HIV-1 Proviral Genome Engineering with CRISPR-Cas9 for Mechanistic Studies
HIV-1 latency remains a barrier to a functional cure because of the ability of virtually silent yet inducible proviruses within reservoir cells to transcriptionally reactivate upon cell stimulation. HIV-1 reactivation occurs through the sequential action of host transcription factors (TFs) during th...
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| Format: | Article |
| Language: | English |
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MDPI AG
2024-02-01
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| Series: | Viruses |
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| Online Access: | https://www.mdpi.com/1999-4915/16/2/287 |
| _version_ | 1827342640207626240 |
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| author | Usman Hyder Ashutosh Shukla Ashwini Challa Iván D’Orso |
| author_facet | Usman Hyder Ashutosh Shukla Ashwini Challa Iván D’Orso |
| author_sort | Usman Hyder |
| collection | DOAJ |
| description | HIV-1 latency remains a barrier to a functional cure because of the ability of virtually silent yet inducible proviruses within reservoir cells to transcriptionally reactivate upon cell stimulation. HIV-1 reactivation occurs through the sequential action of host transcription factors (TFs) during the “host phase” and the viral TF Tat during the “viral phase”, which together facilitate the positive feedback loop required for exponential transcription, replication, and pathogenesis. The sequential action of these TFs poses a challenge to precisely delineate the contributions of the host and viral phases of the transcriptional program to guide future mechanistic and therapeutic studies. To address this limitation, we devised a genome engineering approach to mutate <i>tat</i> and create a genetically matched pair of Jurkat T cell clones harboring HIV-1 at the same integration site with and without Tat expression. By comparing the transcriptional profile of both clones, the transition point between the host and viral phases was defined, providing a system that enables the temporal mechanistic interrogation of HIV-1 transcription prior to and after Tat synthesis. Importantly, this CRISPR method is broadly applicable to knockout individual viral proteins or genomic regulatory elements to delineate their contributions to various aspects of the viral life cycle and ultimately may facilitate therapeutic approaches in our race towards achieving a functional cure. |
| first_indexed | 2024-03-07T22:10:35Z |
| format | Article |
| id | doaj.art-dc3b298c4517474b909c5f1ba2e87aeb |
| institution | Directory Open Access Journal |
| issn | 1999-4915 |
| language | English |
| last_indexed | 2024-03-07T22:10:35Z |
| publishDate | 2024-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Viruses |
| spelling | doaj.art-dc3b298c4517474b909c5f1ba2e87aeb2024-02-23T15:37:42ZengMDPI AGViruses1999-49152024-02-0116228710.3390/v16020287HIV-1 Proviral Genome Engineering with CRISPR-Cas9 for Mechanistic StudiesUsman Hyder0Ashutosh Shukla1Ashwini Challa2Iván D’Orso3Department of Microbiology, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USADepartment of Microbiology, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USADepartment of Microbiology, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USADepartment of Microbiology, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USAHIV-1 latency remains a barrier to a functional cure because of the ability of virtually silent yet inducible proviruses within reservoir cells to transcriptionally reactivate upon cell stimulation. HIV-1 reactivation occurs through the sequential action of host transcription factors (TFs) during the “host phase” and the viral TF Tat during the “viral phase”, which together facilitate the positive feedback loop required for exponential transcription, replication, and pathogenesis. The sequential action of these TFs poses a challenge to precisely delineate the contributions of the host and viral phases of the transcriptional program to guide future mechanistic and therapeutic studies. To address this limitation, we devised a genome engineering approach to mutate <i>tat</i> and create a genetically matched pair of Jurkat T cell clones harboring HIV-1 at the same integration site with and without Tat expression. By comparing the transcriptional profile of both clones, the transition point between the host and viral phases was defined, providing a system that enables the temporal mechanistic interrogation of HIV-1 transcription prior to and after Tat synthesis. Importantly, this CRISPR method is broadly applicable to knockout individual viral proteins or genomic regulatory elements to delineate their contributions to various aspects of the viral life cycle and ultimately may facilitate therapeutic approaches in our race towards achieving a functional cure.https://www.mdpi.com/1999-4915/16/2/287HIV-1TatvirusestranscriptionCRISPRCas9 |
| spellingShingle | Usman Hyder Ashutosh Shukla Ashwini Challa Iván D’Orso HIV-1 Proviral Genome Engineering with CRISPR-Cas9 for Mechanistic Studies Viruses HIV-1 Tat viruses transcription CRISPR Cas9 |
| title | HIV-1 Proviral Genome Engineering with CRISPR-Cas9 for Mechanistic Studies |
| title_full | HIV-1 Proviral Genome Engineering with CRISPR-Cas9 for Mechanistic Studies |
| title_fullStr | HIV-1 Proviral Genome Engineering with CRISPR-Cas9 for Mechanistic Studies |
| title_full_unstemmed | HIV-1 Proviral Genome Engineering with CRISPR-Cas9 for Mechanistic Studies |
| title_short | HIV-1 Proviral Genome Engineering with CRISPR-Cas9 for Mechanistic Studies |
| title_sort | hiv 1 proviral genome engineering with crispr cas9 for mechanistic studies |
| topic | HIV-1 Tat viruses transcription CRISPR Cas9 |
| url | https://www.mdpi.com/1999-4915/16/2/287 |
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