The CRISPR/Cas9 gene editing method is comprised of the guide RNA (gRNA) to target a specific DNA sequence for cleavage and the Cas9 endonuclease for introducing breaks in the double-stranded DNA identified by the gRNA. Co-expression of both a multiplex of HIV-1-specific gRNAs and Cas9 in cells results in the modification and/or excision of the segment of viral DNA, leading to replication-defective virus. In this study, we have personalized the activity of CRISPR/Cas9 by placing the gene encoding Cas9 under the control of a minimal promoter of HIV-1 that is activated by the HIV-1 Tat protein. We demonstrate that functional activation of CRISPR/Cas9 by Tat during the course of viral infection excises the designated segment of the integrated viral DNA and consequently suppresses viral expression. This strategy was also used in a latently infected CD4+ T-cell model after treatment with a variety of HIV-1 stimulating agents including PMA and TSA. Controlled expression of Cas9 by Tat offers a new strategy for safe implementation of the Cas9 technology for ablation of HIV-1 at a very early stage of HIV-1 replication during the course of the acute phase of infection and the reactivation of silent proviral DNA in latently infected cells.

Negative Feedback Regulation of HIV-1 by Gene Editing Strategy / R. Kaminski, Y. Chen, J. Salkind, R. Bella, W.-. Young, P. Ferrante, J. Karn, T. Malcolm, W. Hu, K. Khalili. - In: SCIENTIFIC REPORTS. - ISSN 2045-2322. - 6:1(2016), pp. 31527.1-31527.11. [10.1038/srep31527]

Negative Feedback Regulation of HIV-1 by Gene Editing Strategy

R. Bella;P. Ferrante;
2016

Abstract

The CRISPR/Cas9 gene editing method is comprised of the guide RNA (gRNA) to target a specific DNA sequence for cleavage and the Cas9 endonuclease for introducing breaks in the double-stranded DNA identified by the gRNA. Co-expression of both a multiplex of HIV-1-specific gRNAs and Cas9 in cells results in the modification and/or excision of the segment of viral DNA, leading to replication-defective virus. In this study, we have personalized the activity of CRISPR/Cas9 by placing the gene encoding Cas9 under the control of a minimal promoter of HIV-1 that is activated by the HIV-1 Tat protein. We demonstrate that functional activation of CRISPR/Cas9 by Tat during the course of viral infection excises the designated segment of the integrated viral DNA and consequently suppresses viral expression. This strategy was also used in a latently infected CD4+ T-cell model after treatment with a variety of HIV-1 stimulating agents including PMA and TSA. Controlled expression of Cas9 by Tat offers a new strategy for safe implementation of the Cas9 technology for ablation of HIV-1 at a very early stage of HIV-1 replication during the course of the acute phase of infection and the reactivation of silent proviral DNA in latently infected cells.
3' Untranslated Regions; 5' Untranslated Regions; Base Sequence; CD4-Positive T-Lymphocytes; Clustered Regularly Interspaced Short Palindromic Repeats; Genes, Viral; HIV Long Terminal Repeat; HIV-1; Humans; Jurkat Cells; Promoter Regions, Genetic; Sequence Homology, Nucleic Acid; Virus Replication; Gene Editing; Gene Expression Regulation, Viral
Settore MED/07 - Microbiologia e Microbiologia Clinica
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/659573
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