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

Abstract

Viral latency of human immunodeficiency virus type 1 (HIV-1) has become a major hurdle to a cure in the highly effective antiretroviral therapy (ART) era. The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has successfully been demonstrated to excise or inactivate integrated HIV-1 provirus from infected cells by targeting the long terminal repeat (LTR) region. However, the guide RNAs (gRNAs) have classically avoided transcription factor binding sites (TFBSs) that are readily observed and known to be important in human promoters. Although conventionally thought unfavorable due to potential impact on human promoters, our computational pipeline identified gRNA sequences that were predicted to inactivate HIV-1 transcription by targeting the nuclear factor κB (NF-κB) binding sites (gNFKB0, gNFKB1) with a high safety profile (lack of predicted or observed human edits) and broad-spectrum activity (predicted coverage of known viral sequences). Genome-wide, unbiased identification of double strand breaks (DSBs) enabled by sequencing (GUIDE-seq) showed that the gRNAs targeting NF-κB binding sites had no detectable CRISPR-induced off-target edits in HeLa cells. 5′ LTR-driven HIV-1 transcription was significantly reduced in three HIV-1 reporter cell lines. These results demonstrate a working model to specifically target well-known TFBSs in the HIV-1 LTR that are readily observed in human promoters to reduce HIV-1 transcription with a high-level safety profile and broad-spectrum activity.

Computational Design of gRNAs Targeting Genetic Variants Across HIV-1 Subtypes for CRISPR-Mediated Antiviral Therapy

“…Note that three lead gRNAs identified in this study were predicted to have potential off-target cleavage only at intergenic or intron regions. Further validation with respect to off-target cleavage using genome wide sequencing approach similar to previous study as well as in vitro cell viability test are required to support the safety of proposed gRNAs (Chung C.-H. et al, 2020a). The gRNA search method identified nine gRNAs possessing global patient coverage of 0.9 or higher; seven of the novel gRNA Sequence variant profiles and corresponding CFD score between chosen gRNA and HIV-1 variants.…”

“…g07CF4A held the highest CFD score (0.86) compared to the HXB2 sequence versus g9A8671 (0.61) and gA51706 (0.71) ( Figure 4D). These scores were all higher than the CFD cutoff of 0.569 used to distinguish whether the gRNA was predicted to induce DSBs 95% of the time (Chung C-H et al, 2020). However, only 4.9% of variants in the LANL database possess an identical target site to HXB2 ( Figure 4D).…”

“…The cutting frequency determination (CFD) table, a 16x20 matrix, was later developed to incorporate both position and nucleotide specific mismatch-when assigning an editing efficiency penalty (Doench et al, 2016). We and others have demonstrated that the CFD matrix gives better editingefficiency predictions than the MIT matrix (Cui et al, 2018;Chung C.-H. et al, 2020). Given this observation, the CFD matrix was selected to estimate editing efficiency between candidate gRNAs and HIV-1 genetic variants.…”

Exonic Disruption Facilitates Antiviral CRISPR-Cas9 Activity for Multistrain HIV-1 Elimination

“…With lentiviral transduction at a MOI of 1, the TatDE CRISPR transgene was detected at an average of 33.2 spCas9 RNA copies per AHC2 T cell, with a 94% reduction in RT activity and clear viral excision (Figure 3). In contrast, a single broad spectrum gRNA targeting LTR transduced at this same level still allowed viral rebound in ~20% latently infected JLat cells (36). Other studies have achieved >90% reduction in HIV-1 reactivation and prevention of viral escape when CRISPR-Cas9 was lentivirally transduced in T cells at MOIs 10-15 (21,23,37).…”

“…TatDE's multistrain potential will be further validated against additional HIV-1 strains and clades for antiviral activity. We will also interrogate the lack of off-target editing within the human genome by employing CIRCLE-seq (40,41), Guide-seq (36,42) and whole genome sequencing (29) to our TatDE CRISPR-treated samples. Finally, we plan to further characterize TatDE's mechanism of action in terms of its ability to downregulate full-length viral transcripts due to tat, diminish rev-associated nuclear export, and impede gp41-mediated viral entry.…”