Multiplexed <i>tat</i>-Targeting CRISPR-Cas9 Protects T Cells from Acute HIV-1 Infection with Inhibition of Viral Escape
HIV-1 cure strategy by means of proviral knock-out using CRISPR-Cas9 has been hampered by the emergence of viral resistance against the targeting guide RNA (gRNA). Here, we proposed multiple, concentrated gRNA attacks against HIV-1 regulatory genes to block viral escape. The T cell line were transdu...
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doaj-552a4dbeb9354e1ab4f27a9f8a6f76612020-11-25T03:38:34ZengMDPI AGViruses1999-49152020-10-01121223122310.3390/v12111223Multiplexed <i>tat</i>-Targeting CRISPR-Cas9 Protects T Cells from Acute HIV-1 Infection with Inhibition of Viral EscapeYoudiil Ophinni0Sayaka Miki1Yoshitake Hayashi2Masanori Kameoka3Division of Molecular Medicine and Medical Genetics, Department of Pathology, Kobe University Graduate School of Medicine, Kobe 650-0017 Hyogo, JapanDivision of Global Infectious Diseases, Department of Public Health, Kobe University Graduate School of Health Sciences, Kobe 654-0142 Hyogo, JapanDivision of Molecular Medicine and Medical Genetics, Department of Pathology, Kobe University Graduate School of Medicine, Kobe 650-0017 Hyogo, JapanDivision of Global Infectious Diseases, Department of Public Health, Kobe University Graduate School of Health Sciences, Kobe 654-0142 Hyogo, JapanHIV-1 cure strategy by means of proviral knock-out using CRISPR-Cas9 has been hampered by the emergence of viral resistance against the targeting guide RNA (gRNA). Here, we proposed multiple, concentrated gRNA attacks against HIV-1 regulatory genes to block viral escape. The T cell line were transduced with single and multiple gRNAs targeting HIV-1 <i>tat</i> and <i>rev</i> using lentiviral-based CRISPR-Cas9, followed by replicative HIV-1<sub>NL4-3</sub> challenge in vitro. Viral p24 rebound was observed for almost all gRNAs, but multiplexing three <i>tat</i>-targeting gRNAs maintained p24 suppression and cell viability, indicating the inhibition of viral escape. Multiplexed <i>tat</i> gRNAs inhibited acute viral replication in the 2nd round of infection, abolished cell-associated transmission to unprotected T cells, and maintained protection through 45 days, post-infection (dpi) after a higher dose of HIV-1 infection. Finally, we describe here for the first time the assembly of all-in-one lentiviral vectors containing three and six gRNAs targeting <i>tat</i> and <i>rev</i>. A single-vector <i>tat</i>-targeting construct shows non-inferiority to the <i>tat</i>-targeting multi-vector in low-dose HIV-1 infection. We conclude that Cas9-induced, DNA repair-mediated mutations in <i>tat</i> are sufficiently deleterious and deplete HIV-1 fitness, and multiplexed disruption of <i>tat </i>further limits the possibility of an escape mutant arising, thus elevating the potential of CRISPR-Cas9 to achieve a long-term HIV-1 cure.https://www.mdpi.com/1999-4915/12/11/1223HIV-1cureCRISPR-Casviral escape<i>tat</i> |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Youdiil Ophinni Sayaka Miki Yoshitake Hayashi Masanori Kameoka |
spellingShingle |
Youdiil Ophinni Sayaka Miki Yoshitake Hayashi Masanori Kameoka Multiplexed <i>tat</i>-Targeting CRISPR-Cas9 Protects T Cells from Acute HIV-1 Infection with Inhibition of Viral Escape Viruses HIV-1 cure CRISPR-Cas viral escape <i>tat</i> |
author_facet |
Youdiil Ophinni Sayaka Miki Yoshitake Hayashi Masanori Kameoka |
author_sort |
Youdiil Ophinni |
title |
Multiplexed <i>tat</i>-Targeting CRISPR-Cas9 Protects T Cells from Acute HIV-1 Infection with Inhibition of Viral Escape |
title_short |
Multiplexed <i>tat</i>-Targeting CRISPR-Cas9 Protects T Cells from Acute HIV-1 Infection with Inhibition of Viral Escape |
title_full |
Multiplexed <i>tat</i>-Targeting CRISPR-Cas9 Protects T Cells from Acute HIV-1 Infection with Inhibition of Viral Escape |
title_fullStr |
Multiplexed <i>tat</i>-Targeting CRISPR-Cas9 Protects T Cells from Acute HIV-1 Infection with Inhibition of Viral Escape |
title_full_unstemmed |
Multiplexed <i>tat</i>-Targeting CRISPR-Cas9 Protects T Cells from Acute HIV-1 Infection with Inhibition of Viral Escape |
title_sort |
multiplexed <i>tat</i>-targeting crispr-cas9 protects t cells from acute hiv-1 infection with inhibition of viral escape |
publisher |
MDPI AG |
series |
Viruses |
issn |
1999-4915 |
publishDate |
2020-10-01 |
description |
HIV-1 cure strategy by means of proviral knock-out using CRISPR-Cas9 has been hampered by the emergence of viral resistance against the targeting guide RNA (gRNA). Here, we proposed multiple, concentrated gRNA attacks against HIV-1 regulatory genes to block viral escape. The T cell line were transduced with single and multiple gRNAs targeting HIV-1 <i>tat</i> and <i>rev</i> using lentiviral-based CRISPR-Cas9, followed by replicative HIV-1<sub>NL4-3</sub> challenge in vitro. Viral p24 rebound was observed for almost all gRNAs, but multiplexing three <i>tat</i>-targeting gRNAs maintained p24 suppression and cell viability, indicating the inhibition of viral escape. Multiplexed <i>tat</i> gRNAs inhibited acute viral replication in the 2nd round of infection, abolished cell-associated transmission to unprotected T cells, and maintained protection through 45 days, post-infection (dpi) after a higher dose of HIV-1 infection. Finally, we describe here for the first time the assembly of all-in-one lentiviral vectors containing three and six gRNAs targeting <i>tat</i> and <i>rev</i>. A single-vector <i>tat</i>-targeting construct shows non-inferiority to the <i>tat</i>-targeting multi-vector in low-dose HIV-1 infection. We conclude that Cas9-induced, DNA repair-mediated mutations in <i>tat</i> are sufficiently deleterious and deplete HIV-1 fitness, and multiplexed disruption of <i>tat </i>further limits the possibility of an escape mutant arising, thus elevating the potential of CRISPR-Cas9 to achieve a long-term HIV-1 cure. |
topic |
HIV-1 cure CRISPR-Cas viral escape <i>tat</i> |
url |
https://www.mdpi.com/1999-4915/12/11/1223 |
work_keys_str_mv |
AT youdiilophinni multiplexeditatitargetingcrisprcas9protectstcellsfromacutehiv1infectionwithinhibitionofviralescape AT sayakamiki multiplexeditatitargetingcrisprcas9protectstcellsfromacutehiv1infectionwithinhibitionofviralescape AT yoshitakehayashi multiplexeditatitargetingcrisprcas9protectstcellsfromacutehiv1infectionwithinhibitionofviralescape AT masanorikameoka multiplexeditatitargetingcrisprcas9protectstcellsfromacutehiv1infectionwithinhibitionofviralescape |
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1724541648160948224 |