Viruses, Vol. 16, Pages 287: HIV-1 Proviral Genome Engineering with CRISPR-Cas9 for Mechanistic Studies

Viruses, Vol. 16, Pages 287: HIV-1 Proviral Genome Engineering with CRISPR-Cas9 for Mechanistic Studies Viruses doi: 10.3390/v16020287 Authors: Usman Hyder Ashutosh Shukla Ashwini Challa Iván D’Orso 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 tat 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 kn...
Source: Viruses - Category: Virology Authors: Tags: Article Source Type: research
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