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CCR5 Gene Disruption via Lentiviral Vectors Expressing Cas9 and Single Guided RNA Renders Cells Resistant to HIV-1 Infection.

Authors :
Wang, Weiming
Ye, Chaobaihui
Liu, Jingjing
Zhang, Di
Kimata, Jason T.
Zhou, Paul
Source :
PLoS ONE. Dec2014, Vol. 9 Issue 12, p1-26. 26p.
Publication Year :
2014

Abstract

CCR5, a coreceptor for HIV-1 entry, is a major target for drug and genetic intervention against HIV-1. Genetic intervention strategies have knocked down CCR5 expression levels by shRNA or disrupted the CCR5 gene using zinc finger nucleases (ZFN) or Transcription activator-like effector nuclease (TALEN). In the present study, we silenced CCR5 via CRISPR associated protein 9 (Cas9) and single guided RNAs (sgRNAs). We constructed lentiviral vectors expressing Cas9 and CCR5 sgRNAs. We show that a single round transduction of lentiviral vectors expressing Cas9 and CCR5 sgRNAs into HIV-1 susceptible human CD4+ cells yields high frequencies of CCR5 gene disruption. CCR5 gene-disrupted cells are not only resistant to R5-tropic HIV-1, including transmitted/founder (T/F) HIV-1 isolates, but also have selective advantage over CCR5 gene-undisrupted cells during R5-tropic HIV-1 infection. Importantly, using T7 endonuclease I assay we did not detect genome mutations at potential off-target sites that are highly homologous to these CCR5 sgRNAs in stably transduced cells even at 84 days post transduction. Thus we conclude that silencing of CCR5 via Cas9 and CCR5-specific sgRNAs could be a viable alternative strategy for engineering resistance against HIV-1. [ABSTRACT FROM AUTHOR]

Details

Language :
English
ISSN :
19326203
Volume :
9
Issue :
12
Database :
Academic Search Index
Journal :
PLoS ONE
Publication Type :
Academic Journal
Accession number :
100187759
Full Text :
https://doi.org/10.1371/journal.pone.0115987