Computational analysis of cas proteins unlocks new potential in HIV-1 targeted gene therapy

Introduction: The human immunodeficiency virus type 1 (HIV-1) pandemic has been slowed with the advent of anti-retroviral therapy (ART). However, ART is not a cure and as such has pushed the disease into a chronic infection. One potential cure strategy that has shown promise is the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas gene editing system. It has recently been shown to successfully edit and/or excise the integrated provirus from infected cells and inhibit HIV-1 in vitro, ex vivo, and in vivo. These studies have primarily been conducted with SpCas9 or SaCas9. However, additional Cas proteins are discovered regularly and modifications to these known proteins are being engineered. The alternative Cas molecules have different requirements for protospacer adjacent motifs (PAMs) which impact the possible targetable regions of HIV-1. Other modifications to the Cas protein or gRNA handle impact the tolerance for mismatches between gRNA and the target. While reducing off-target risk, this impacts the ability to fully account for HIV-1 genetic variability.

Methods: This manuscript strives to examine these parameter choices using a computational approach for surveying the suitability of a Cas editor for HIV-1 gene editing. The Nominate, Diversify, Narrow, Filter (NDNF) pipeline measures the safety, broadness, and effectiveness of a pool of potential gRNAs for any PAM. This technique was used to evaluate 46 different potential Cas editors for their HIV therapeutic potential.

Results: Our examination revealed that broader PAMs that improve the targeting potential of editors like SaCas9 and LbCas12a have larger pools of useful gRNAs, while broader PAMs reduced the pool of useful SpCas9 gRNAs yet increased the breadth of targetable locations. Investigation of the mismatch tolerance of Cas editors indicates a 2-missmatch tolerance is an ideal balance between on-target sensitivity and off-target specificity. Of all of the Cas editors examined, SpCas-NG and SPRY-Cas9 had the highest number of overall safe, broad, and effective gRNAs against HIV.

Discussion: Currently, larger proteins and wider PAMs lead to better targeting capacity. This implies that research should either be targeted towards delivering longer payloads or towards increasing the breadth of currently available small Cas editors. With the discovery and adoption of additional Cas editors, it is important for researchers in the HIV-1 gene editing field to explore the wider world of Cas editors.