Abstract
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.
Highlights
Combination antiretroviral therapy increases survival and quality of life of HIV-1 infected patients
To evaluate the breadth of these dual-guide RNA (gRNA) clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 therapies, we set out to test their capacity to inhibit replication of diverse HIV-1 isolates, including viruses corresponding to other subtypes (A, C, D and CRF01_AE; listed in Figure 1b), which were expected to show sequence variation in the gRNA targets
To evaluate whether HIV-1 genetic variation may compromise a CRISPR-Cas9-based cure attempt, we tested two potent dual-gRNA combinations for their capacity to inhibit the replication of diverse
Summary
Combination antiretroviral therapy (cART) increases survival and quality of life of HIV-1 infected patients. CART is not curative and HIV-1 persists in viral reservoirs [1]. These reservoirs, defined as a cell type or anatomical site where a replication-competent form of the virus persists during therapy, are established during the first days of infection [2,3,4]. The integrated proviral DNA in the reservoir cells can give rise to infectious virus production upon activation by various stimuli, leading to viral rebound when cART is interrupted [1,4,8,9]. Therapeutic strategies that eliminate these proviruses in the reservoir cells may lead to a cure
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