The CRISPR/Cas9 system inactivates latent HIV-1 proviral DNA
© Zhu et al.; licensee BioMed Central. 2015
Received: 25 November 2014
Accepted: 9 February 2015
Published: 27 February 2015
Highly active antiretroviral therapy (HAART) has transformed HIV-1 infection from a deadly disease to a manageable chronic illness, albeit does not provide a cure. The recently developed genome editing system called CRISPR/Cas9 offers a new tool to inactivate the integrated latent HIV-1 DNA and may serve as a new avenue toward cure.
We tested 10 sites in HIV-1 DNA that can be targeted by CRISPR/Cas9. The engineered CRISPR/Cas9 system was introduced into the JLat10.6 cells that are latently infected by HIV-1. The sequencing results showed that each target site in HIV-1 DNA was efficiently mutated by CRISPR/Cas9 with the target site in the second exon of Rev (called T10) exhibiting the highest degree of mutation. As a result, HIV-1 gene expression and virus production were significantly diminished with T10 causing a 20-fold reduction.
The CRISPR/Cas9 complex efficiently mutates and deactivates HIV-1 proviral DNA in latently infected Jurkat cells. Our results also revealed a highly efficient Cas9 target site within the second exon of Rev that represents a promising target to be further explored in the CRISPR/Cas9-based cure strategy.
KeywordsHIV-1 Provirus Reservoir CRISPR/Cas9 Genome editing
Current antiretroviral therapy (ART) enables control of HIV infection at both the individual level and on a global scale. As a result, steady declines have been seen in both numbers of new HIV infections as well as in HIV mortality rates [1,2]. Unfortunately, a cure of HIV infection has not yet been attained. One block to this ultimate goal is the persistence of HIV reservoirs that cannot be cleared by current ART . The establishment of viral reservoirs requires the integration of HIV DNA into cellular genome . Theoretically, deleting or deactivating proviral DNA should eliminate the source of HIV persistence, and may thus provide a valuable tool toward cure.
Two approaches have been explored in this context. One is based on the zinc-finger nucleases (ZFNs) that were engineered to recognize and cleave specific HIV DNA sequences . The second approach is called TALENS (transcription activator-like effector nucleases) that program transcription factors to recognize specific DNA sequences . Both approaches exploit the principle of protein-DNA recognition that often involves a certain degree of ambiguity. A recent development in genome editing is the CRISPR (Type II microbial clustered regularly interspaced short palindromic repeat)/Cas9 system that utilizes a guide RNA strand to recognize and mutate target DNA; this therefore now offers a new strategy to inactivate HIV DNA [7-10].
This CRISPR/Cas9 system arms a DNA endonuclease called Cas9 with an RNA component that utilizes a 20-nucleotide guide sequence (gRNA) to recognize a specific DNA site [7,8]. Cleavage of DNA by Cas9 generates double-stranded DNA breaks that, upon non-homologous end joining (NHEJ) repair, cause insertions or deletions at the DNA target sites. This approach has been demonstrated highly specific and efficient in targeting multiple cellular genes and has been further developed for genome-wide screens to study gene function [11-14]. In this study, we have tested the efficiency of the CRISPR/Cas9 system in inactivating the integrated HIV-1 DNA in a HIV-GFP Jurkat cell line called JLat10.6 that was developed to study HIV latency .
In JLat10.6 cells, without any external stimulation, HIV-1 DNA is transcription silent as a result of the inaccessibility of cellular transcriptional machinery to HIV-1 LTR promoter [15,16]. We suspect that this inaccessibility, likely a result of modified chromatin structure, may hinder the ability of the CRISPR/Cas9 complex to target HIV-1 DNA. Were this the case, then pretreatment with HDAC inhibitors such as SAHA may deem necessary to activate HIV-1 gene expression in order to enhance the effectiveness of CRISPR/Cas9 system. We therefore treated the JLat10.6 cells with TNF-α for 16 hours prior to nucleotransfection with the gRNA and hCas9 plasmid DNA. In contrary to our expectation, activation of HIV-1 transcription by TNF-α did not render HIV-1 DNA more susceptible to inhibition by the CRISPR/Cas9 machinery (Figure 3E and F). This observation suggests that the CRISPR/Cas9 system has the ability to access transcription silent HIV-1 DNA without the need to activate viral gene expression by TNF-α or related agents such as SAHA. This property of the CRISPR/Cas9 system supports its possible utility in eradication of integrated HIV-1 DNA from latently infected immune cells such as CD4+ T cells and macrophages, in which HIV-1 transcription is also inert. Given that these HIV-1 latently-infected cells are often non-cycling, further studies are warranted to assess whether cell cycling may affect the efficacy of CRISPR/Cas9.
We next tested whether combinations of different gRNAs further increased the potency of inhibition. This combination strategy is also expected to diminish the chances of HIV-1 to escape from gRNA targeting. Indeed, certain combinations, such as T1/T6/T9, T2/T4/T10 and T3/T6/T9, reduced the numbers of GFP-positive cells and the yields of viruses by more than 24-fold (Figure 3G and H). It is noted that all three combinations contain gRNAs that target tat/rev, which indicates that tat and rev sequences may represent vulnerable sites for CRISPR/Cas9 targeting.
In conclusion, results of this study demonstrate the great potency of the CRISPR/Cas9 system to target and inactivate HIV-1 proviral DNA in the latent JLat10.6 cell line. Since JLat10.6 is a clonal cell line in which HIV-1 is integrated at a specific site in cellular DNA, results in this study cannot conclude whether the CRISRP/Cas9 system may target HIV-1 DNA that is integrated at different cellular DNA loci with different efficiency. Since the CRISPR/Cas9 complex targets both the transcription inert and the transcription active HIV-1 DNA with similar efficiency, CRISPR/Cas9 should be effective in eliminating HIV-1 reservoirs in which HIV-1 exists in a latent state. In support of our data, recent studies reported suppression of the HIV-based vectors using the CRISPR/Cas9 system [19,20]. Both studies tested gRNAs targeting HIV-1 LTR. We have herein tested 10 target sites across HIV-1 genome and identified one site in the second exon of Rev (called T10) that exhibits the highest level of mutation by Cas9. In a separate study, the CRISPR/Cas9 system was exploited to successfully generate the CCR5Δ32 deletion in pluripotent stem cells with 33% efficiency, as compared to 14% efficiency when the TALENS method was employed . Together, all these findings support the utility of the CRISPR/Cas9 system as a new genome editing approach to eradicate HIV-1. Challenges also exist to target a highly mutable virus like HIV-1 using the CRISPR/Cas9 system whose efficacy is largely dependent on how well the gRNA matches the target viral DNA sequence. One solution can be a personalized approach in which the gRNA is designed to match the HIV-1 sequences that are archived in the reservoir of the individual patient. This effort may especially pay off if such an approach can be used to cure infected individuals. A second strategy may involve exploiting multiple gRNAs to target several relatively conserved sites in the HIV-1 genome in order to maximize efficacy and minimize virus escape. With the CRISPR/Cas9 field quickly evolving, new tools will hopefully emerge to help eradicate HIV in clinical settings.
We thank Li li and Yan Xiao for their technical assistance, Dr. George M Church for providing the gRNA and hCas9 plasmids. This study was supported by the National 12th 5-year project 2012ZX10001009, basic scientific research grant 2012IPB301 from the Institute of Pathogen Biology, 2012C01 and a grant for a distinguished visiting professor from the Chinese Academy of Medical Sciences & Peking Union Medical College, the Program for Changjiang Scholars and Innovative Research Team in University (IRT13007), the Ministry of Science and Technology of China (2012CB911103, 2012ZX10001006-003 and 2013ZX10001005-002), as well as the Canadian Institutes of Health Research.
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