CRISPR/Cas9 Ablation of Integrated HIV-1 Accumulates Proviral DNA Circles with Reformed Long Terminal Repeats.

Michele Lai,Guido Antonelli,Eyal Maori,Stefania Crucitta,Fabrizio Maggi,Giulia Freer,Giulia Matteoli,Marco Sgarbanti,Paola Quaranta,Simone Pacini,Jonathan L Heeney,Ombretta Turriziani,Mauro Pistello,Viviana Simon

doi:10.1128/jvi.01358-21

Abstract

ABSTRACTGene editing may be used to excise the human immunodeficiency virus type 1 (HIV-1) provirus from the host cell genome, possibly eradicating the infection. Here, using cells acutely or latently infected by HIV-1 and treated with long terminal repeat (LTR)-targeting CRISPR/Cas9, we show that the excised HIV-1 provirus persists for a few weeks and may rearrange in circular molecules. Although circular proviral DNA is naturally formed during HIV-1 replication, we observed that gene editing might increase proviral DNA circles with restored LTRs. These extrachromosomal elements were recovered and probed for residual activity through their transfection in uninfected cells. We discovered that they can be transcriptionally active in the presence of Tat and Rev. Although confirming that gene editing is a powerful tool to eradicate HIV-1 infection, this work highlights that, to achieve this goal, the LTRs must be cleaved in several pieces to avoid residual activity and minimize the risk of reintegration in the context of genomic instability, possibly caused by the off-target activity of Cas9.IMPORTANCE The excision of HIV-1 provirus from the host cell genome has proven feasible in vitro and, to some extent, in vivo. Among the different approaches, CRISPR/Cas9 is the most promising tool for gene editing. The present study underlines the remarkable effectiveness of CRISPR/Cas9 in removing the HIV-1 provirus from infected cells and investigates the fate of the excised HIV-1 genome. This study demonstrates that the free provirus may persist in the cell after editing and in appropriate circumstances may reactivate. As an episome, it might be transcriptionally active, especially in the presence of Tat and Rev. The persistence of the HIV-1 episome was strongly decreased by gene editing with multiple targets. Although gene editing has the potential to eradicate HIV-1 infection, this work highlights a potential issue that warrants further investigation.

Highlights

Gene editing may be used to excise the human immunodeficiency virus type 1 (HIV-1) provirus from the host cell genome, possibly eradicating the infection
Experiments were conducted in human embryonic kidney 293T cells bearing integrated, labeled HIV-1pseudotyped vectors, and we extended our observations to human T cell leukemia cells actively replicating HIV-1, as occurs during acute infection, or latently infected J-Lat cells, as observed in the asymptomatic phase
The results show that the excised provirus persists in the nucleus for a prolonged period of time; depending on the number of copies per cell, it closes as a single molecule or with another, yielding circular elements that can, even if at a very low frequency, form complete long terminal repeat (LTR) again, thereby reducing the efficiency of HIV-1 eradication by gene editing

Summary

Introduction

Gene editing may be used to excise the human immunodeficiency virus type 1 (HIV-1) provirus from the host cell genome, possibly eradicating the infection. While these LTR circles are often considered dead ends of the viral life cycle [13, 14], we hypothesize that in the context of genomic instability caused by either on-target or off-target DNA cleavage, these elements might have a second chance of integrating through complementation or homology-directed repair (HDR) induced by gene editing [16] For this purpose, we used a single CRISPR/Cas guide RNA targeting both LTRs. Experiments were conducted in human embryonic kidney 293T cells bearing integrated, labeled HIV-1pseudotyped vectors, and we extended our observations to human T cell leukemia cells actively replicating HIV-1, as occurs during acute infection, or latently infected J-Lat cells, as observed in the asymptomatic phase. Once LTRs are targeted with multiple guides, these elements lose their ability to be recovered by Tat and Rev, and more importantly, they cannot be active even if reintegrated through HDR

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