About: The CRISPR technology not only can knock out target genes by using the RNA-guided Cas9 nuclease but also can activate their expression when a nuclease-deficient Cas9 (dCas9) is employed. Using the latter function, we here show the effect of the CRISPR-mediated pinpoint activation of endogenous expression of BST-2 (also known as tetherin), a virus restriction factor with a broad antiviral spectrum. Single-guide RNA (sgRNA) sequences targeting the BST-2 promoter were selected by promoter assays. Potential sgRNAs and dCas9 fused to the VP64 transactivation domain, along with an accessory transcriptional activator complex, were introduced into cells by lentiviral transduction. Increased expression of BST-2 mRNA in transduced cells was confirmed by real-time RT-PCR. Cells in which BST-2 expression was highly enhanced showed the effective inhibition of HIV-1 production and replication even in the presence of the viral antagonist Vpu against BST-2. These findings confirm that the physiological stoichiometry between host restriction factors and viral antagonists may determine the outcome of the battle with viruses.   Goto Sponge  NotDistinct  Permalink

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  • The CRISPR technology not only can knock out target genes by using the RNA-guided Cas9 nuclease but also can activate their expression when a nuclease-deficient Cas9 (dCas9) is employed. Using the latter function, we here show the effect of the CRISPR-mediated pinpoint activation of endogenous expression of BST-2 (also known as tetherin), a virus restriction factor with a broad antiviral spectrum. Single-guide RNA (sgRNA) sequences targeting the BST-2 promoter were selected by promoter assays. Potential sgRNAs and dCas9 fused to the VP64 transactivation domain, along with an accessory transcriptional activator complex, were introduced into cells by lentiviral transduction. Increased expression of BST-2 mRNA in transduced cells was confirmed by real-time RT-PCR. Cells in which BST-2 expression was highly enhanced showed the effective inhibition of HIV-1 production and replication even in the presence of the viral antagonist Vpu against BST-2. These findings confirm that the physiological stoichiometry between host restriction factors and viral antagonists may determine the outcome of the battle with viruses.
subject
  • Virology
  • Immune system
  • Genome editing
  • Genetic engineering
  • Molecular biology
  • Repetitive DNA sequences
  • Virus genera
  • Daylight saving time in the United Kingdom
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