17RNA-guided CRISPR/Cas9 systems can be designed to mutate or excise the integrated HIV 18 genome from latently infected cells and have therefore been proposed as a curative approach 19 for HIV. However, most studies to date have focused on molecular clones with ideal target site 20 recognition and do not account for target site variability observed within and between patients. 21For clinical success and broad applicability, guide RNA (gRNA) selection must account for 22 circulating strain diversity and incorporate the within-host diversity of HIV. To address this, we 23 identified a set of gRNAs targeting HIV LTR, gag and pol using publicly available sequences for 24 these genes. We ranked gRNAs according to global conservation across HIV-1 group M and 25 within subtypes A-C. By considering paired and triplet combinations of gRNAs, we found triplet 26 sets of target sites such that at least one of the gRNAs in the set was present in over 98% of all 27 globally-available sequences. We then selected 59 gRNAs from our list of highly-conserved LTR 28 target sites and evaluated in vitro activity using a loss-of-function LTR-GFP fusion reporter. We 29 achieved efficient GFP knockdown with multiple gRNAs and found clustering of highly active 30 gRNA target sites near the middle of the LTR. Using published deep-sequence data from HIV-31 infected patients, we found that globally conserved sites also had greater within-host target 32 conservation. Lastly, we developed a mathematical model based on varying distributions of 33 within-host HIV sequence diversity and enzyme efficacy. We used the model to estimate the 34 number of doses required to deplete the latent reservoir and achieve functional cure thresholds. 35Our modeling results highlight the importance of within-host target site conservation. While 36 increased doses may overcome low target cleavage efficiency, inadequate targeting of rare 37 strains is predicted to lead to rebound upon ART cessation even with many doses. 38 39 40 All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/255133 doi: bioRxiv preprint first posted online Jan. 27, 2018; Author summary 41The field of genome engineering has exploded over the last decade with the discovery of 42 targeted endonucleases such as CRISPR/Cas9. Endonucleases are now being used to develop 43 a wide range of therapeutics and their use has expanded into antiviral therapy against latent 44 viral infections like HIV. The idea is to induce mutations in latent viral genomes that will render 45 them replication-incompetent, thereby producing a functional cure. Although a great deal of 46 progress has been made, most studies to date have relied on molecular clones that represent 47 "ideal" targets. For clinical success and broad applicability, these therapies need to account for 48 viral genetic diversity within and between...