Dataset Information

Repression of HIV-1 reactivation mediated by CRISPR/dCas9-KRAB in lymphoid and myeloid cell models.

ABSTRACT:

Background

Despite antiretroviral treatment efficacy, it does not lead to the complete eradication of HIV infection. Consequently, reactivation of the virus from latently infected cell reservoirs is a major challenge toward cure efforts. Two strategies targeting viral latency are currently under investigation: the "shock and kill" and the "block and lock." The "Block and Lock" methodology aims to control HIV-1 latency reactivation, promoting a functional cure. We utilized the CRISPR/dCas9-KRAB platform, which was initially developed to suppress cellular genes transcription, to block drug-induced HIV-1 reactivation in latently infected T cells and myeloid cells.

Results

We identified a set of five sgRNAs targeting the HIV-1 proviral genome (LTR1-LTR5), having the lowest nominated off-target activity, and transduced them into the latently infected lymphoid (J-Lat 10.6) and myeloid (U1) cell lines. One of the sgRNAs (LTR5), which binds specifically in the HIV-1 LTR NFκB binding site, was able to promote robust repression of HIV-1 reactivation in latently infected T cells stimulated with Phorbol 12-Myristate 13-Acetate (PMA) and Ingenol B (IngB), both potent protein kinase C (PKC) stimulators. Reactivation with HDAC inhibitors, such as SAHA and Panobinostat, showed the same strong inhibition of reactivation. Additionally, we observed a hundred times reduction of HIV-1 RNA expression levels in the latently infected myeloid cell line, U1 induced with IngB.

Conclusion

Taken together, our results show that the KRAB fused CRISPR/dCas9 system can robustly prevent the HIV-1 latency reactivation process, mediated by PMA or IngB and SAHA or Panobinostat, both in myeloid and lymphoid HIV-1 latently infected cells. In addition, we demonstrated that KRAB repressor protein is crucial to reactivation resistance phenotype, and we have identified some useful hotspots sequences in HIV-1 LTR for the design sgRNAs.

SUBMITTER: da Costa LC

PROVIDER: S-EPMC9215058 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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Publications

Repression of HIV-1 reactivation mediated by CRISPR/dCas9-KRAB in lymphoid and myeloid cell models.

da Costa Lendel Correia LC Bomfim Larissa Maciel LM Dittz Uilla Victoria Torres UVT Velozo Camila de Almeida CA da Cunha Rodrigo Delvecchio RD Tanuri Amilcar A

Retrovirology 20220622 1

<h4>Background</h4>Despite antiretroviral treatment efficacy, it does not lead to the complete eradication of HIV infection. Consequently, reactivation of the virus from latently infected cell reservoirs is a major challenge toward cure efforts. Two strategies targeting viral latency are currently under investigation: the "shock and kill" and the "block and lock." The "Block and Lock" methodology aims to control HIV-1 latency reactivation, promoting a functional cure. We utilized the CRISPR/dCas ...[more]

PMID: 35733180

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Project description:BackgroundCRISPR/CAS9 (epi)genome editing revolutionized the field of gene and cell therapy. Our previous study demonstrated that a rapid and robust reactivation of the HIV latent reservoir by a catalytically-deficient Cas9 (dCas9)-synergistic activation mediator (SAM) via HIV long terminal repeat (LTR)-specific MS2-mediated single guide RNAs (msgRNAs) directly induces cellular suicide without additional immunotherapy. However, potential off-target effect remains a concern for any clinical application of Cas9 genome editing and dCas9 epigenome editing. After dCas9 treatment, potential off-target responses have been analyzed through different strategies such as mRNA sequence analysis, and functional screening. In this study, a comprehensive analysis of the host transcriptome including mRNA, lncRNA, and alternative splicing was performed using human cell lines expressing dCas9-SAM and HIV-targeting msgRNAs.ResultsThe control scrambled msgRNA (LTR_Zero), and two LTR-specific msgRNAs (LTR_L and LTR_O) groups show very similar expression profiles of the whole transcriptome. Among 839 identified lncRNAs, none exhibited significantly different expression in LTR_L vs. LTR_Zero group. In LTR_O group, only TERC and scaRNA2 lncRNAs were significantly decreased. Among 142,791 mRNAs, four genes were differentially expressed in LTR_L vs. LTR_Zero group. There were 21 genes significantly downregulated in LTR_O vs. either LTR_Zero or LTR_L group and one third of them are histone related. The distributions of different types of alternative splicing were very similar either within or between groups. There were no apparent changes in all the lncRNA and mRNA transcripts between the LTR_L and LTR_Zero groups.ConclusionThis is an extremely comprehensive study demonstrating the rare off-target effects of the HIV-specific dCas9-SAM system in human cells. This finding is encouraging for the safe application of dCas9-SAM technology to induce target-specific reactivation of latent HIV for an effective "shock-and-kill" strategy.

Project description:Magnaporthe oryzae and Ustilaginoidea virens are two filamentous fungal pathogens that threaten rice production worldwide. Genetic tools that permit fast gene deletion and silencing are of great interest for functional genomics of fungal pathogens. As a revolutionary genome editing tool, clustered regularly interspaced palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) enable many innovative applications. Here, we developed a CRISPR interference (CRISPRi) toolkit using nuclease activity dead Cas9 (dCas9) to silence genes of interest in M. oryzae and U. virens. We optimized the components of CRISPRi vectors, including transcriptional repression domains, dCas9 promoters, and guide RNA (gRNA) promoters. The CRISPRi tool was tested using nine gRNAs to target the promoters of MoATG3, MoATG7, and UvPal1. The results indicated that a single gRNA could direct the dCas9-fused transcriptional repression domain to efficiently silence the target gene in M. oryzae and U. virens. In both fungi, the target genes were repressed >100-fold, and desired phenotypes were observed in CRISPRi strains. Importantly, we showed that multiple genes could be easily silenced using polycistronic tRNA-gRNA in CRISPRi. Furthermore, gRNAs that bind different promoter regions displayed variable repression levels of target genes, highlighting the importance of gRNA design for CRISPRi efficiency. Together, this study provides an efficient and robust CRISPRi tool for targeted gene silencing in M. oryzae and U. virens. Owing to its simplicity and multiplexity, CRISPRi will be a useful tool for gene function discovery in fungal pathogens. IMPORTANCE Many devastating plant diseases are caused by fungal pathogens that evolve rapidly to adapt to host resistance and environmental changes. Therefore, genetic tools that enable fast gene function discovery are needed to study the pathogenicity and stress adaptation of fungal pathogens. In this study, we adopted the CRISPR/Cas9 system to silence genes in Magnaporthe oryzae and Ustilaginoidea virens, which are two dominant fungal pathogens that threaten rice production worldwide. We present a versatile and robust CRISPRi toolkit that represses target gene expression >100-fold using a single gRNA. We also demonstrated that CRISPRi could simultaneously silence multiple genes using the tRNA-gRNA strategy. The CRISPRi technologies described in this study would accelerate the functional genomics of fungal pathogens.

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Dataset Information

Repression of HIV-1 reactivation mediated by CRISPR/dCas9-KRAB in lymphoid and myeloid cell models.

Background

Results

Conclusion

Publications

Repression of HIV-1 reactivation mediated by CRISPR/dCas9-KRAB in lymphoid and myeloid cell models.

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