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Assessment of anti-HIV-1 guide RNA efficacy in cells containing the viral target sequence, corresponding gRNA, and CRISPR/Cas9

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 gene editing system has been shown to be effective at inhibiting human immunodeficiency virus type 1 (HIV-1). Studies have not consistently used a trackable dual reporter system to determine what cells received the Cas9/gRNA...

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Autores principales: Allen, Alexander G., Chung, Cheng-Han, Worrell, Stephen D., Nwaozo, Glad, Madrid, Rebekah, Mele, Anthony R., Dampier, Will, Nonnemacher, Michael R., Wigdahl, Brian
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10134072/
https://www.ncbi.nlm.nih.gov/pubmed/37124096
http://dx.doi.org/10.3389/fgeed.2023.1101483
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author Allen, Alexander G.
Chung, Cheng-Han
Worrell, Stephen D.
Nwaozo, Glad
Madrid, Rebekah
Mele, Anthony R.
Dampier, Will
Nonnemacher, Michael R.
Wigdahl, Brian
author_facet Allen, Alexander G.
Chung, Cheng-Han
Worrell, Stephen D.
Nwaozo, Glad
Madrid, Rebekah
Mele, Anthony R.
Dampier, Will
Nonnemacher, Michael R.
Wigdahl, Brian
author_sort Allen, Alexander G.
collection PubMed
description The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 gene editing system has been shown to be effective at inhibiting human immunodeficiency virus type 1 (HIV-1). Studies have not consistently used a trackable dual reporter system to determine what cells received the Cas9/gRNA to determine the overall knockdown of HIV. Some studies have used stably transduced cells under drug selection to accomplish this goal. Here a two-color system was used that allows tracking of viral protein expression and which cells received the CRISPR/Cas9 system. These experiments ensured that each gRNA used was a perfect match to the intended target to remove this variable. The data showed that gRNAs targeting the transactivation response element (TAR) region or other highly conserved regions of the HIV-1 genome were effective at stopping viral gene expression, with multiple assays demonstrating greater than 95 percent reduction. Conversely, gRNAs targeting conserved sites of the 5’ portion of the U3 region were largely ineffective, demonstrating that the location of edits in the long terminal repeat (LTR) matter with respect to function. In addition, it was observed that a gRNA targeting Tat was effective in a T-cell model of HIV-1 latency. Taken together, these studies demonstrated gRNAs designed to highly conserved functional regions have near 100% efficacy in vitro in cells known to have received the Cas9/gRNA pair.
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spelling pubmed-101340722023-04-28 Assessment of anti-HIV-1 guide RNA efficacy in cells containing the viral target sequence, corresponding gRNA, and CRISPR/Cas9 Allen, Alexander G. Chung, Cheng-Han Worrell, Stephen D. Nwaozo, Glad Madrid, Rebekah Mele, Anthony R. Dampier, Will Nonnemacher, Michael R. Wigdahl, Brian Front Genome Ed Genome Editing The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 gene editing system has been shown to be effective at inhibiting human immunodeficiency virus type 1 (HIV-1). Studies have not consistently used a trackable dual reporter system to determine what cells received the Cas9/gRNA to determine the overall knockdown of HIV. Some studies have used stably transduced cells under drug selection to accomplish this goal. Here a two-color system was used that allows tracking of viral protein expression and which cells received the CRISPR/Cas9 system. These experiments ensured that each gRNA used was a perfect match to the intended target to remove this variable. The data showed that gRNAs targeting the transactivation response element (TAR) region or other highly conserved regions of the HIV-1 genome were effective at stopping viral gene expression, with multiple assays demonstrating greater than 95 percent reduction. Conversely, gRNAs targeting conserved sites of the 5’ portion of the U3 region were largely ineffective, demonstrating that the location of edits in the long terminal repeat (LTR) matter with respect to function. In addition, it was observed that a gRNA targeting Tat was effective in a T-cell model of HIV-1 latency. Taken together, these studies demonstrated gRNAs designed to highly conserved functional regions have near 100% efficacy in vitro in cells known to have received the Cas9/gRNA pair. Frontiers Media S.A. 2023-04-13 /pmc/articles/PMC10134072/ /pubmed/37124096 http://dx.doi.org/10.3389/fgeed.2023.1101483 Text en Copyright © 2023 Allen, Chung, Worrell, Nwaozo, Madrid, Mele, Dampier, Nonnemacher and Wigdahl. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Genome Editing
Allen, Alexander G.
Chung, Cheng-Han
Worrell, Stephen D.
Nwaozo, Glad
Madrid, Rebekah
Mele, Anthony R.
Dampier, Will
Nonnemacher, Michael R.
Wigdahl, Brian
Assessment of anti-HIV-1 guide RNA efficacy in cells containing the viral target sequence, corresponding gRNA, and CRISPR/Cas9
title Assessment of anti-HIV-1 guide RNA efficacy in cells containing the viral target sequence, corresponding gRNA, and CRISPR/Cas9
title_full Assessment of anti-HIV-1 guide RNA efficacy in cells containing the viral target sequence, corresponding gRNA, and CRISPR/Cas9
title_fullStr Assessment of anti-HIV-1 guide RNA efficacy in cells containing the viral target sequence, corresponding gRNA, and CRISPR/Cas9
title_full_unstemmed Assessment of anti-HIV-1 guide RNA efficacy in cells containing the viral target sequence, corresponding gRNA, and CRISPR/Cas9
title_short Assessment of anti-HIV-1 guide RNA efficacy in cells containing the viral target sequence, corresponding gRNA, and CRISPR/Cas9
title_sort assessment of anti-hiv-1 guide rna efficacy in cells containing the viral target sequence, corresponding grna, and crispr/cas9
topic Genome Editing
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10134072/
https://www.ncbi.nlm.nih.gov/pubmed/37124096
http://dx.doi.org/10.3389/fgeed.2023.1101483
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