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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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Schematic of the CRISPR library construction and HCV screening.(a) The structure of the lentiviral plasmid expressing OCT1 and Cas9. (b) Indels induced by the lentivirus-delivered sgRNA (5-TTGGCCAGACTTGCATCCG-3) targeting the CSPG4 gene in the indicated cells were assayed by T7E1 digestion. Genomic DNA from HeLaOC-SC31 was used as a positive control, and the wild type (WT) Huh7.5(NIrD) was used as a negative control. (c) Schematic of the sgRNA library screening. sgRNAs were delivered into Huh7.5(NIrD)OC-SC cells by lentiviral infection with a MOI of 0.1. Three replicates of the libraries were challenged with 3–4 rounds of HCVcc, followed by FACS sorting to enrich the mCherry-negative clones. A comparison of the abundance of sgRNAs between the treated and untreated populations through high-throughput sequencing analysis was conducted following the same procedure as previously reported31.
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f3: Schematic of the CRISPR library construction and HCV screening.(a) The structure of the lentiviral plasmid expressing OCT1 and Cas9. (b) Indels induced by the lentivirus-delivered sgRNA (5-TTGGCCAGACTTGCATCCG-3) targeting the CSPG4 gene in the indicated cells were assayed by T7E1 digestion. Genomic DNA from HeLaOC-SC31 was used as a positive control, and the wild type (WT) Huh7.5(NIrD) was used as a negative control. (c) Schematic of the sgRNA library screening. sgRNAs were delivered into Huh7.5(NIrD)OC-SC cells by lentiviral infection with a MOI of 0.1. Three replicates of the libraries were challenged with 3–4 rounds of HCVcc, followed by FACS sorting to enrich the mCherry-negative clones. A comparison of the abundance of sgRNAs between the treated and untreated populations through high-throughput sequencing analysis was conducted following the same procedure as previously reported31.

Mentions: Next, we investigated whether the NIrD system could be used for large-scale screening purposes. Taking advantage of a functional screening technique that we have recently developed31, we screened a focused CRISPR library in Huh7.5(NIrD) cells, in which OCT1-Cas9 expression was pre-established through lentiviral infection and neomycin selection (Fig. 3a). One particular clone, designated as Huh7.5(NIrd)OC-SC14, was selected to construct the library because it showed the highest level of efficiency in creating indels using CSPG4-targeting sgRNA31 and increased its efficiency in causing DNA double-strand breaks (DSBs) with prolonged culturing, as expected (Fig. 3b).


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)

Schematic of the CRISPR library construction and HCV screening.(a) The structure of the lentiviral plasmid expressing OCT1 and Cas9. (b) Indels induced by the lentivirus-delivered sgRNA (5-TTGGCCAGACTTGCATCCG-3) targeting the CSPG4 gene in the indicated cells were assayed by T7E1 digestion. Genomic DNA from HeLaOC-SC31 was used as a positive control, and the wild type (WT) Huh7.5(NIrD) was used as a negative control. (c) Schematic of the sgRNA library screening. sgRNAs were delivered into Huh7.5(NIrD)OC-SC cells by lentiviral infection with a MOI of 0.1. Three replicates of the libraries were challenged with 3–4 rounds of HCVcc, followed by FACS sorting to enrich the mCherry-negative clones. A comparison of the abundance of sgRNAs between the treated and untreated populations through high-throughput sequencing analysis was conducted following the same procedure as previously reported31.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4352851&req=5

f3: Schematic of the CRISPR library construction and HCV screening.(a) The structure of the lentiviral plasmid expressing OCT1 and Cas9. (b) Indels induced by the lentivirus-delivered sgRNA (5-TTGGCCAGACTTGCATCCG-3) targeting the CSPG4 gene in the indicated cells were assayed by T7E1 digestion. Genomic DNA from HeLaOC-SC31 was used as a positive control, and the wild type (WT) Huh7.5(NIrD) was used as a negative control. (c) Schematic of the sgRNA library screening. sgRNAs were delivered into Huh7.5(NIrD)OC-SC cells by lentiviral infection with a MOI of 0.1. Three replicates of the libraries were challenged with 3–4 rounds of HCVcc, followed by FACS sorting to enrich the mCherry-negative clones. A comparison of the abundance of sgRNAs between the treated and untreated populations through high-throughput sequencing analysis was conducted following the same procedure as previously reported31.
Mentions: Next, we investigated whether the NIrD system could be used for large-scale screening purposes. Taking advantage of a functional screening technique that we have recently developed31, we screened a focused CRISPR library in Huh7.5(NIrD) cells, in which OCT1-Cas9 expression was pre-established through lentiviral infection and neomycin selection (Fig. 3a). One particular clone, designated as Huh7.5(NIrd)OC-SC14, was selected to construct the library because it showed the highest level of efficiency in creating indels using CSPG4-targeting sgRNA31 and increased its efficiency in causing DNA double-strand breaks (DSBs) with prolonged culturing, as expected (Fig. 3b).

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