Safe CRISPR-Cas9 Inhibition of HIV-1 with High Specificity and Broad-Spectrum Activity by Targeting LTR NF-κB Binding Sites.
CRISPR-Cas9
GUIDE-Seq
HIV-1 reservoir
NF-κB
computational biology
gene therapy
Journal
Molecular therapy. Nucleic acids
ISSN: 2162-2531
Titre abrégé: Mol Ther Nucleic Acids
Pays: United States
ID NLM: 101581621
Informations de publication
Date de publication:
04 Sep 2020
04 Sep 2020
Historique:
received:
08
06
2020
revised:
22
06
2020
accepted:
08
07
2020
pubmed:
21
8
2020
medline:
21
8
2020
entrez:
21
8
2020
Statut:
ppublish
Résumé
Viral latency of human immunodeficiency virus type 1 (HIV-1) has become a major hurdle to a cure in the highly effective antiretroviral therapy (ART) era. The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has successfully been demonstrated to excise or inactivate integrated HIV-1 provirus from infected cells by targeting the long terminal repeat (LTR) region. However, the guide RNAs (gRNAs) have classically avoided transcription factor binding sites (TFBSs) that are readily observed and known to be important in human promoters. Although conventionally thought unfavorable due to potential impact on human promoters, our computational pipeline identified gRNA sequences that were predicted to inactivate HIV-1 transcription by targeting the nuclear factor κB (NF-κB) binding sites (gNFKB0, gNFKB1) with a high safety profile (lack of predicted or observed human edits) and broad-spectrum activity (predicted coverage of known viral sequences). Genome-wide, unbiased identification of double strand breaks (DSBs) enabled by sequencing (GUIDE-seq) showed that the gRNAs targeting NF-κB binding sites had no detectable CRISPR-induced off-target edits in HeLa cells. 5' LTR-driven HIV-1 transcription was significantly reduced in three HIV-1 reporter cell lines. These results demonstrate a working model to specifically target well-known TFBSs in the HIV-1 LTR that are readily observed in human promoters to reduce HIV-1 transcription with a high-level safety profile and broad-spectrum activity.
Identifiants
pubmed: 32818921
pii: S2162-2531(20)30205-5
doi: 10.1016/j.omtn.2020.07.016
pmc: PMC7452136
pii:
doi:
Types de publication
Journal Article
Langues
eng
Pagination
965-982Informations de copyright
Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.
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