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Inhibition of HIV-1 infection of primary CD4+ T-cells by gene editing of CCR5 using adenovirus-delivered CRISPR/Cas9
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 resistanc…
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“Li et al. created HIV-resistant primary CD4+ T cells by adding cell membrane CCR5Δ32 variant using AdV-delivered CRISPR/Cas9 [42].”
From Viral Delivery Systems for CRISPR (2019) · cites this paper as [42]
Citations
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Metadata
- Authors
- Li, C., Guan, X., Du, T., Jin, W., Wu, B., Liu, Y., Wang, P., Hu, B., Griffin, G. E., Shattock, R. J., & Hu, Q.
- Affiliations
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, PR China
- Department of General Surgery, Wuhan No.1 Hospital, Wuhan 430022, PR China
- Institute for Infection and Immunity, St George's University of London, London SW17 0RE, UK
- Section of Infectious Diseases, Faculty of Medicine, Imperial College London, St Mary's Campus, London W2 1PG, UK
- Venue
- Journal of General Virology
- Publisher
- Microbiology Society
- Published
- 2015-08-01
- Volume
- 96
- Issue
- 8
- Pages
- 2381-2393
- ISSN
- 0022-1317
- 1465-2099
- Cited by
- 192
- References
- 16
- Language
- en
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