Broad-Spectrum and Personalized Guide RNAs for CRISPR/Cas9 HIV-1 Therapeutics

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas9 system has been used to excise the HIV-1 proviral genome from latently infected cells, potentially offering a cure for HIV-infected patients. Recent studies have shown that most published HIV-1 guide RNAs (gRNAs) do not account for the diverse viral quasispecies within or among patients, which continue to diversify with time even in long-term antiretroviral therapy (ART)-suppressed patients. Given this observation, proviral genomes were deep sequenced from 23 HIV-1-infected patients in the Drexel Medicine CNS AIDS Research and Eradication Study cohort at two different visits. Based on the spectrum of integrated proviral DNA polymorphisms observed, three gRNA design strategies were explored: based on the patient's own HIV-1 sequences (personalized), based on consensus sequences from a large sample of patients [broad-spectrum (BS)], or a combination of both approaches. Using a bioinformatic algorithm, the personalized gRNA design was predicted to cut 46 of 48 patient samples at 90% efficiency, whereas the top 4 BS gRNAs (BS4) were predicted to excise provirus from 44 of 48 patient samples with 90% efficiency. Using a mixed design with the top three BS gRNAs plus one personalized gRNA (BS3 + PS1) resulted in predicted excision of provirus from 45 of 48 patient samples with 90% efficiency. In summary, these studies used an algorithmic design strategy to identify potential BS gRNAs to target a spectrum of HIV-1 long teriminal repeat (LTR) quasispecies for use with a small HIV-1-infected population. This approach should advance CRISPR/Cas9 excision technology taking into account the extensive molecular heterogeneity of HIV-1 that persists in situ after prolonged ART.

Keywords: CRISPR/Cas9; HIV-1; excision; gRNA; gRNA pipeline; patient-derived sequence.

FIG. 1.

gRNAs were designed against patient-derived HIV-1 quasispecies. The position-specific penalties associated with mismatches within the protospacer used the MIT penalty score (A) as described by the equation (B) derived by Hsu et al.. W[e] is a penalty function that evaluates to 0 if position e is a match between the gRNA and the target and the position-specific penalty M in the event of a mismatch at position e. d is the arithmetic mean of pairwise distance between gRNA and target across the entire 20-mer and n represents the number of mismatches between the target and the gRNA. The MIT penalty score ranges from 1, in the case of a perfect match, to 0, in the case of a complete mismatch, and is interpreted as the percentage of strands that would be predicted to be cleaved. (C) All patient quasispecies sequences with a frequency >1% from sample A0010-R06 spanning the target of the gRNA (HXB2 561–581); mutations from the ideal target are marked in bold. (D) For each of the quasispecies of sample A0010-R06, the MIT penalty score is shown in black, the relative abundance of the sequence is shown in white, and the fraction of the quasispecies expected to be cut by the gRNA is shown in gray. For example, the most abundant quasispecies accounts for 40% of the total and the gRNA exactly matches the sequence (row 1). As such, the entire quasispecies has been assumed to be cut. The second most abundant has a penalty score of 0.9, as such only 36% of that fraction was predicted to be removed. gRNA, guide RNA; MIT, Massachusetts Institute of Technology.

FIG. 2.

Personalized gRNAs can target the quasispecies with longitudinal efficacy. (A) The number of gRNAs needed to cleave the fraction of the viral quasispecies for each patient. Most patients require three to eight gRNAs at the current level of NGS detection (1:10,000). The three lines to the right of the arrow indicate the level of detection of the three that are not captured by the three to eight gRNAs. (B) A histogram indicating the number of samples that pass a particular cleavage threshold when tested with a package of four gRNAs personalized to the sample. (C) The samples from 23 patients were examined longitudinally by designing a package of four personalized gRNAs against one visit and evaluating the effectiveness on a subsequent visit from the same patient. Solid lines indicate instances in which the treatment has become less effective and dotted lines indicate the treatment has either been maintained or improved in effectiveness. The effectiveness was limited by the level of detection (10⁻⁴). NGS, next generation sequencing.

FIG. 3.

A package of four gRNAs has potential BS cutting efficiency. (A) All five gRNAs have >10% predicted cutting fraction of patient-derived sequences from the CNS AIDS Research and Eradication Study cohort. (B) The top two BS gRNAs were compared with gRNAs LTR-A/B to determine how many samples would be predicted to be cleaved to a particular cutoff. The results obtained with the top two gRNAs (Top-2) are shown as dotted line and the results obtained with LTR-A/B gRNAs are shown in black. The graph is extended to include predicted cuts <10%, so all 48 samples are presented. Results obtained with cutting efficiencies of 90% and 99% are indicated. BS, broad-spectrum.

FIG. 4.

Combining three BS gRNAs with a single personalized gRNA (BS3 + PS1) is predicted to cleave the majority of the patient quasispecies. On the left, the positions of each gRNA for each patient are shown with the shade indicating the weighted average of the quasispecies that was predicted to be cleaved. On the right, the combined weighted cleavage fraction was calculated for each patient assuming each position was independent.