Viral diversity is an obligate consideration in CRISPR/Cas9 designs for targeting the HIV reservoir

Abstract

Background: RNA-guided CRISPR/Cas9 systems can be designed to mutate or excise the integrated HIV genome from latently infected cells and have therefore been proposed as a curative approach for HIV. However, most studies to date have focused on molecular clones with ideal target site recognition and do not account for target site variability observed within and between patients. For clinical success and broad applicability, guide RNA (gRNA) selection must account for circulating strain diversity and incorporate the within-host diversity of HIV.

Results: We identified a set of gRNAs targeting HIV LTR, gag, and pol using publicly available sequences for these genes and ranked gRNAs according to global conservation across HIV-1 group M and within subtypes A-C. By considering paired and triplet combinations of gRNAs, we found triplet sets of target sites such that at least one of the gRNAs in the set was present in over 98% of all globally available sequences. We then selected 59 gRNAs from our list of highly conserved LTR target sites and evaluated in vitro activity using a loss-of-function LTR-GFP fusion reporter. We achieved efficient GFP knockdown with multiple gRNAs and found clustering of highly active gRNA target sites near the middle of the LTR. Using published deep-sequence data from HIV-infected patients, we found that globally conserved sites also had greater within-host target conservation. Lastly, we developed a mathematical model based on varying distributions of within-host HIV sequence diversity and enzyme efficacy. We used the model to estimate the number of doses required to deplete the latent reservoir and achieve functional cure thresholds. Our modeling results highlight the importance of within-host target site conservation. While increased doses may overcome low target cleavage efficiency, inadequate targeting of rare strains is predicted to lead to rebound upon cART cessation even with many doses.

Conclusions: Target site selection must account for global and within host viral genetic diversity. Globally conserved target sites are good starting points for design, but multiplexing is essential for depleting quasispecies and preventing viral load rebound upon therapy cessation.

Keywords: CRISPR/Cas9; Computational biology; Endonucleases; Gene editing; Gene therapy; Genomics; HIV; Latent reservoir; Mathematical modeling; Viral genetic diversity.

Fig. 1

Top 100 gRNA target sites in HIV LTR (a), gag (b), and pol (c) ranked by prevalence (bottom to top) within an alignment of available sequences within group M for each genomic region. The x-axis shows the percentage of all sequences in group M that contain an exact match to the target site. Within each horizontal bar, shading indicates what percentage of sequences with target sites hits belong to each subtype. Inset bar plots show the total number of sequences of each subtype in the alignment

Fig. 2

a Histogram of predicted activity of all gRNAs identified in LTR, gag, and pol across all four consensus sequences (group M, subtypes A–C) for each gene. b Predicted activity score vs. target site conservation for individual gRNAs grouped by subtype and gene. Red triangles indicate gRNAs excluded due to predicted off-target activity. Numbers in blue represent the total number of guides with predicted activity score > 0.2 and where target sites occur in more than 50% of sequences in the group or subtype alignment

Fig. 3

a LTR-GFP fusion reporter to test gRNAs for activity in vitro. b Activity was measured in terms of percent knockdown of median GFP fluorescence intensity relative to negative controls. We found positive but statistically non-significant correlation between computationally predicted activity scores and measured activity. c We achieved reduction of GFP fluorescence intensity (positive activity) with a majority of gRNA designs and observed clustering of tested target sites in two areas of the LTR with the most active guides being clustered around the center of the LTR. With a small number of gRNAs, we observed negative activity (increase in GFP fluorescence). Lower panel shows residue conservation (in 0–2 bits) across the LTR for alignments of subtype sequences or all sequences in group M

Fig. 4

a Number of previously identified target sites from global consensus sequences of group M and subtypes A–C that were present in each patient’s HIV consensus sequence. b Within-host target site conservation for each identified target site using deep-sequence data for 4 patients, summarized using box plots. Black dots indicate outlier target sites (outside 1.5 × IQR), and target sites are grouped and colored according to which consensus sequence they were identified from (the group- or subtype-level consensus from LANL alignments, or from the patient’s HIV consensus sequence)

Fig. 5

Simulated reservoir depletion with anti-HIV CRISPR therapy. a Example simulation based on predicted target site conservation (“potency,” ρ = 0.5) and enzyme efficacy to each target site (ϵ = 0.5). CRISPR therapy is dosed weekly, and the average strain contains 100 infected cells (μ_s = 100). Thin colored lines represent single strains, L_s(t), and the thick black line represents the total reservoir, L(t) = ∑_sL_s(t). Strains targeted by CRISPR are cleared rapidly, but untargeted strains remain unaffected and the total reservoir size does not decrease below estimated depletion thresholds for functional cure. The dashed line represents a stringent threshold for latent reservoir reduction where patients are expected to remain suppressed for years without cART [15, 16]. See Additional file 4: Figure S3 for simulations varying all parameters. b If 100% coverage (ρ = 1) of target sites can be achieved (either through multiplexing of targets or due to a target site that is highly conserved), enzyme efficacy becomes relevant, dictating the number of doses to cure. At or better than predicted efficacy ϵ > 0.5, doses range between 1 and 5 doses for a median 1 year remission and 5–10 doses for a potentially lifelong absence of viral rebound based on previously estimated thresholds. However, even for 100% coverage, efficacy at 10% or less per dose requires substantial dosing (> 30) to achieve thresholds