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

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.

CRISPR-Cas9 Dual-gRNA Attack Causes Mutation, Excision and Inversion of the HIV-1 Proviral DNA

“…Other prominent issues relate to off-target effects due to Cas9 cleavage at non-target sites in the cellular DNA and immune responses against the non-human Cas9 protein [54,55]. The high genetic diversity of HIV can be another complicating factor, as even single nucleotide mismatches between the gRNA and target sequence can prevent Cas9 recognition [56]. The simultaneous use of multiple gRNAs that target highly conserved sequences seems the best strategy to circumvent this problem, although exotic HIV isolates with divergent target sequences may require specific adaptation of the gRNA molecules [56][57][58][59].…”

Safe CRISPR-Cas9 Inhibition of HIV-1 with High Specificity and Broad-Spectrum Activity by Targeting LTR NF-κB Binding Sites

“…Darcis et al. 45 demonstrated the direct effect of genetic variation using seven distinct isolates to perform an efficacy test with three gRNAs. They found in long-term viral infection culture that even a single mismatch between gRNA and the intended target site on single molecular clones could result in viral escape.…”

“… 33 , 36 Other studies also emphasized the consideration of genetic variability during gRNA design and found that mismatches in target sites reduced the efficiency on HIV-1 inactivation, as well as facilitated the occurrence of escape mutants. 39 , 43 , 44 , 45 …”

Designing Safer CRISPR/Cas9 Therapeutics for HIV: Defining Factors That Regulate and Technologies Used to Detect Off-Target Editing

“…CRISPR/Cas9-based anti-HIV-1 technology has been developing rapidly, but few studies have focused on mitigating the potential for off-target events outside preliminary bioinformatic predictions (Roychoudhury et al, 2018;Darcis et al, 2019) with the exception of whole genome sequencing (WGS). An optimal therapeutic strategy would be both highly effective against the virus and safe for patients.…”