Novel gRNA design pipeline to develop broad-spectrum CRISPR/Cas9 gRNAs for safe targeting of the HIV-1 quasispecies in patients

Abstract

The CRISPR/Cas9 system has been proposed as a cure strategy for HIV. However, few published guide RNAs (gRNAs) are predicted to cleave the majority of HIV-1 viral quasispecies (vQS) observed within and among patients. We report the design of a novel pipeline to identify gRNAs that target HIV across a large number of infected individuals. Next generation sequencing (NGS) of LTRs from 269 HIV-1-infected samples in the Drexel CARES Cohort was used to select gRNAs with predicted broad-spectrum activity. In silico, D-LTR-P4-227913 (package of the top 4 gRNAs) accounted for all detectable genetic variation within the vQS of the 269 samples and the Los Alamos National Laboratory HIV database. In silico secondary structure analyses from NGS indicated extensive TAR stem-loop malformations predicted to inactivate proviral transcription, which was confirmed by reduced viral gene expression in TZM-bl or P4R5 cells. Similarly, a high sensitivity in vitro CRISPR/Cas9 cleavage assay showed that the top-ranked gRNA was the most effective at cleaving patient-derived HIV-1 LTRs from five patients. Furthermore, the D-LTR-P4-227913 was predicted to cleave a median of 96.1% of patient-derived sequences from other HIV subtypes. These results demonstrate that the gRNAs possess broad-spectrum cutting activity and could contribute to an HIV cure.

CRISPR-Cas9 Dual-gRNA Attack Causes Mutation, Excision and Inversion of the HIV-1 Proviral DNA

“…The high genetic diversity of HIV can be another complicating factor, as even single nucleotide mismatches between the gRNA and target sequence can prevent Cas9 recognition [56]. The simultaneous use of multiple gRNAs that target highly conserved sequences seems the best strategy to circumvent this problem, although exotic HIV isolates with divergent target sequences may require specific adaptation of the gRNA molecules [56][57][58][59].…”

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

“…The top ranked gRNAs in this region have been shown to be very effective at reducing HIV-1 transcription as we and others have previously published. 22 , 24 , 26 , 29 , 33 , 35 , 51 , 52 , 53 , 54 Effective sequence mutations and correlated functional changes in TAR implies that this low diversity region was under evolutionary constraints due to the functional importance of those sequence domains. An obvious reduction of diversity after position 615 of the LTR was due to lack of coverage for the calculation of diversity.…”

“…A number of recent studies have designed gRNAs based on sequence conservation observed in HIV-1 sequence databases. 33 , 36 , 38 , 39 , 40 However, a conserved sequence does not guarantee a likely CRISPR-mediated cleaving site, since CRISPR possesses asymmetric tolerance for mismatches. The loss of CRISPR-mediated cleavage activity due to a single nucleotide change was quantified using gRNA mutants that targeted the same DNA.…”

“…The MIT matrix was previously used to identify the gRNAs that were predicted to cleave most of the HIV-1 genetic variants that were known in patient-derived sequences in Drexel CNS AIDS Research and Eradication Study (CARES) cohort. 33 , 36 Other studies also emphasized the consideration of genetic variability during gRNA design and found that mismatches in target sites reduced the efficiency on HIV-1 inactivation, as well as facilitated the occurrence of escape mutants. 39 , 43 , 44 , 45 …”

Designing Safer CRISPR/Cas9 Therapeutics for HIV: Defining Factors That Regulate and Technologies Used to Detect Off-Target Editing

“…In this case, the Cas9 system would be more likely to bind and cleave, even though there is a mismatch. This tolerance to mismatch was used to allow investigators to design gRNAs that target more HIV viral variants ( Dampier et al, 2014 , 2016 , 2018 ; Sullivan et al, 2019 ). However, mismatch-position-based off-target prediction has raised concerns due to low predictive accuracy ( Doench et al, 2016 ).…”