Gene Therapy (2017) 24, 377–384

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved 0969-7128/17
www.nature.com/gt

REVIEW
Current application of CRISPR/Cas9 gene-editing technique
to eradication of HIV/AIDS
Z Huang, A Tomitaka, A Raymond and M Nair

Human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) remains a major health hazard despite
significant advances in prevention and treatment of HIV infection. The major reason for the persistence of HIV/AIDS is the inability
of existing treatments to clear or eradicate the multiple HIV reservoirs that exist in the human body. To suppress the virus
replication and rebound, HIV/AIDS patients must take life-long antiviral medications. The clustered regularly interspaced
palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) system is an emerging gene-editing technique with the potential
to eliminate or disrupt HIV-integrated genomes or HIV-infected cells from multiple HIV reservoirs, which could result in the
complete cure of HIV/AIDS. Encouraging progress has already been reported for the application of the CRISPR/Cas9 technique to
HIV/AIDS treatment and prevention, both in vitro in human patient cells and in vivo in animal model experiments. In this review, we
will summarize the most recent progress in the application of the CRISPR/Cas9 gene-editing technique to HIV/AIDS therapy and
elimination. Future directions and trends of such applications are also discussed.

Gene Therapy (2017) 24, 377–384; doi:10.1038/gt.2017.35

INTRODUCTION human brain. Neurons are believed to be resistant to HIV

Human immunodeficiency virus (HIV)/acquired immunodefi-
ciency syndrome (AIDS) is one of the most severe pandemic
diseases in modern history. In 2015, according to the World
Health Organization (WHO), about 36.7 million people world-
wide were living with HIV, and roughly 2.2 million people died of
AIDS, while more than 2.1 million new patients were diagnosed
with HIV infection. Of all people diagnosed with HIV infections, it
is estimated that only 17 million (46%) are on antiretroviral
therapy (WHO). With breakthroughs in HIV/AIDS prevention,
diagnosis, and treatment, the morbidity and mortality of HIV/
AIDS has decreased significantly. However, HIV/AIDS remains an
incurable, chronic disease due to the multiple HIV latent
reservoirs in patients’ bodies. These reservoirs primarily contain
very small numbers (for example, about 1 per million memory
CD4+ T cells) of latently infected cells1–4 which are located in the
brain,5–7 peripheral blood,2,3,8,9 lymphoid tissue,1,10,11 gastro-
intestinal tract,10,11 and other locations.12,13 Complicated
mechanisms underlie the persistence of HIV reservoirs, including
the stability of reservoir cells;4 homeostatic proliferation of
reservoir cells;14 replenishment of reservoir cells;15 and low
levels of antiviral drug penetration.16–18 Moreover, even under
antiretroviral therapy (ART), about 30–50% of AIDS patients
eventually develop HIV-associated neurological disorders
(HAND),19,20 which are cognitive, motor and/or behavioral
impairments caused by HIV infection in the brain.6,20–23 HAND
can further be grouped into asymptomatic neurocognitive
impairment (ANI), minor neurocognitive disorder (MND) and
the most severe, HIV-associated dementia (HAD).21 The mechan-
ism of HAND remains to be elucidated, but it is generally
accepted that HAND is tightly correlated with HIV infection of
astrocytes,22,24–26 microglial cells5,26 and macrophages5,6 in the
infection; however, the neurotoxic products released from HIV-
infected brain cells significantly dysregulate neuronal function
and homeostasis.26
The ultimate cure for HIV/AIDS will be the removal or disruption
of integrated HIV provirus from latently infected cells or the
elimination of these latent cells completely. However, until
recently, gene therapy for HIV/AIDS has progressed very slowly.
The breakthrough of gene-editing technology raises the hope for
eradication of HIV as the provirus can be removed from the host
cell genome through the newly developed technique of clustered
regularly interspaced short palindromic repeat (CRISPR)/CRISPR-
associated protein 9 (Cas9) gene-editing.27–30 Furthermore,
CRISPR/Cas9 system can be utilized to induce apoptosis in
reservoir cells.31,32 Originally, an adaptive immune response
mechanism in bacteria,33,34 the CRISPR/Cas9 system has emerged
as a popular gene-editing system with proven efficiency in
mammalian cells. As early as 2013, soon after the CRISPR/Cas9
technique emerged, its potential applications for treating
HIV/AIDS were considered.28 This mini-review addresses the use
of the CRISPR/Cas9 system as a powerful gene-editing tool to
delete or disrupt HIV provirus from latently infected cell genomes
or to knock out CXCR4 and CCR5 receptors for HIV infection
treatment and prevention. The elimination or disruption of HIV
provirus in reservoir cells or the elimination of reservoir cells may
result in a cure for HIV/AIDS.
OVERVIEW OF HIV PRODUCTIVE AND LATENT INFECTION
HIV is a retrovirus that belongs to the lentivirus genus. Its DNA
genome has two long terminal repeat (LTR) structures flanking the
whole virus genome, which consists of nine overlapping genes:

Department of Immunology, Institute of NeuroImmune Pharmacology, Center for Personalized Nanomedicine, Herbert Wertheim College of Medicine, Florida International
University, Miami, FL, USA. Correspondence: Professor M Nair or Dr Z Huang, Department of Immunology, Institute of NeuroImmune Pharmacology, Herbert Wertheim College of
Medicine, Florida International University, 11200 SW 8th Street, Miami, FL 33199, USA.
E-mail: nairm@fiu.edu or zahuan@fiu.edu
Received 25 August 2016; revised 25 January 2017; accepted 1 February 2017; accepted article preview online 4 May 2017; advance online publication, 1 June 2017
 Application of CRISPR/Cas9 gene-editing technique to eradication of HIV
Z Huang et al
378
gag, pol, env, tat, rev, nef, vif, vpr, vpu (Figure 1a). HIV is the
causative agent of AIDS in human patients, and it can be
transmitted through contact with infected blood or other body
fluids through broken skin or mucous membranes. The two most
common ways to spread HIV/AIDS are unprotected sex and needle
sharing for intravenous drug injection.
HIV targets CD4+ human immune cells, including T cells,
dendritic cells and monocytes, as well as cells within the central
nervous system (CNS), such as perivascular macrophages, astro-
cytes, and microglial cells. The life cycle of HIV is quite complex
(Figures 1b and c). HIV establishes two types of infection in target
cells—active infection (Figure 1b) or latent infection (Figure 1c).

Figure 1. HIV life cycle. (a) HIV DNA genome HIV-1 DNA genome structure is illustrated above and each gene is drawn in ratio. The viral
genome is ~ 10 kb long and contains nine viral genes. All the viral genes’ locations are identified. In addition, tat and rev have two exons. TAT
protein is expressed in a minor form (72 amino acids) and a major forms (86 amino acids), while REV protein is expressed as one form of 116
amino acids. Genes of gag and env encode structural proteins; gene of pol encodes enzymes and all other genes encode regulatory proteins.
(b). HIV active infection: In this illustration, HIV life cycle is divided into seven stages: Binding, fusion and entry (1 and 2): HIV binds to immune
cell surface receptors, including CD4 and CXCR4 or CD4 and CCR5. The binding causes conformation changes and results in the membrane
fusion between HIV and cell membrane. HIV enters the cell, disintegrates and releases the viral RNA and enzymes. Reverse transcription (3):
HIV viral reverse transcriptase converting viral RNA into doubled strand viral DNA. Integration (4): HIV DNA enters host cell nucleus and
integrates into host cell genome with the help of viral integrase. Replication (5): Integrated HIV DNA uses host cell machinery to translate viral
proteins and transcribe viral RNA. Assembly (6): the components of HIV, including the HIV RNA and proteins move to the cell membrane to
form an immature viral particle. Budding (7): The new immature viral particles are released from cell membrane and become mature and
infectious after viral protease action. (c). HIV latent infection: In HIV latent infection, HIV life cycle stops at stage 4 after HIV DNA genome
integration. However, under certain conditions, the integrated HIV genome can be activated and the whole life cycle described in 1B is
accomplished.

Gene Therapy (2017) 377 – 384 © 2017 Macmillan Publishers Limited, part of Springer Nature.

Active infection occurs in most cells, while latent infection occurs
in much fewer cells1,2 and at very early stages of HIV infection.9,35
In active infection, HIV provirus is active and HIV virus particles are
actively replicated; and the infected cells continuously release viral
progeny; while in latent infection, HIV provirus is transcriptionally
silenced and no viral progeny is produced. However, under certain
conditions, such as normal immunologic responses to various
recall antigens or routine vaccination,36 or the induction of
cytokines,2,35 latently infected cells are reactivated to produce
virus;2 subsequently, a pool of new latent cells are generated by
these reactivated latent cells.35 These reservoirs ultimately hinder
HIV/AIDS eradication. The mechanisms of how latent HIV infection
occurs remain elusive, but evidence suggests that the establish-
ment of an HIV reservoir is related to the following factors: HIV
provirus integration site;37–39 chromatin environment;40,41 acces-
sibility of transcription factors41–43 and RNA interference.44–46
Latently infected cells are long-lived and mainly reside in reservoir
sites where antiviral drugs may not penetrate, such as the brain,5–7
lymphoid tissue1,10,11 and gastrointestinal tract.10,11 Latently
infected cell populations include memory CD4+ T cells,1–4,10
macrophages,5,6,11 microglial cells5,26 and astrocytes.22,24–26 The
establishment of an HIV reservoir occurs soon after infection, and
early application of antiviral drugs typically only diminishes rather
than eliminates the reservoir.9,35
To eliminate the viral reservoirs, current ‘shock and kill’ efforts
seek to reactivate the latent reservoirs (the shock) and destroy the
reactivated cells (the kill) through an intensifying combination of
antiretroviral therapy (cART) and host immune responses.47
Research efforts have shown that using SAHA, a histone
deacetylase inhibitor, in conjunction with cART reactivates latent
HIV in infected CD4+ T cells, making these cells susceptible to
specific CD8 T-cell killing in vitro.48,49 However, none of the clinical
studies demonstrated a significant therapeutic effect as measured
by HIV DNA or quantitative viral outgrowth assay, which means
they are ineffective.48,49 Now ‘shock and kill’ efforts have
expanded to include testing and optimizing an increasing number
of latency-reversing compounds combined with host immuno-
modulatory strategies.47 The setbacks of ‘shock and kill’ have
encouraged researchers to look for alternative approaches to
eradicate HIV reservoirs. One approach to complete HIV eradica-
tion would involve the actual removal or disruption of the provirus
from HIV reservoir cells. This type of gene therapy approach has
recently been investigated using CRISPR/Cas9 gene-editing
system with promising results.27–30
OVERVIEW OF CRISPR/CAS9 TECHNOLOGY
The ultimate goal of gene-editing is gene repair. Several gene-
editing technologies recently demonstrated strong potential for
therapeutic uses. These technologies include zinc-finger nucleases
(ZFNs),50–52 transcription activator–like effector nucleases
(TALENs),53–55 and CRISPR/Cas9 system.34,56–59 In principle, ZFNs
and TALENs fuse DNA nuclease and DNA-binding domains
together to break DNA at specific loci, while Cas9 nucleases work
with guide RNA to break DNA at specific loci. Both ZFNs and
TALENs techniques have significant applications and studies in
HIV/AIDS prevention and therapy.60–63 However, this review will
be focusing solely on the CRISPR/Cas9 system due to the
limited scope.
Compared to the initial CRISPR/Cas9 system in bacteria, the
current CRISPR/Cas9 system has been simplified, modified and
adapted for mammalian genome editing and bioengineered for
better nucleus localization and mammalian cell expression.57,59
The current CRISPR/Cas9 system includes a single guide RNA
(sgRNA) consisting of crRNA and tracRNA57,59 and Cas9, which
excises the target DNA.57,59 Cas9 is a nuclease of 1368 amino acids
with two nuclease activity domains named HNH (residues 775–
908) and RuvC (residues 1–59, 718–769 and 909–1098)59,64–66
© 2017 Macmillan Publishers Limited, part of Springer Nature.
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(Figure 2a). Each domain can cleave a DNA strand directed by a
sgRNA complementary to the target DNA sequence (generally 20
nucleotides long). The prerequisite to be a target sequence is the
presence of a NGG sequence (N: any nucleotides; G: guanine
nucleotide, also called protospacer adjacent motifs (PAMs))
downstream (3′ end) of the target site.59,64–66 In the nucleus,
Cas9, sgRNA and target DNA form a complex, then HNH and RuvC
domains each cleave a DNA strand59,64–66 (Figure 2b). The double-
strand DNA breaks are consequently repaired by two different
approaches. One is non-homologous end-joining (NHEJ) when
there is no template and the other is homology-directed repair
(HDR) when there is a homologous nucleotide template present
such as ssDNA or dsDNA. This template must possess homology
sequences (arms) to the two regions that flank the Cas9 excision
site. If the two homologous arms flank a correct sequence, which
can be used as a template to ‘repair’ the genomic sequences.59,67
NHEJ usually results in an insertion or deletion in a DNA sequence,
in total called indel, while HDR results in correct repair as directed
by the template.59,67 CRISPR/Cas9 system can also be used in
biological functions other than gene-editing. For example,
catalytically inactive Cas9 (dCas9) has no active nuclease domains
and is often applied to be a site-specific DNA-binding factor.68 The
fusion protein of dCas9 and transcription activator or repressor
domain are often used to regulate gene expressions.59,68 In
summary, CRISPR/Cas9 system is a full developed multifunction
gene-editing and regulating system which has unlimited
potential.59,67
APPLICATION OF CRISPR/CAS9 SYSTEM TO HIV/AIDS
PREVENTION AND TREATMENTS
The first attempt to apply CRISPR/Cas9 system to HIV/AIDS
eradication was conducted in Japan in 2013. Ebina et al.28 tried
to eliminate HIV provirus from T cells by targeting the LTR region
of HIV provirus. CRISPR/Cas9 system was applied to target TAR
sequence of the R region and NF-κB-binding sequence in the U3
region. The indel formation by CRISPR/Cas9 cleavage of TAR
region is more effective to disrupt provirus transcription and
replication.28 The indel formation by CRISPR/Cas9 cleavage of LTR
resulted in inhibition of active provirus expression and decreased
reactivation of latent provirus.28
For HIV prevention, many investigators focus on applying the
CRISPR/Cas9 technique to the deletion or disruption of the HIV
receptors’ expression on human cells. Since CD4 is the essential
primary receptor in human T cells and is indispensable for normal
cell functions, HIV secondary receptors of CXCR4 and CCR5 are
often candidates for the deletion and disruption by CRISPR/Cas9
technique. T-cell genome engineering holds great promise for
cell-based therapies for HIV eradication due to the rapid
development of gene-editing. Hou et al.69 efficiently disrupted
the CXCR4 gene with CRISPR/Cas9 technology in human primary
CD4+ T cells. Ten sites within the CXCR4 gene were targeted and
CXCR4 expression decreased by ~ 30% as confirmed by western
blot.69 The engineered T cells showed resistance to HIV infection
and p24 production reduced over 60%.69 More importantly, this
technology indicated high specificity and no off-target effects,
while cell division and propagation remained normal.69 Schumann
et al.70 used Cas9/sgRNA ribonucleoproteins (Cas9 RNPs) to ablate
CXCR4 gene expression in human T cells, and about 40% of cells
lost surface expression of CXCR4. When paired with repair
template, Cas9 RNPs produced a desired genome modification
in human primary T cells.70 Deep sequencing confirmed that Cas9
RNPs achieved about 20% efficiency of knockin genome
modifications.70 Because of the success of the Berlin Patient case,
Ye et al.71 tried to produce CCR5Δ32-induced pluripotent stem
cells (iPSCs) using CRISPR/Cas9 system. Close to 100% efficiency
was achieved for homologous recombination of a single allele and
up to 33% of double alleles.71 As expected, monocytes and
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 Application of CRISPR/Cas9 gene-editing technique to eradication of HIV
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Figure 2. CRISPR/Cas9 protein structure and working mechanism. (a) CRISP/Cas9 protein domain structure: CRISPR/Cas9 protein structure is
illustrated above. The number of amino acids are labeled above the protein schema, and the domain names are listed below the protein
schema. HNH nuclease domain is labeled in green, and RuvC nuclease domain consists of three sub-domains, which are labeled in red.
(b) CRISPR/Cas9 working mechanism: In detail, Cas9 has two nuclease domains (HNH and RuvC) represented by two grey oval shapes in the
figure. Each nuclease domain can cleave a DNA strand directed by a sgRNA (In this figure, a sgRNA is in a black box), which is complementary
to the target DNA sequence. The prerequisite to be a target sequence is the presence of a NGG (In this figure, it is CGG) sequence downstream
of the target site (3’ end). The length of the target sequence is generally 20 nucleotides (In this figure the target sequence is in green). In cell
nucleus, Cas9, sgRNA and double-strand target DNA form a complex. Each nuclease domain cleave a DNA strand at the third nucleotide from
the NGG (indicated by a thin red line). The double-strand breaks are consequently repaired mainly by either non-homologous end-joining
(NHEJ) or homology-directed repair (HDR) when there is a homologous repair template (not shown). NHEJ is error prone and often results in
deletions and insertions while HDR is a high-fidelity repair according to the homologous repair template. In this figure, an insertion NHEJ is
shown in underlined bold black and a faithful HDR is shown in underlined bold green.

macrophages derived from such engineered iPSCs were found to
be resistant to HIV infection.71 Wang et al.72 applied CRISPR/Cas9
technology to block CCR5 expression in human CD4+ cells.
Lentiviral vectors were used to target three sites of the CCR5
gene.72 The disruption ratio of CCR5 was high, and those cells
were resistant to R5 tropic HIV with negligible off-target effect.72
However, attempts to disrupt CCR5 gene in human primary T cells
with the same approach failed for unknown reasons.72 In another
study, Li et al.73 applied adenoviral vectors to deliver CRISPR/Cas9
system to target multiple sites of the fourth exon of CCR5 gene to
block CCR5 expression. First, the TZM-b1 cell line was tested and
two important target sites, which knockout over 60% of the gene
were identified. Secondly, the results were confirmed in a CHO
and a human T-cell lines. Thirdly, CRISPR/Cas9 technique
conferred TZM-B1 cell the resistance to HIV infection. Finally, a
chimeric Ad5/F35 adenoviral vector was used to deliver the
CRISPR/Cas9 system to human primary CD4+ T cells to disrupt the
CCR5 gene, which provided the primary T-cells the resistance to
HIV infection.73
Besides the deletion or disruption of HIV receptors on human
cells, many scientists have applied the CRISPR/Cas9 technique to
remove or disrupt HIV provirus in latently infected human cells
(Figure 3). The Drexel Medicine CNS AIDS Research and
Eradication Study (CARES) cohort studies27 demonstrated the
following: (1) The proviral LTR sequences within the peripheral
blood mononuclear cells (PBMC) decreased variations every year
with ART; (2) HIV LTR sequences continued genetic changes for a
minimum of 6 years even with effective suppressive ART; (3) Next-
generation sequencing (NGS) analysis found that a regimen of
sgRNAs could be designed to target all known HIV quasispecies in
50% of the analyzed patients; and (4) HAART therapy might
reduce the required sgRNA scale to eradicate provirus. For the first
time, the study demonstrated the feasibility of HIV provirus
elimination with CRISPR/Cas9 technology in a single patient.27 Zhu
et al.74 targeted 10 sites of the HIV provirus to eradicate HIV DNA
from Jukat cell genome. These sites include three within LTR
region, five within pol gene, and two within rev gene.74 The
second exon of rev ranked as the best targeting site, and the
targeting at this site caused a 20-fold reduction in HIV
production.74 To eradicate the latent HIV reservoirs and achieve
a complete cure of HIV/AIDS, the ‘shock and kill’ strategy is often
applied to reactivate the latent HIV reservoirs and then kill those
activated reservoir cells by ART.47–49 However, the efficiency,
specificity and toxicity of current drugs turned out to be
disappointing.47–49 Now CRISPR/Cas9 system is utilized to execute
the ‘shock and kill’ strategy, in detail, catalytically deficient Cas9
(dCas9)-transcription activator fusion protein/sgRNA is applied to
specifically activate HIV latent reservoirs.31,32 With this technique,
Zhang et al.32 targeted 20 sites within the LTR U3 region of HIV
provirus and found that the two best activation sites are at or near
the NF-κB-binding site. This targeted activation potently reacti-
vated several HIV cell lines, including HIV-1 latent TZM-bI
epithelial, Jurkat T cells and CHME5 microglial cells.32 Moreover,
the reactivation resulted in cell death of Jurkat T cells and CHME5
microglial cells due to the accumulation of viral proteins of TAT,
NEF, REV and so on.32 Almost at the same time, similarly, Saayman
et al.31 targeted 23 sites in the LTR U3 region of the HIV provirus
and identified the best activation site as at near the NF-κB-binding
sites. The targeting system worked in many different in vitro latent
T-cell models to reactivate provirus, and these reactivations were

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Figure 3. Application of CRISPR/Cas9 technology to eradicate HIV reservoir cells. The major application of CRISPR/Cas9 technology to
eradicate HIV latent reservoir cells (a) is to eliminate (b) or disrupt the HIV provirus (c) from host reservoir cells genome. In detail, several
sgRNAs, which target two or more sites of HIV provirus are applied to HIV reservoir cells with Cas9. This action results in multiple cuts at the
multi-sites of HIV provirus, thus provirus is removed from host cell DNA (b) or provirus is disrupted (c). The DNA of host HIV reservoir cells is
further repaired by DNA non-homologous end-joining or homology-directed repair when the homologous repair template is available (not
shown).

consistent and more efficient than latency breaking reagents such
as prostratin and SAHA.31 More importantly, the activation was
specific to the provirus, while latency breaking reagents caused
widespread side effects. In addition, the reactivation also blocked
the NF-κB reactivation.31 Hu et al.29 tried using the CRISPR/Cas9
system to eradicate HIV provirus in microglial cells, pro-
monocytes, and T cells. The LTR U3 region was tested for four
sites individually or in combination. It turned out that two sites
combined approach achieved the best eradication results.29
Furthermore, no off-target effects were found, and the stable
expression of Cas9 and sgRNA conferred cells with the ability to
resist new HIV infections.29 Kaminski et al.30 applied a similar
approach to remove the entire HIV provirus from latently infected
human CD4 T cells. No off-target effects were found and cell
health parameters were not affected.30 The persistent expression
of Cas9 and sgRNA protected the human CD4 T cells from new HIV
infection.30 The delivery of Cas9 and sgRNA using lentivirus vector
could reduce HIV replication in HIV-infected primary human T cells
and decrease viral load in ex vivo patient CD4+ T cells.30 Kaminski
et al.75 has also applied a recombinant adeno-associated virus 9
(rAAV9) vector expressing saCas9 (a smaller version of Cas9
derived from Staphylococcus aureus to fit AAV vectors) and sgRNAs
to remove integrated HIV DNA in a mouse transgenic model. A
978 bp DNA fragment was removed from integrated HIV DNA
from lymphocytes and cells in multiple organs, including brain,
heart, kidney, liver, lung and spleen.75 Similar results were found in
transgenic rat experiments.75 This is the first report of such studies
in transgenic animal models. These encouraging progress
demonstrated the great promise of CRISPR/Cas9 technique in
eradication of HIV/AIDS. But there were scientific findings that
pointed out some pitfalls of CRISPR/Cas9 application to HIV/AIDS
eradication. Wang et al.76 demonstrated that Cas9/sgRNA can
inhibit HIV replication initially, but soon HIV-1 escape variants
were produced due to the NHEJ repair, and the escape variants
contained mutations around the Cas9 cleavage sites. Interestingly,
the more conservative the Cas9/gRNA targeting sequences were,
the longer it took for the HIV escape variants to appear.76 In
another research group, Wang et al.77 found that: CRISPR/Cas9
could derive mutant HIV which could resist Cas9 and sgRNA
reagents due to the host cell DNA repair. Yoder et al.78 recently
reported research results with the similar data and conclusions.
These negative findings suggest the need for careful design and
cautious evaluations of the application of the CRISPR/Cas9
technique to HIV/AIDS eradication. Multiplex gRNAs and several
excision sites of a provirus may be necessary to forever eliminate
or disrupt HIV provirus in reservoir cells.
CRISPR/CAS9 SYSTEM DELIVERY APPROACH
The major challenge of curing HIV/AIDS lies in the persistence of
multiple reservoirs in several drug- inaccessible parts of the
human body. Moreover, while current HIV/AIDS patients can take
medications to suppress symptoms, the side effects of these drugs
may contribute to high morbidity in these patients. The complete
cure is the elimination or disruption of HIV provirus from the
patient’s reservoir cells’ genomes or the elimination of patient
reservoir cells. As stated above, gene-editing technology makes it
possible to manipulate DNA at the cell genomic level. Since its
discovery, CRISPPR/Cas9 system has been a premier tool in gene-
editing. As mentioned before, many experimental efforts and
clinical trials are underway for the application of the CRISPR/Cas9
system for eradication of HIV/AIDS. The most substantive progress
to date has been reported are ex vivo studies. Future efforts will be
directed towards efficient delivery of CRISPR/Cas9 system
components to latently infected cells.
Currently, the delivery methods of CRISPR/Cas9 system are
divided into viral delivery and non-viral delivery. The CRISPR/Cas9
system is very flexible and can be delivered in the form of DNA,
RNA or protein/RNA complex. The most popular approach is the
DNA form as one or several plasmids. In the viral delivery
methods, lentivirus,29,79,80 baculovirus81 and recombinant adeno-
associated virus (rAAV) have been tried.75,80 Among these virus
vectors, rAAV has the advantages of high safety, flexible
administration routes and low or no immune response, but it
has the disadvantage of limited cargo size. The major administra-
tion approaches of rAAV include stereotactic injection, intranasal
and intratracheal administration, intravenous injection, intraper-
itoneal Injection and intramuscular Injection. In the non-viral

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Figure 4. Brain targeting delivery by magnetic or ligand-labeled nanoparticles. Drug and reagents loaded nanoparticles are systematically
administered and target the CNS cells in human brain. In magnetic targeting (a), nanoparticles complexes target brain cells and cross the BBB
under the force of magnetic field. In Receptor-mediated transcytosis (b), nanoparticle complexes target brain cells and cross BBB by brain-
specific receptor-mediated transcytosis. After crossing the BBB, drugs are released to conduct the therapeutic procedure.
delivery approach, vectors such as cationic polymer polyethyle-
neimine (PEI),82 liposomes,58,80 lipid nanoparticles,83,84 virus-like
particles derived from bacteriophage,85 and self-assembled DNA
nanoparticles86 have been applied for the delivery of the CRISPR/
Cas9 system. Since viral and non-viral approaches have relative
advantages and disadvantages, some investigators started trying
the combined approach. For example, lipid nanoparticle-mediated
delivery of Cas9 mRNA has been combined with adeno-associated
viruses encoding a sgRNA and a repair template.87 This delivery
strategy successfully repaired a disease gene in an adult mouse
model.87
No matter which delivery strategy is being used, the emphases
are biocompatible and targetable carriers, controllable reagents
release, and low-invasive administration. As for HIV/AIDS eradica-
tion, in order to establish more efficient evaluation methods, HIV/
AIDS primate models will be the center of the next phase of study.
Drug brain delivery is the key for the eradication of the HIV
reservoir in patients’ CNS systems. The obstacle for drug brain
delivery of the CRISPR/Cas9 system is the existence of the blood–
brain barrier (BBB). The tight junction between capillary endothe-
lial cells block transportation of large molecules and only limited
molecules with molecular weight smaller than 400 Da and
appropriate lipophilicity can cross. All large molecules and most
of the small molecules cannot cross the BBB. Great efforts have
been made to improve BBB permeability for therapeutic agents
for the treatment of various brain diseases and HAND. However,
currently available procedures are mainly invasive, including
intracerebroventricular (ICV) infusion, intra-cerebral injection.
Although these approaches have shown successful disruption of
the BBB, the invasiveness of the technique and the risk of damage
to the brain remain significant concerns. To overcome these
problems, the use of nanomaterials as drug delivery carriers to the
brain has been gaining appreciation.
The most popular approach for brain targeting using nanoma-
terials is receptor-mediated transcytosis (Figure 4). The endothelial
cells that form the BBB are known to express receptors, including
the transferrin receptor and insulin receptor.88–90 Ligand functio-
nalized nanoparticles can promote efficient brain delivery through
the binding of ligands to the receptors on endothelial cells.
Various nanoparticles have been developed for brain targeting
using this receptor-mediated transcytosis such as transferrin,91,92
lactoferrin93,94 or monoclonal antibody-conjugated liposomes,93
Gene Therapy (2017) 377 – 384
polymer nanoparticles,95 gold nanoparticles91 and iron oxide
nanoparticles.94,96
Magnetic targeting is another approach to improve BBB
transmigration of nanoparticles (Figure 4). Nair et al.92,97–100 have
made important progress in brain delivery using magnetic
nanoparticles and magneto-electric nanoparticles. Magneto-
electric nanoparticles have been used as carrier molecules, which
could cross the BBB and mediate controlled release of the loaded
drugs. These nanoparticles were able to cross the BBB using
magnetic force induced on the nanoparticles by the magnetic
field gradient.97,98 To date, these nanoparticles have not been
applied to the delivery of the CRISPR/Cas9 system nor have they
been fully tested in animal models. However, the potential for
these nanoparticles to deliver CRISPR/Cas9 system and target HIV
reservoir in brain seems high due to the nanoparticles’ capacities
of specific targeting, crossing the BBB, constant or controlled
release and non-invasive application.
CONCLUSION AND FUTURE DIRECTIONS
For the future of applying CRISPR/Cas9 technique to eradicate HIV
reservoirs, safety, efficiency and specificity will be the main
emphases. Research efforts using the CRISPR/Cas9 technique will
now be focused on primate models and phase I clinical trials. From
the point of this review, the CRISPR/Cas9 technique holds great
promise for the eradication of HIV/AIDS. Since the FDA has already
approved use of CRISPR/Cas9 technique in several clinical trials, it
is surely not long before the CRISPR/Cas9 system can be applied
for clinical therapeutics and prevention on HIV/AIDS.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
ACKNOWLEDGEMENTS
This work was supported by grants (R01DA037838 and R01DA034547) from National
Institutes of Health, US. We also thank Maureen Pelham and Courtney Myhr for their
help in English editing.
© 2017 Macmillan Publishers Limited, part of Springer Nature.

Z Huang et al
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