Advance in Genome Editing of HIV-1 through CRISPR Technique

Jiaqi Liu

Davidson School of Chemical Engineering, University of Purdue, West Lafayette, IN 47907, U.S.A.

Keywords: Human Immunodeficiency Virus, Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)

/CRISPR-Associated Nuclease 9 (Cas9) System, Life Cycle, Genomic Editing, Integrated Viral Genes.

Abstract: Although tons of great efforts and improvement have been made to treat HIV-1 patients, HIV-1/AIDS remain

a big threat to global health. Although antiretroviral treatment (AT) yielded outstanding results in terms of its

impressive effect in the suppression of HIV-1 replication and expression, latent viral reservoirs in HIV-1

patients still exist and potentially activate to disrupt human health in the future. Recent advance in the

clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9)

system has been well-engineered with finely designed single-guide RNA (sgRNA) to effectively target

cellular co-receptors CCR5 and CXCR4 or HIV-1 genome so that this genomic editing system can reduce

HIV-1 reduction and eradicate integrated provirus. Such a versatile genome editing technology has been

applied to several types of human cells or animal models to testify its value in the prevention of HIV-1. Here,

the progress of the CRISPR/Cas9 -based approach in recent decades will be covered and discussed. In

addition, its potential drawbacks are also analyzed for future perspectives of its full application in the HIV-1

treatment regimen.

1 INTRODUCTION

For centuries, human beings have been struggling

against troubles from the human immunodeficiency

virus (HIV), which causes acquired

immunodeficiency syndrome (AIDS). At present, it

has become one of the world’s most severe public

health issues in terms of its high incidence and

prevalence. HIV is normally spread through wounds,

injured skin, or blood containing HIV and sexual

intercourse, etc., which makes it omnipresent in daily

life. Indeed, its nearly ubiquitous presence has caused

a lot of death and fear around the world. Such a

depressing impact continues inflicting both

psychological and physiological pains on everyone

else in recent years, especially in the developing

countries in Africa and the Asia Pacific. In 2018 an

estimated 37.9 million people were living with HIV

(including 1.7 million children), with a global HIV

prevalence of 18% among adults. Roughly 21% of

those people do not realize that they carry the virus

(Global HIV and AIDS Statistics 2020). Statistically,

an estimated 74.9 million people have become

infected with HIV and 32 million people have died of

https://orcid.org/0000-0002-7206-5327

HIV-related illnesses since the start of the epidemic

(Global HIV and AIDS Statistics 2020). According

to the data gathered by Kaiser Family Foundation

(KFF), which focused on adults ages 15-49, global

prevalence has leveled since 2001 and was 0.7% in

2019 (Global HIV and AIDS Statistics 2020). A lot

of countries are still suffering from the pains it has

brought. Of those countries, Eastern and Southern

African countries are populated by most people living

with HIV, which is about 20.7 million, accounting for

54% of the total global amount. Although Asia and

the Pacific, Western and Central Europe, and North

America do not have the same amount of infected

population as those African countries, the situation is

far from being good or optimistic since they

collectively account for 21% of the infected global

population (Global HIV and AIDS Statistics 2020).

As stated above, the situation in developed

countries like the U.S. is not optimistic as well. In the

U.S., roughly 1.2 million people are living with HIV

today. About 14 percent of those people are not aware

of it and need testing (U.S. Statistics 2021). It is

obvious that HIV is hard to be tested in the early stage

and can lay dormant for several months or a couple

Liu, J.

Advance in Genome Editing of HIV-1 through CRISPR Technique.

DOI: 10.5220/0011378700003443

In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 1131-1137

ISBN: 978-989-758-595-1

1131

of years. This is indeed the case; most people are

living with HIV before being diagnosed, and others

are diagnosed right after HIV infection. According to

the latest CDC data, in 2018, 37,968 people received

an HIV diagnosis in the United States and dependent

areas. From 2014 through 2018, the annual number

and rate of diagnoses of HIV infection in the United

States decreased. However, trends are mostly

disparate due to different groups of people (U.S.

Statistics 2021). In 2018, the gay or bisexual

population are the most vulnerable groups since they

accounted for 69% of new HIV diagnoses.

Heterosexual people accounted for 24% and the

remaining part all comes from people who inject

drugs (U.S. Statistics 2021). By race, the Blacks or

African American population is 13% of the U.S.

population in 2018, but they sadly contributed to 42%

of new HIV diagnoses. When it comes to age, it

reveals that people aged 25-44 held the highest

number of new HIV diagnoses (U.S. Statistics 2021).

As stated above, it is not quite hard to realize that HIV

prefers certain groups of people. It reminds people to

be careful of their choices on occupations, daily

habits, or ethnicity. Furthermore, there were 15,820

deaths among adults and adolescents with diagnosed

HIV in the U.S. in 2018 (U.S. Statistics 2021).

Given those discouraging statistical data,

scientists and pharmacists have been doing tons of

tests and related research to search for a

pharmacologically or medically efficacious regime to

tackle the problems it causes. To date, there is still no

efficacious cure for HIV-related diseases like AIDS.

Antiretroviral therapy (ART) is being incorporated

into the treatment of AIDS, along with other daily

medications. This treatment regimen yields

outstanding results due to its observable effect in

declining HIV-related deaths and simultaneously

protecting CD4 cells. It normally suppresses the viral

load to an undetectable level in the peripheral blood

of HIV patients. However, ART cannot eradicate the

integrated HIV-1 genome from latently infected cells.

This means that ART is not a desirable cure for HIV

infection and necessitates a lifelong dependence on

this therapy (Prevention 2021, HIV treatment

overview 2021). Accordingly, a promising gene-

editing tool, which is CRISPR/CAS9, has been tested

and investigated both in vivo and in vitro to resolve

those problems in the ART treatment regimen.

2 THE CRISPR/CAS9 ADVANCE

IN HIV-1 TREATMENT

2.1 Life Cycle of HIV

Based on the previous findings, two types of HIV

viruses are identified, which are HIV-1 and HIV-2.

They both can give rise to AIDS in human bodies, but

they do have a lot of distinctions regarding

pervasiveness, geographical distribution, and

lethality. Specifically, HIV-1 is the most common

HIV seen in clinical practices, and it is more fatal and

spreading more quickly than HIV-2. Therefore, HIV-

1 has been regarded as a focal point for the treatment

of AIDS. Here, all the materials and discussion

centers around it. Below is the shared mechanism by

which both HIV-1 and HIV-2 make their invasion

come true.

Here, as shown in Figure 1, In the first stage, HIV

normally enters the human body, and it strives to flow

around CD4 T lymphocytes (CD4 + cells) and

prepares for an invasion by binding its glycoprotein

120 (gp120) to the CD4 receptor on the surface of

CD4 cells, and subsequently to the co-receptors C-C

chemokine receptor type 5 (CCR5) or C-X-C

chemokine receptor type 4 (CXCR4). In the second

stage, the binding from the first stage normally gives

rise to a fusion between HIV and cell membrane,

which provides easy access for HIV to enter and

release its viral RNA. In the third stage, HIV viral

RNA was converted into double-stranded DNA

(dsDNA) with the help of reverse transcriptase. This

stage is also named after reverse transcription. The

conversion of HIV RNA to HIV DNA makes HIV

genetic material penetrate easily into the nucleus of

CD4 cells, and subsequently, HIV DNA goes through

the fourth stage where it employs integrase to embed

its viral DNA into the genome of CD4 cells. Then,

new HIV RNA, which is derived from proviral HIV

DNA, can serve as genomic RNA to manufacture

viral proteins. In the fifth stage, those viral proteins

will aggregate and fuse with HIV viral RNA, and

then they come to the cell surface to generate

immature (noninfectious) HIV particles. Then, it

comes to the last stage of its life cycle, which is

budding. During budding, immature HIV particles

are released out of CD4 cells, and simultaneously it

releases a type of HIV protease, which will generate

several mature (infectious) viruses by breaking the

long protein chains of those immature HIV particles.

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Figure 1: Life Cycle of HIV.

2.2

CRISPR/Cas9 Technology

On earth, to defend against the outside attack from

bacterial and archaeal viruses, prokaryotes have

developed a new defensive immune system clustered

regularly interspaced short palindromic repeats &

CRISPR-associated proteins (CRISPR-Cas). Given

its viral genomic editing ability, such a defense

system caught the attention of the scientific

community, especially the pharmaceutical and

medical field. Its biological value is highly evaluated

not only because of its adaptive nature, but also its

therapeutic potential. In medical applications, it

serves as a gene-editing tool by recognizing and

cleaving foreign RNA or DNA in a targeted

sequencing manner. This defense system consists of

three stages, which are adaptation, crRNA biogenesis,

and target interference, respectively.

Figure 2: CRISPR/Cas9 Technology in Genomic Editing of HIV-1.

Advance in Genome Editing of HIV-1 through CRISPR Technique

1133

As shown in Figure 2, there are three essential

stages CRISPR systems must go through, which are

spacer integration (adaptation), expression, and

interference. In the first stage, the bacteriophage

invades prokaryotic cells and releases its viral gene.

To defend against such an attack, prokaryotic cells

give that bacteriophage permission to get access to its

DNA sequence, and simultaneously cell itself

integrates such an invading nucleic acid into its DNA

sequence. Such a foreign viral gene is normally

derived from a phage of plasmid DNA, which is

called a spacer. By structure, CRISPR is comprised

of arrays of short repeating units interspaced by

spacers. The acquisition of foreign spacer paves a

way for prokaryotic cells in further defense against

invasion by phages or plasmids carrying similar

sequences.

In the expression stage, the CRISPR arrays,

which are generated from the spacer acquisition stage,

turn themselves into long primary transcripts via

transcription. Those transcripts are precursor crRNAs

(pre-crRNAs), which play a vital role in subsequent

stages. Subsequently, Pre-crRNA matures into

crRNA by virtue of Cas9 proteins, tracrRNA, and

RNase III. TracrRNA is a trans-encoded small RNA

base-paired with the repeating region located on the

Pre-crRNA.

Then, in the interference stage, an effector

complex is formed by binding of Cas9 proteins into a

single guide RNA (sgRNA), which is formed by the

linkage between tracrRNA and targeting crRNA. The

formation of this effector complex can directly

initiate and control target DNA cleavage in the

Protospacer adjacent motifs (PAMs). Such cleavage

is directed by two essential domains within Cas9

nuclease, which are the histidine-asparagine-

histidine (HNH) domain, and RuvC domain,

respectively. Target DNA strand, which matches the

base pairs with sgRNA, is normally cleaved by the

HNH domain, while the non-target DNA strand is

broken apart by the RuvC domain. Such a cleavage

will give rise to double-stranded DNA break (DSB),

which is subsequently going to be repaired by non-

homologous end-joining (NHEJ) without any

homologous template or homology-directed repair

(HDR).

2.3 CRISPR/CAS9 Technique in

Genome Editing of HIV-1

Currently, ART is still being used as a major

treatment regimen for HIV-1/AIDS patients in the

clinic. However, it cannot easily eradicate latent viral

reservoirs in HIV-1 patients. To achieve this, more

and more attention was paid to recently popular new

genome editing technology, which is the CRISPR

system. Such a functional system, along with

nuclease 9 (Cas9), is engineered to target certain

genomic regions in the HIV genome or cellular co-

factors, which can give rise to a big reduction in HIV

infection and an elimination of internal viral genes.

Based on the life cycle of HIV-1, there are two

pathways to reach our goal. The first one is to directly

target the HIV-1 genome to inactivate and eliminate

the HIV provirus. The second one is to disrupt co-

receptors CCR5 and CXCR4 by CRISPR/Cas9

technology. Tests or assays have been conducted to

verify the feasibility and effectiveness of

CRISPR/Cas9 in the suppression of HIV-1

expression and elimination of provirus within the

host cell. The CRISPR/Cas9-based approach was

first tested in HIV-1/AIDS treatment in 2013 (Ebina

et al. 2013). Their test results revealed that the

CRISPR/Cas9 system successfully targets HIV long

terminal repeat (LTR) and then suppresses the

expression of HIV genes. The target sites in their tests

were the NV-kB binding cassettes and TAE

sequences, which were situated in the U3 region of

LTR and R region, respectively. By targeting those

sites, HIV-1 provirus transcription and replication

were effectively restrained (Ebina et al. 2013). Most

importantly, the ability of CRISPR/Cas9 to eliminate

internal integrated provirus from the host cell genome

was proved. Such a desired ability is not something

we cannot see from ART treatment. The HIV-1

patients treated with ART mostly have a risk of HIV-

1 rebound since it cannot eradicate the latent provirus

inside the host cells. That is why such a functional

elimination of internal viral genes is really valued in

the clinic. Soon afterwards, Wenhui Hu and his

colleagues conducted another research to apply

CRISPR/Cas9 based approach to excise genome of

HIV-1, and they succeeded in getting some good

results showing that inactivating viral gene

expression and suppression of viral replication in a

HIV-1 latently infected T cell line, microglial cell

line with minor genotoxicity, pro-monocytic cell line,

and no detectable level of off target genomic editing,

was achieved (Hu et al. 2014). In their research, they

designed four different LTR Guide RNA (gRNA) to

separately test out their specificity and efficacy in

editing HIV-target genome. It turned out all those

gRNA worked well with different cell lines during

the assay, but not all of them were functional, which

indicates their individual specificity in genomic

editing.

Later, Liao Hsin-Kai and his group members also

selected several potential gRNA target sites located

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

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inside the genome of HIV-1, and they identified

optimal target sites that can provide the human body

with long-term and effective protection against HIV-

1. The host cells they used here were primary T cells

and human pluripotent stem cells (hPSC). By

analyzing mean fluorescence intensity (MFI) of

enhanced green fluorescent protein (EGFP) via

FACS analysis and the percentage of green

fluorescence protein (GFP) cells, it indicated

targeting LTR sequences in the R region was more

efficient than the other gRNA target sites (Liao et al.

2015). Then, they experimentally concluded that

different targeting/disruption strategies and

efficiencies in different stages (pre-integration and

provirus) and different targeting sites as well (coding

and non-coding regions) (Liao et al. 2015).

When double-stranded breaks (DSBs) happen,

exonucleases inside host cells are capable to degrade

the viral genome near the DSBs, and the Coding

region in the genome of HIV-1 was targeted and its

viral genes were disrupted via non-homologous end

joining (NHEJ), which includes replacement,

insertion, and deletion. On the contrary, the structural

disruption can be triggered in a pre-integration stage

when targeting non-coding regions like LTR regions

Most importantly, they found out multiplexed

CRISPR/Cas9 systems, which involve different

gRNAs, can increase the level of disruption and

excision of the pre-integrated proviral genome.

Additionally, targeting multiple conserved sites

instead of single one can effectively disrupt the

genome of HIV-1 while clearing the concern about

HIV-1 variants forming a resistance to singly guided

CRISPR/Cas9 (Liao et al. 2015).

Besides directly targeting the genome of the HIV-

1 virus, CRISPR/Cas9 can also serve as a useful tool

to prevent HIV-1 from invading primary T cells or

other cell lines by editing of co-receptor, which are

CCR5 and CXCR4. As stated above, HIV-1 makes

its entry into CD4 T lymphocytes by binding to the

CD4 receptor, as well as CCR5 and CXCR4. If either

CCR5 or CXCR4 was genetically modified or

engineered, a negative impact on entry of HIV-1 will

be expected. In the research of Liu et al, they

designed two types of sgRNAs, which served as a

tool to target CXCR4 and CCR5 at the same time.

What they were trying to do was apply CRISPR-

sgRNAs-Cas9 to trigger the genomic editing of

CXCR4 and CCR5 in different cell lines including

CD4 T cells (Liu et al. 2017). Then, by off-target and

apoptosis assays to figure out if such editing can

result in any non-specific editing or cytotoxic effect

on cell viability. The final result turned out to be very

impressive because both CXCR4 modified and

CCR5 modified cells exhibited a selective advantage

over unmodified cells during HIV-1 infection. Most

importantly, there weren’t any nonspecific editing or

cytotoxic symptoms, which concluded that

CRISPR/Cas9 based approach in editing both co-

receptors are quite safe and effective to suppress X4-

or/and R5-tropic HIV-1 infection (Liu et al. 2017).

Overall, the CRISPR/Cas9 systems have an

apparent advantage over the other gene-editing

techniques, such as ZFN and TALEN. CRISPR/Cas9

can change the cleavage target by simply changing

the gRNA sequence. ZFN and TALEN systems need

a redesigned protein binding domain to make

changing of cleavage target sequences occur (Gaj et

al. 2012, Khalili et al. 2017, Ousterout et al. 2016).

Such an advantage makes CRISPR/CAS9 impressive

from a design perspective. Besides, Yin Chaoran et al

demonstrated the effective excision of HIV-1

proviral DNA from the host-host genome in pre-

clinical animal models using saCas9 and multiplex

sgRNAs by AAV-DJ/8 vector (Yin et al. 2017). Their

research further supports the application of

CRISPR/CAS9 in HIV treatment by animal model

validation, which really lowers the sounds of those

skeptics.

Despite the promising aspects of genomic editing

of CRISPR/CAS9, challenges still exist. At present,

one major concern is probably the off-target effect,

which may give rise to genetic mutation or chromosol

translocation. If CRISPR/Cas9 enters the clinic field,

the reduction of the off-target effect would be

necessary and urgent. Some researchers have already

proved that the off-target effect found in

CRISPR/Cas9 system was very limited compared

with other editing techniques such as TALENs, ZFNs,

or homing endonucleases by ChIP-seq (Duan et al.

2014). However, improvement is still needed for

CRISPR/Cas9 to eliminate mismatches as many as

possible.

Another challenge would be what kind of vectors

should be utilized to deliver CRISPR/Cas9. As we

know, CRISPR/Cas9 system is normally introduced

into host cells through transfection. Normally, a

successful transfection does need an appropriate

delivering vector. The main vector being used

consists of lentiviral, adenoviral, and adeno-

associated viral vectors. Of those vectors, lentiviral

vectors are widely utilized to deliver CRISPR

systems with high efficiency and stable expression,

but simultaneously the chances of off-target effect

can be even higher (Wang et al. 2014). It is apparent

that finding a vector with less off-target would be a

big concern we should always keep in mind.

Advance in Genome Editing of HIV-1 through CRISPR Technique

1135

Also, HIV-genetic variation is highly likely to

badly influence the efficacy of a CRISPR/Cas9 based

treatment regimen. To resolve this problem,

researchers designed dual-gRNA/Cas9 strategy, and

their results demonstrated that although such a

strategy can cure T cells infected by distinct HIV-1

isolates, the efficacy of this strategy is really

compromised by sequence variation of the target sites

in HIV-1 (Darcis et al. 2014). Later on, a quadruplex

sgRNAs/saCas9 strategy was applied and tested. The

results demonstrated maximization of the possibility

of multiple indel mutations on six on-target sites and

fragmental deletions among these sites Furthermore,

this strategy can offer additional advantages, such as

reducing the potential of HIV-1 escape (Wang et al.

2016), the high possibility of HIV-1 excision despite

the continuous proviral mutation in the clinical HIV-

1 patient population, a reliable loss-of-function

achievement due to removal of a substantial portion

of the target gene or genome (Bauer et al. 2015), and

optimal efficiency of excision (Yin et al. 2016).

3 CONCLUSIONS

Currently, ART is still being used as the major

treatment regimen for HIV-1/AIDS patients in the

clinic. By the long-term effect of ART, there would

be an obvious decrease in HIV-1 expression and

symptoms can be alleviated to an undetectable level.

However, meanwhile, a lot of side effects, such as

appetite loss, or lipodystrophy, become a big concern.

Although there has been a lot of improvement in ART

to minimize its possible side effects, some severe side

effects are still out there. Additionally, ART does

require a long-term intake regularly. If patients forget

or skip one of the doses of the day or the week, the

HIV-1 virus would take full advantage of this chance

to replicate and copy itself in their bodies again. At

worst, it could result in drug-resistant, and then there

would be few feasible HIV-1 treatment regimens for

HIV patients to choose from. Hence, genomic editing

technologies start to get more and more attention due

to their potential in the suppression of HIV-1 viral

gene and elimination of integrated HIV-1 viral gene

within CD4 lymphocytes. CRISPR/Cas9 is one of

them and the one with no cytotoxic effect and less

off-target effect. However, there is still a long way

ahead until this technique can be fully applied to the

clinic field. Off-target and delivery vectors need

more time and effort to deal with and eventually this

technique is going to be more mature and feasible to

be clinically qualified enough in the near future.

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