f4: Screen of host genes essential for an HCV infection.(a) Primary HCVcc screening data. The count of every sgRNA is the number of reads that match the sgRNA target sequence. (b) sgRNA ranking was based on the fold change of normalised counts of every sgRNA in the HCV treated and untreated populations. (c) The FDR (false discovery rate) of every gene in the library was calculated by MAGeCK32 based on the counts and kinds of sgRNAs in the three replicates. (d) Effects of the gene knockout of CLND1, OCLN and CD81 on cell-free entry of HCVcc. All cells indicated carry the NIrD system. Light and fluorescence images were taken 72 h post-HCVcc infection in the presence of Dox (2 μg/ml). Scale bar, 100 μm. (e) Effects of the gene knockout of CLND1, OCLN and CD81 on cell-to-cell transmission of HCVcc. Huh7.5(NΙrD) cells with the indicated background (WT, CLDN1−/−, CLDN1−/−/CLDN1, OCLN−/−, OCLN−/−/OCLN, CD81−/− or CD81−/−/CD81) were co-cultured with HCVcc pre-infected (24 h prior) Huh7.5 cells. HCVcc carries the EGFP gene in its genome28, resulting in a punctuated green fluorescence pattern in the cells. OCLN−/− and CD81−/− knockout cells expressed diffused green fluorescence because they were derived from cells expressing EGFP. The light and fluorescence (green and red) images were taken 72 h following the co-culturing of the cells. Scale bar, 100 μm.
Mentions: High-throughput sequencing analysis revealed a total of 912 sgRNA sequences (99.3% of the 918 designed) from the original library (Supplementary Table 2). We compared the abundance of each sgRNA between the final enriched Huh7.5(NIrD)OC cells that no longer responded to HCV inoculation and the untreated populations and calculated a score for each sgRNA or gene using different algorithms (Fig. 4a–c). Three genes, CLDN1, OCLN and CD81, were confirmed to encode receptor proteins essential for HCV infections, based on false discovery rate (FDR) calculations32. It remains to be determined whether the other genes in the library are minimally involved in HCV infections or whether the number of designed sgRNAs targeting these genes were simply too small to produce data with statistical significance.

Bottom Line: The hepatitis C virus (HCV) is one of the leading causes of chronic hepatitis, liver cirrhosis and hepatocellular carcinomas and infects approximately 170 million people worldwide.Using the NIrD system and a focused CRISPR/Cas9 library, we identified CLDN1, OCLN and CD81 as essential genes for both the cell-free entry and the cell-to-cell transmission of HCV.The combination of this ultra-sensitive reporter system and the CRISPR knockout screening provides a powerful and high-throughput strategy for the identification of critical host components for HCV infections.

View Article: PubMed Central - PubMed

Affiliation: Biodynamic Optical Imaging Center (BIOPIC), Peking-Tsinghua Center for Life Sciences, State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing 100871, China.

ABSTRACT
The hepatitis C virus (HCV) is one of the leading causes of chronic hepatitis, liver cirrhosis and hepatocellular carcinomas and infects approximately 170 million people worldwide. Although several reporter systems have been developed, many shortcomings limit their use in the assessment of HCV infections. Here, we report a real-time live-cell reporter, termed the NIrD (NS3-4A Inducible rtTA-mediated Dual-reporter) system, which provides an on-off switch specifically in response to an HCV infection. Using the NIrD system and a focused CRISPR/Cas9 library, we identified CLDN1, OCLN and CD81 as essential genes for both the cell-free entry and the cell-to-cell transmission of HCV. The combination of this ultra-sensitive reporter system and the CRISPR knockout screening provides a powerful and high-throughput strategy for the identification of critical host components for HCV infections.

Show MeSH
Related in: MedlinePlus