A Combinatorial CRISPR-Cas9 Attack on HIV-1 DNA Extinguishes All Infectious Provirus in Infected T Cell Cultures

Publication date: 13 December 2016
Source:Cell Reports, Volume 17, Issue 11
Author(s): Gang Wang, Na Zhao, Ben Berkhout, Atze T. Das
Current drug therapies effectively suppress HIV-1 replication but do not inactivate the provirus that persists in latent reservoirs. Recent studies have found that the guide RNA (gRNA)-directed CRISPR/Cas9 system can be used for sequence-specific attack on this proviral DNA. Although potent inhibition of virus replication was reported, HIV-1 can escape from a single antiviral gRNA by mutation of the target sequence. Here, we demonstrate that combinations of two antiviral gRNAs delay viral escape, and identify two gRNA combinations that durably block virus replication. When viral escape is prevented, repeated Cas9 cleavage leads to saturation of major mutations in the conserved target sequences that encode critical proteins. This hypermutation coincides with the loss of replication-competent virus as scored in sensitive co-cultures with unprotected cells, demonstrating complete virus inactivation. These results provide a proof-of-principle that HIV-1-infected cells can be functionally cured by dual-gRNA CRISPR/Cas9 treatment.

Graphical abstract

Teaser

Although antiviral drug therapy is a life-saving treatment for HIV-infected individuals, a cure is never reached because the virus persists. Using CRISPR-Cas9 gene editing, Wang et al. find that complete virus inactivation can be achieved in cultured T cells, suggesting an avenue toward a functional cure.


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