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Open AccessArticle

The Impact of HIV-1 Genetic Diversity on CRISPR-Cas9 Antiviral Activity and Viral Escape

1
Laboratory of Experimental Virology, Department of Medical Microbiology, Amsterdam University Medical Centers, 1105AZ Amsterdam, The Netherlands
2
Infectious Diseases Department, Liège University Hospital, 4000 Liège, Belgium
*
Authors to whom correspondence should be addressed.
Viruses 2019, 11(3), 255; https://doi.org/10.3390/v11030255
Received: 18 January 2019 / Revised: 8 March 2019 / Accepted: 9 March 2019 / Published: 13 March 2019
(This article belongs to the Special Issue Applications of CRISPR Technology in Virology 2018)
The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system is widely explored for sequence-specific attack on HIV-1 proviral DNA. We recently identified dual-guide RNA (dual-gRNA) combinations that can block HIV-1 replication permanently in infected cell cultures and prevent viral escape. Although the gRNAs were designed to target highly conserved viral sequences, their efficacy may be challenged by high genetic variation in the HIV-1 genome. We therefore evaluated the breadth of these dual-gRNA combinations against distinct HIV-1 isolates, including several subtypes. Replication of nearly all virus isolates could be prevented by at least one gRNA combination, which caused inactivation of the proviral genomes and the gradual loss of replication-competent virus over time. The dual-gRNA efficacy was not affected by most single nucleotide (nt) mismatches between gRNA and the viral target. However, 1-nt mismatches at the Cas9 cleavage site and two mismatches anywhere in the viral target sequence significantly reduced the inhibitory effect. Accordingly, sequence analysis of viruses upon breakthrough replication revealed the acquisition of escape mutations in perfectly matching and most 1-nt mismatching targets, but not in targets with a mismatch at the Cas9 cleavage site or with two mismatches. These results demonstrate that combinatorial CRISPR-Cas9 treatment can cure T cells infected by distinct HIV-1 isolates, but even minor sequence variation in conserved viral target sites can affect the efficacy of this strategy. Successful cure attempts against isolates with divergent target sequences may therefore require adaptation of the gRNAs. View Full-Text
Keywords: HIV-1; subtypes; isolates; diversity; CRISPR-Cas9; dual-gRNA; escape; cure HIV-1; subtypes; isolates; diversity; CRISPR-Cas9; dual-gRNA; escape; cure
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MDPI and ACS Style

Darcis, G.; Binda, C.S.; Klaver, B.; Herrera-Carrillo, E.; Berkhout, B.; Das, A.T. The Impact of HIV-1 Genetic Diversity on CRISPR-Cas9 Antiviral Activity and Viral Escape. Viruses 2019, 11, 255. https://doi.org/10.3390/v11030255

AMA Style

Darcis G, Binda CS, Klaver B, Herrera-Carrillo E, Berkhout B, Das AT. The Impact of HIV-1 Genetic Diversity on CRISPR-Cas9 Antiviral Activity and Viral Escape. Viruses. 2019; 11(3):255. https://doi.org/10.3390/v11030255

Chicago/Turabian Style

Darcis, Gilles; Binda, Caroline S.; Klaver, Bep; Herrera-Carrillo, Elena; Berkhout, Ben; Das, Atze T. 2019. "The Impact of HIV-1 Genetic Diversity on CRISPR-Cas9 Antiviral Activity and Viral Escape" Viruses 11, no. 3: 255. https://doi.org/10.3390/v11030255

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