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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| Format: | Article |
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MDPI AG
2020-10-01
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| Series: | Viruses |
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| Online Access: | https://www.mdpi.com/1999-4915/12/11/1223 |
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| author | Youdiil Ophinni Sayaka Miki Yoshitake Hayashi Masanori Kameoka |
| author_facet | Youdiil Ophinni Sayaka Miki Yoshitake Hayashi Masanori Kameoka |
| author_sort | Youdiil Ophinni |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-03-10T15:15:45Z |
| format | Article |
| id | doaj.art-552a4dbeb9354e1ab4f27a9f8a6f7661 |
| institution | Directory Open Access Journal |
| issn | 1999-4915 |
| language | English |
| last_indexed | 2024-03-10T15:15:45Z |
| publishDate | 2020-10-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Viruses |
| spelling | doaj.art-552a4dbeb9354e1ab4f27a9f8a6f76612023-11-20T18:55:14ZengMDPI AGViruses1999-49152020-10-011211122310.3390/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> |
| 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> |
| title | 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_short | 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 |
| topic | HIV-1 cure CRISPR-Cas viral escape <i>tat</i> |
| url | https://www.mdpi.com/1999-4915/12/11/1223 |
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