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. 2014 Aug 5;111(31):11461-6.
doi: 10.1073/pnas.1405186111. Epub 2014 Jul 21.

RNA-directed Gene Editing Specifically Eradicates Latent and Prevents New HIV-1 Infection

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Free PMC article

RNA-directed Gene Editing Specifically Eradicates Latent and Prevents New HIV-1 Infection

Wenhui Hu et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

AIDS remains incurable due to the permanent integration of HIV-1 into the host genome, imparting risk of viral reactivation even after antiretroviral therapy. New strategies are needed to ablate the viral genome from latently infected cells, because current methods are too inefficient and prone to adverse off-target effects. To eliminate the integrated HIV-1 genome, we used the Cas9/guide RNA (gRNA) system, in single and multiplex configurations. We identified highly specific targets within the HIV-1 LTR U3 region that were efficiently edited by Cas9/gRNA, inactivating viral gene expression and replication in latently infected microglial, promonocytic, and T cells. Cas9/gRNAs caused neither genotoxicity nor off-target editing to the host cells, and completely excised a 9,709-bp fragment of integrated proviral DNA that spanned from its 5' to 3' LTRs. Furthermore, the presence of multiplex gRNAs within Cas9-expressing cells prevented HIV-1 infection. Our results suggest that Cas9/gRNA can be engineered to provide a specific, efficacious prophylactic and therapeutic approach against AIDS.

Keywords: CRISPR/Cas9; genome editing; latency; reservoir; retrovirus.

Conflict of interest statement

Conflict of interest statement: A patent application has been filed relating to this work.

Figures

Fig. 1.
Fig. 1.
Cas9/LTR-gRNA suppresses HIV-1 reporter virus production in CHME5 microglial cells latently infected with HIV-1. (A) Representative gating diagram of EGFP flow cytometry shows a dramatic reduction in TSA-induced reactivation of latent pNL4-3-ΔGag-d2EGFP reporter virus by stably expressed Cas9 plus LTR-A or -B, vs. empty U6-driven gRNA expression vector (U6-CAG). (B) SURVEYOR Cel-I nuclease assay of PCR product (−453 to +43 within LTR) from selected LTR-A- or -B-expressing stable clones shows dramatic indel mutation patterns (arrows). (C and D) PCR fragment analysis shows a precise deletion of 190-bp region between LTR-A and -B cutting sites (red arrowhead and arrow), leaving 306-bp fragment (black arrow) validated by TA-cloning and sequencing results. (EG) Subcloning of LTR-A/B stable clones reveals complete loss of reporter reactivation determined by EGFP flow cytometry (E) and elimination of pNL4-3-ΔGag-d2EGFP proviral genome detected by standard (F) and real-time (G) PCR amplification of genomic DNA for EGFP and HIV-1 Rev response element (RRE); β-actin is a DNA purification and loading control. (H) PCR genotyping of LTR-A/B subclones (#8, #13) using primers to amplify DNA fragment covering HIV-1 LTR U3/R/U5 regions (−411 to +129) shows indels (a, deletion; c, insertion) and “intact” or combined LTR (b).
Fig. 2.
Fig. 2.
Cas9/LTR-gRNA efficiently eradicates latent HIV-1 virus from U1 monocytic cells. (A) (Right) Diagram showing excision of HIV-1 entire genome in chromosome Xp11.4. HIV-1 integration sites were identified using a Genome-Walker link PCR kit. (Left) Analysis of PCR amplicon lengths using a primer pair (P1/P2) targeting chromosome X integration site-flanking sequence reveals elimination of the entire HIV-1 genome (9,709 bp), leaving two fragments (833 and 670 bp). (B) (Upper) TA cloning and sequencing of the LTR fragment (833 bp) showing the host genomic sequence (small letters, 226 bp) and the partial sequences (634 − 27 = 607 bp) of 5′ LTR (green) and 3′ LTR (red) with a 27-bp deletion around the LTR A targeting site (underlined). (Lower) Two indel alleles identified from 15 sequenced clonal amplicons. The 670-bp fragment consists of a host sequence (226 bp) and the remaining LTR sequence (634-190 = 444 bp) after 190-bp excision by simultaneous cutting at LTR-A and -B target sites. The underlined and green-highlighted sequences indicate the gRNA LTR-A target site and PAM. (C) Functional analysis of LTR-A/B-induced eradication of HIV-1 genome, showing substantial blockade of p24 virion release induced by TSA/phorbol myristate acetate (PMA) treatment. U1 cells were transfected with pX260-LTRs A, B, or A/B. After 2-wk puromycin selection, cells were treated with TSA (250 nM)/PMA for 2 d before p24 Gag ELISA was performed.
Fig. 3.
Fig. 3.
Stable expression of Cas9 plus LTR-A/B vaccinates TZM-bI cells against new HIV-1 virus infection. (A) Immunocytochemistry (ICC) and Western blot (WB) analyses with anti-Flag antibody confirm the expression of Flag-Cas9 in TZM-bI stable clones puromycin (1 µg/mL) selected for 2 wk. (B) PCR genotyping of Cas9/LTR-A/B stable clones (c1−c7) reveals a close correlation of LTR excision with repression of LTR luciferase reporter activation. Fold changes represent TSA/PMA-induced levels over corresponding noninduction levels. (C) Stable Cas9/LTR-A/B-expressing cells (c4) were infected with pseudotyped-pNL4-3-Nef-EGFP lentivirus at indicated multiplicity of infection (MOI), and infection efficiency was measured by EGFP flow cytometry, 2 d postinfection. (D) Representative phase-contrast/fluorescence micrographs show that LTR-A/B stable but not control (U6-CAG) cells are resistant to new infection by pNL4-3-ΔE-EGFP HIV-1 reporter virus (green).
Fig. 4.
Fig. 4.
Off-target effects of Cas9/LTR-A/B on human genome. (A) SURVEYOR assay shows no indel mutations in predicted/potential off-target regions in human TZM-bI and U1 cells. LTR-A on-target region (A) was used as a positive control and empty U6-CAG vector (U6) as a negative control. (BD) Whole-genome sequencing of LTR-A/B stable TZM-bI subclone showing the numbers of called indels in the U6-CAG control and LTR-A/B samples (B), detailed information on 10 called indels near gRNA target sites in both samples (C), and distribution of off-target called indels (D).

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