CRISPR-Cas9-based Genome Engineering to Generate Jurkat Reporter Models for HIV-1 Infection with Selected Proviral Integration Sites

Białek, J; Walther, Thomas; Hauber, Joachim; Lange, Ulrike

doi:10.3791/58572

Cited by 3 publications

(2 citation statements)

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“…In this context, can primary models of latency be created in the presence of ART, without the ectopic expression of anti-apoptotic factors but allowing their long-term culturing without causing T cell exhaustion? With the advent of genome editing tools [280], can we engineer "anatomically" correct in vitro models of latency (in immortalized but noncancerous cells or naïve healthy donor cells) in where proviruses are directed to the same integration sites as those found in patient samples? Would this approach potentially remove the roadblock of limited number of cells?…”

Section: The Ephemeral Nature Of Ideas and Considerations For Future Researchmentioning

confidence: 99%

HIV-1 Proviral Transcription and Latency in the New Era

Shukla

Ramirez

D’Orso

2020

Viruses

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Three decades of extensive work in the HIV field have revealed key viral and host cell factors controlling proviral transcription. Various models of transcriptional regulation have emerged based on the collective information from in vitro assays and work in both immortalized and primary cell-based models. Here, we provide a recount of the past and current literature, highlight key regulatory aspects, and further describe potential limitations of previous studies. We particularly delve into critical steps of HIV gene expression including the role of the integration site, nucleosome positioning and epigenomics, and the transition from initiation to pausing and pause release. We also discuss open questions in the field concerning the generality of previous regulatory models to the control of HIV transcription in patients under suppressive therapy, including the role of the heterogeneous integration landscape, clonal expansion, and bottlenecks to eradicate viral persistence. Finally, we propose that building upon previous discoveries and improved or yet-to-be discovered technologies will unravel molecular mechanisms of latency establishment and reactivation in a “new era”.

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Section: The Ephemeral Nature Of Ideas and Considerations For Future Researchmentioning

confidence: 99%

HIV-1 Proviral Transcription and Latency in the New Era

Shukla

Ramirez

D’Orso

2020

Viruses

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“…The most common strategy of 'knock-in' relies on exploiting homologous recombination to repair the induced DSBs using a transgene template, hence resulting in targeted integration [49]. This strategy of targeted integration has recently been proposed by the team of Ulrike Lange to generate reporter models for HIV-1 infection with selected proviral integration sites in BACH2 (Figure 2b) [50 ]. So far, the proposed protocol is time-consuming; however, it paves the way for the use of CRISPR/Cas9 targeted integration in mechanistic studies on position effects of HIV-1 integration in latency and clonal expansion.…”

Section: Role Of the Integration Site In Hiv-1 Reservoir Persistence mentioning

confidence: 99%

Applications of CRISPR/Cas9 tools in deciphering the mechanisms of HIV-1 persistence

Verdikt

Darcis²,

Aït-Ammar

et al. 2019

Current Opinion in Virology

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HIV-1 infection can be controlled but not cured by combination antiretroviral therapy. Indeed, the virus persists in treated individuals in viral reservoirs, the best described of which consisting in latently infected central memory CD4 + T cells. However, other cell types in other body compartments than in the peripheral blood contribute to HIV-1 persistence. Addressing the molecular mechanisms of HIV-1 persistence and their cell-specific and tissue-specific variations is thus crucial to develop HIV-1 curative strategies. CRISPR/Cas9 editing technologies have revolutionized genetic engineering by their high specificity and their versatility. Multiple applications now allow to investigate the molecular mechanisms of HIV-1 persistence. Here, we review recent advances in CRISPR-based technologies in deciphering HIV-1 gene expression regulation during persistence.

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