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. 2020 Oct;36(10):862-874.
doi: 10.1089/AID.2020.0055. Epub 2020 Aug 26.

Analysis of CRISPR/Cas9 Guide RNA Efficiency and Specificity Against Genetically Diverse HIV-1 Isolates

Katherine J Sessions  1 , Yun Yue Chen  2   3 , Christine A Hodge  2   3 , Taylor R Hudson  1 , Susan K Eszterhas  1   4 , Matthew S Hayden  2   3 , Alexandra L Howell  1   4   5
Affiliations

Analysis of CRISPR/Cas9 Guide RNA Efficiency and Specificity Against Genetically Diverse HIV-1 Isolates

Katherine J Sessions et al. AIDS Res Hum Retroviruses. 2020 Oct.

Abstract

Gene editing approaches using CRISPR/Cas9 are being developed as a means for targeting the integrated HIV-1 provirus. Enthusiasm for the use of gene editing as an anti-HIV-1 therapeutic has been tempered by concerns about the specificity and efficacy of this approach. Guide RNAs (gRNAs) that target conserved sequences across a wide range of genetically diverse HIV-1 isolates will have greater clinical utility. However, on-target efficacy should be considered in the context of off-target cleavage events as these may comprise an essential safety parameter for CRISPR-based therapeutics. We analyzed a panel of Streptococcus pyogenes Cas9 (SpCas9) gRNAs directed to the 5' and 3' long terminal repeat (LTR) regions of HIV-1. We used in vitro cleavage assays with genetically diverse HIV-1 LTR sequences to determine gRNA activity across HIV-1 clades. Lipid-based transfection of gRNA/Cas9 ribonucleoproteins was used to assess targeting of the integrated HIV-1 proviral sequence in cells (in vivo). For both the in vitro and in vivo experiments, we observed increased efficiency of sequence disruption through the simultaneous use of two distinct gRNAs. Next, CIRCLE-Seq was utilized to identify off-target cleavage events using genomic DNA from cells with integrated HIV-1 proviral DNA. We identified a gRNA targeting the U3 region of the LTR (termed SpCas9-127HBX2) with broad cleavage efficiency against sequences from genetically diverse HIV-1 strains. Based on these results, we propose a workflow for identification and development of anti-HIV CRISPR therapeutics.

Keywords: CRIPSR; Cas9; HIV-1; HIV-1 LTR; off-target analysis.

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Conflict of interest statement

No competing financial interests exist.

Figures

FIG. 1.
FIG. 1.
U3 region of 5′ LTR sequences. The sequence conservation of the four different target regions for the gRNAs was derived by web alignments using the Los Alamos National Laboratory HIV Sequence Database (https://www.hiv.lanl.gov/content/index) utilizing 1,242 complete HIV sequences from all clades (available in 2017). The logo for each target region indicates the probability of each nucleotide at a specific position. The PAM sequence is boxed, and gray boxes denote sites of sequence insertions and deletions in multiple clades where alignment is lost, but becomes re-established downstream. The consensus sequence (shown in bold font) from 1,242 analyzed HIV-1 sequences was aligned with the sequence of NL4-3 (GenBank AF324493.2), the source of LTRs in the plasmid used in Figure 2. The target region of the four guides is underlined, and the PAM sequence is in red font. The numbers in parentheses are coordinates with reference to the HIV-1 HXB2 sequence. gRNAs, guide RNAs; LTR, long terminal repeat; PAM, protospacer adjacent motif. Color images are available online.
FIG. 2.
FIG. 2.
Maps of the two plasmids used in the in vitro cleavage assays. (A) Schematic representation of plasmid pNL-GFP with 5′ and 3′ LTRs (HIV-1 LTRs derived from NL4-3, shown as blue arrows) flanking the green fluorescent protein gene (eGFP in green). Noted are the restriction enzyme sites for XmnI and KpnI that cleave the plasmid into three fragments, with the anticipated fragment sizes indicated. Target sites of the LTR guides are noted by the numerical value that represents the nucleotide closest to the PAM. (B) Schematic representation of plasmid pBlue3′LTR-luc-B. This plasmid contains the LAI HIV-1 3′ LTR derived from pBluescript KS(+), shown as a blue arrow. This plasmid was used as the backbone for each clade, interchanging the 3′ LTR to correspond appropriately. Noted are the restriction enzyme sites for XmnI and EcoRI that cleave the plasmid into two fragments, and the target sites of the LTR gRNA 127 and gRNA 363. Color images are available online.
FIG. 3.
FIG. 3.
In vitro analysis of fragmented pNL-GFP cleaved with single or double LTR gRNAs. Left panels: gel images of fragmented pNL-GFP cleaved by (A) single or (B) double LTR guides directed to the 5′ and 3′ LTRs. Right panels: cleavage efficiency of each guide tested was determined for the 5′ and 3′ LTR independently. Data are the mean ± SEM from three experiments and analyzed using an unpaired two-tailed t-test; *p ≤ .05, **p ≤ .01, ***p ≤ .001, ****p ≤ .0001. Results show cleavage of pNL-GFP cleaved by one LTR guide (right panel, A) and two LTR guides (right panel, B) at the 5′ and 3′ LTRs. SEM, standard error of the mean.
FIG. 4.
FIG. 4.
Cleavage by individual gRNAs of the 3′ LTR from multiple HIV-1 clades. (A) Gel images of gRNA 127 (A, top panel) and gRNA 363 (A, bottom panel) cleavage of the 3′ LTR from pBlue 3′LTR-luc-A through G. (B) Cleavage efficiency of each clade against gRNA 127 (B, top panel) or gRNA 363 (B, bottom panel) was quantified. Data are the mean ± SEM from three experiments and statistical analysis was determined using a one-way ANOVA with Tukey's HSD post hoc test. All means were compared with one another. p Values are found in the table for clades cleaved with guide 363; ****p ≤ .0001. No significant differences were found between clades cleaved with gRNA 127. (C) DNA sequence differences (shown in red) between target sequence of gRNA 127 (top) and gRNA 363 (bottom) against the HIV-1 reference sequence, HXB2 (Target sequence). (D) CFD was calculated using Python version 2.7 with packages pickle, re, and NumPy. Original code was obtained from Doench et al. ANOVA, analysis of variance; CFD, Cutting Frequency Determination. Color images are available online.
FIG. 5.
FIG. 5.
CRISPR/Cas9 cleavage of 5′LTR in TZM-bl cells with one or more LTR gRNAs. Gene modification was analyzed using T7EI assay. (A) Example gel image yielded by T7E1 assay, which showed positive gene modification to the amplified 5′LTR and HPRT gene by gRNAs 363 and 127, and in combination. (B) The expected fragment size of PCR product after T7E1 digestion. (C) Percent gene modification for each gRNA treatment was determined using the following formula: 100 × [(1 − (1-fraction cleaved))1/2]. Mean data ± SEM from six experiments. (D) Luciferase reporter assay mean data ± SEM from triplicates. Statistical analysis was determined for both (C, D) using one-way ANOVA with Tukey's HSD post hoc test. All means were compared with one another; *p ≤ .05, **p ≤ .01, ***p ≤ .001. HPRT, hypoxanthine phosphoribosyltransferase; PCR, polymerase chain reaction; T7E1, T7 endonuclease 1 mutation detection.
FIG. 6.
FIG. 6.
Assessment of on- and off-target cleavage events using CIRCLE-Seq. CIRCLE-Seq was performed using DNA from TZM-bl cells and RNPs formed with recombinant SpCas9 and in vitro transcribed sgRNA 363 (A) and sgRNA 127 (B). Analysis of the resulting sequencing data identified the indicated cleavage events with the indicated numbers of read counts. (C) Annotation of cleavage events identified by CIRCLE-Seq. No off-target events were identified within the exons of protein coding genes. Off-target cleavage events occurred within both intergenic and intronic regions as indicated. RNP, ribonucleoprotein; sgRNA, single-guide RNA; SpCas9, Streptococcus pyogenes Cas9. Color images are available online.
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