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

Pavitra Roychoudhury,Keith R Jerome,Daniel Stone,Bryan T Mayer,Harshana De Silva Feelixge,Joshua T Schiffer,Daniel Reeves

doi:10.1186/s12915-018-0544-1

Pavitra Roychoudhury, Keith R Jerome + Show 5 more

Open Access

https://doi.org/10.1186/s12915-018-0544-1

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Abstract

BackgroundRNA-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.ResultsWe 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.ConclusionsTarget 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.

Highlights

RNA-guided CRISPR/Cas9 systems can be designed to mutate or excise the integrated HIV genome from latently infected cells and have been proposed as a curative approach for HIV
Targeting spCas9 guide RNA (gRNA) against HIV gag, pol, and LTR We performed a screen to identify globally conserved target sites for Streptococcus pyogenes in LTR, gag, and pol using alignments for these regions obtained from the HIV Los Alamos National Laboratory (LANL) database
LTR was chosen for its utility in excision of the provirus [8, 20, 21], while gag and pol were chosen based on their conservation between HIV strains [22]

Summary

Introduction

RNA-guided CRISPR/Cas systems can be designed to mutate or excise the integrated HIV genome from latently infected cells and have been proposed as a curative approach for HIV. For clinical success and broad applicability, guide RNA (gRNA) selection must account for circulating strain diversity and incorporate the within-host diversity of HIV. Despite the success of combination antiretroviral therapy (cART) in suppressing HIV viremia, reservoirs of latently infected cells remain the major barrier for HIV cure [1]. A promising therapeutic strategy for achieving cure involves depleting the reservoir by direct disruption of proviral genomes using engineered DNA-editing enzymes such as CRISPR/Cas nucleases. Viral reservoirs are highly diverse, even in well-suppressed individuals [13, 14], and this diversity remains a major challenge for the application of genome editing strategies towards an HIV cure. Effective targeting of all viral genetic variants within an infected individual will be crucial for achieving sufficient reservoir reduction to prevent viral rebound upon cART cessation [15, 16] and preventing the emergence of resistance to this therapy [11]

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