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Volume 103, Issue 6

Systematic HIV-1 promoter targeting with CRISPR/dCas9-VPR reveals optimal region for activation of the latent provirus No Access

Abstract

CRISPR/dCas9-based activation systems (CRISPRa) enable sequence-specific gene activation and are therefore of particular interest for the ‘shock and kill’ cure approach against HIV-1 infections. This approach aims to activate the latent HIV-1 proviruses in infected cells and subsequently kill these cells. Several CRISPRa systems have been shown to specifically and effectively activate latent HIV-1 when targeted to the HIV-1 5′LTR promoter, making them a promising ‘shock’ strategy. Here, we aimed to evaluate the dCas9-VPR system for its applicability in reversing HIV-1 latency and identify the optimal gRNA target site in the HIV-1 5′LTR promoter leading to the strongest activation of the provirus with this system. We systematically screened the HIV-1 promoter by selecting 14 specific gRNAs that cover almost half of the HIV-1 promoter from the 3′ half of the U3 until the beginning of the R region. Screening in several latently HIV-1 infected cell lines showed that dCas9-VPR leads to a high activation of HIV-1 and that gRNA-V and -VII induce the strongest activation of replication competent latent provirus. This data indicates that the optimal activation region in the HIV-1 promoter for the dCas9-VPR system is located −165 to −106 bp from the transcription start site and that it is consistent with the optimal activation region reported for other CRISPRa systems. Our data demonstrates that the dCas9-VPR system is a powerful tool for HIV-1 activation and could be harnessed for the ‘shock and kill’ cure approach.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): CRISPR , HIV-1 , HIV-1 reservoir , Latency , shock and kill and viral activation
Funding
This study was supported by the:
  • Gilead Sciences (Award 00408)
    • Principle Award Recipient: KarinJ. Metzner
© 2022 The Authors
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/content/journal/jgv/10.1099/jgv.0.001754
2022-06-07
2024-04-19
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Systematic HIV-1 promoter targeting with CRISPR/dCas9-VPR reveals optimal region for activation of the latent provirus
J. Gen. Virol. 103, 001754 (2022); https://doi.org/10.1099/jgv.0.001754
/content/journal/jgv/10.1099/jgv.0.001754
/content/journal/jgv/10.1099/jgv.0.001754
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