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Safe CRISPR-Cas9 Inhibition of HIV-1 with High Specificity and Broad-Spectrum Activity by Targeting LTR NF-κB Binding Sites

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 int...

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Autores principales: Chung, Cheng-Han, Allen, Alexander G., Atkins, Andrew J., Sullivan, Neil T., Homan, Greg, Costello, Robert, Madrid, Rebekah, Nonnemacher, Michael R., Dampier, Will, Wigdahl, Brian
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society of Gene & Cell Therapy 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7452136/
https://www.ncbi.nlm.nih.gov/pubmed/32818921
http://dx.doi.org/10.1016/j.omtn.2020.07.016
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author Chung, Cheng-Han
Allen, Alexander G.
Atkins, Andrew J.
Sullivan, Neil T.
Homan, Greg
Costello, Robert
Madrid, Rebekah
Nonnemacher, Michael R.
Dampier, Will
Wigdahl, Brian
author_facet Chung, Cheng-Han
Allen, Alexander G.
Atkins, Andrew J.
Sullivan, Neil T.
Homan, Greg
Costello, Robert
Madrid, Rebekah
Nonnemacher, Michael R.
Dampier, Will
Wigdahl, Brian
author_sort Chung, Cheng-Han
collection PubMed
description 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.
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spelling pubmed-74521362020-09-09 Safe CRISPR-Cas9 Inhibition of HIV-1 with High Specificity and Broad-Spectrum Activity by Targeting LTR NF-κB Binding Sites Chung, Cheng-Han Allen, Alexander G. Atkins, Andrew J. Sullivan, Neil T. Homan, Greg Costello, Robert Madrid, Rebekah Nonnemacher, Michael R. Dampier, Will Wigdahl, Brian Mol Ther Nucleic Acids Article 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. American Society of Gene & Cell Therapy 2020-07-15 /pmc/articles/PMC7452136/ /pubmed/32818921 http://dx.doi.org/10.1016/j.omtn.2020.07.016 Text en © 2020. http://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Article
Chung, Cheng-Han
Allen, Alexander G.
Atkins, Andrew J.
Sullivan, Neil T.
Homan, Greg
Costello, Robert
Madrid, Rebekah
Nonnemacher, Michael R.
Dampier, Will
Wigdahl, Brian
Safe CRISPR-Cas9 Inhibition of HIV-1 with High Specificity and Broad-Spectrum Activity by Targeting LTR NF-κB Binding Sites
title Safe CRISPR-Cas9 Inhibition of HIV-1 with High Specificity and Broad-Spectrum Activity by Targeting LTR NF-κB Binding Sites
title_full Safe CRISPR-Cas9 Inhibition of HIV-1 with High Specificity and Broad-Spectrum Activity by Targeting LTR NF-κB Binding Sites
title_fullStr Safe CRISPR-Cas9 Inhibition of HIV-1 with High Specificity and Broad-Spectrum Activity by Targeting LTR NF-κB Binding Sites
title_full_unstemmed Safe CRISPR-Cas9 Inhibition of HIV-1 with High Specificity and Broad-Spectrum Activity by Targeting LTR NF-κB Binding Sites
title_short Safe CRISPR-Cas9 Inhibition of HIV-1 with High Specificity and Broad-Spectrum Activity by Targeting LTR NF-κB Binding Sites
title_sort safe crispr-cas9 inhibition of hiv-1 with high specificity and broad-spectrum activity by targeting ltr nf-κb binding sites
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7452136/
https://www.ncbi.nlm.nih.gov/pubmed/32818921
http://dx.doi.org/10.1016/j.omtn.2020.07.016
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