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J Gen Virol. 2015 Aug;96(8):2381-93. doi: 10.1099/vir.0.000139. Epub 2015 Apr 8.

Inhibition of HIV-1 infection of primary CD4+ T-cells by gene editing of CCR5 using adenovirus-delivered CRISPR/Cas9.

Author information

1
1​ State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, PR China 2​ University of Chinese Academy of Sciences, Beijing 100049, PR China.
2
1​ State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, PR China.
3
3​ Department of General Surgery, Wuhan No.1 Hospital, Wuhan 430022, PR China.
4
4​ Institute for Infection and Immunity, St George's University of London, London SW17 0RE, UK.
5
5​ Section of Infectious Diseases, Faculty of Medicine, Imperial College London, St Mary's Campus, London W2 1PG, UK.
6
1​ State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, PR China 4​ Institute for Infection and Immunity, St George's University of London, London SW17 0RE, UK.

Abstract

CCR5 serves as an essential coreceptor for human immunodeficiency virus type 1 (HIV-1) entry, and individuals with a CCR5(Δ32) variant appear to be healthy, making CCR5 an attractive target for control of HIV-1 infection. The CRISPR/Cas9, which functions as a naturally existing adaptive immune system in prokaryotes, has been recently harnessed as a novel nuclease system for genome editing in mammalian cells. Although CRISPR/Cas9 can be readily delivered into cell lines, due to the large size of the Cas9 protein, efficient delivery of CCR5-targeting CRISPR/Cas9 components into primary cells, including CD4(+) T-cells, the primary target for HIV-1 infection in vivo, remains a challenge. In the current study, following design of a panel of top-ranked single-guided RNAs (sgRNAs) targeting the ORF of CCR5, we demonstrate that CRISPR/Cas9 can efficiently mediate the editing of the CCR5 locus in cell lines, resulting in the knockout of CCR5 expression on the cell surface. Next-generation sequencing revealed that various mutations were introduced around the predicted cleavage site of CCR5. For each of the three most effective sgRNAs that we analysed, no significant off-target effects were detected at the 15 top-scoring potential sites. More importantly, by constructing chimeric Ad5F35 adenoviruses carrying CRISPR/Cas9 components, we efficiently transduced primary CD4(+) T-lymphocytes and disrupted CCR5 expression, and the positively transduced cells were conferred with HIV-1 resistance. To our knowledge, this is the first study establishing HIV-1 resistance in primary CD4(+) T-cells utilizing adenovirus-delivered CRISPR/Cas9.

PMID:
25854553
DOI:
10.1099/vir.0.000139
[Indexed for MEDLINE]

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