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CRISPR-Cas based strategies against HIV-1

Wang, G.

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Citation for published version (APA):Wang, G. (2018). CRISPR-Cas based strategies against HIV-1.

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Gang Wang


ISBN: 978-94-6299-816-2

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CRISPR-CAS BASED STRATEGIES AGAINST HIV-1

ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad van doctor aan de

Universiteit van Amsterdam

op gezag van de Rector Magnificus

prof. dr. ir. K.I.J. Maex

ten overstaan van een door het College voor Promoties ingestelde commissie,

in het openbaar te verdedigen in de Agnietenkapel

op donderdag 18 januari 2018, te 12:00 uur

door Gang Wang

geboren te Shaanxi, China

Promotiecommissie:

Promotor: Prof. dr. B. Berkhout AMC-UvA Copromotor: Dr. A.T. Das AMC-UvA

Overige leden: Prof. dr. J. Ananworanich AMC-UvA Prof. dr. V.M. Christoffels AMC-UvA Dr. P. Konstantinova uniQure Prof. dr. R.W. Sanders AMC-UvA Prof. dr. E.J.H.J. Wiertz Universiteit Utrecht Prof. dr. H.L. Zaaijer AMC-UvA

Faculteit der Geneeskunde

Table of contents

Chapter 1 General Introduction 7

Chapter 2 CRISPR-Cas9 can inhibit HIV-1 replication but NHEJ 18

repair facilitates virus escape

Chapter 3 Strategies to prevent NHEJ-mediated HIV-1 escape from 41

CRISPR-Cas9 attack

Chapter 4 A combinatorial CRISPR-Cas9 attack on HIV-1 DNA extinguishes 47

all infectious provirus in infected T cell cultures

Chapter 5 Combinatorial CRISPR-Cas9 and RNAi attack on HIV-1 DNA 74

and RNA can lead to cross-resistance

Chapter 6 CRISPR-Cas based antiviral strategies against HIV-1 91

Chapter 7 Addendum 121

Summary 122

Samenvatting 125

Acknowledgments 128

PhD Portfolio 129

List of Publications 131

132

133

Author Affiliations

Curriculum Vitae

Words of Thanks 134

Chapter 1

General Introduction

Scope of this thesis

7

HIV and AIDS

The lentivirus HIV-1 is the causative agent of acquired immunodeficiency syndrome (AIDS)1.

Over the last few decades, many advances have been made in the treatment of this chronic

virus infection. A combination of several drugs, known as combined Antiretroviral Therapy

(cART), is able to keep HIV-1 replication under control and to stop disease progression, but a

cure is never reached. One of the reasons why HIV-1 is so difficult to cure is that the DNA

genome of this virus, which is called the provirus, can be present within the host genome in a

dormant state, which remains invisible to the immune system. If therapy is discontinued, such

latently infected cells can produce infectious virus particles and reignite the spreading infection2.

Thus, novel anti-HIV strategies are urgently needed to either eradicate or inactivate the

integrated HIV genome in the latent reservoir. Engineered nucleases including zinc finger

nuclease (ZFN)3 and transcription activator-like effector nuclease (TALEN)4, 5 have been

designed to eradicate the integrated HIV DNA. In this thesis, I used the new genome editing

system CRISPR-Cas9 to attack the HIV genome.

The HIV genome

HIV-1 virus particles carry two identical 9.2 kb single-stranded RNA molecules, whereas the

persistent form of the HIV-1 genome is proviral double-stranded DNA integrated into a

chromosome of the infected cells (Fig. 1). The HIV-1 genome encodes nine genes, which can

be subdivided into three classes according to the function of the encoded proteins: structural

proteins (gag, pol and env genes), regulatory proteins (tat and rev genes) and accessory

proteins (vif, vpr, vpu and nef genes). To produce the full range of viral proteins, the HIV-1

primary transcript undergoes extensive and complex alternative splicing 6, 7. In the early phase of

HIV-1 gene expression, HIV-1 produces only short multiply spliced mRNAs encoding Tat, Rev

and Nef proteins. When the Tat and Rev proteins accumulate, transcription increases sharply,

and singly and unspliced mRNAs are produced. The singly spliced mRNAs express Env, Vif, Vpr,

and Vpu, whereas the full-length unspliced transcripts act both as virion genomic RNA and as

mRNA for synthesis of the Gag and Gag-Pol polyprotein.

Common to all retroviruses, HIV-1 encodes three core proteins: Gag, Pol and Env proteins8, 9.

The gag gene codes for the Gag precursor protein that plays a major role in building the virus

particle structure. Gag is processed by the viral protease into matrix (MA) , capsid ( CA) and

nucleocapsid (NC) during virus maturation. The env gene codes for the gp160 envelope (Env)

protein that is cleaved into gp120 and gp41 by the host cell protease Furin. Trimers of these Env

8

proteins are exposed on the viral membrane and enable HIV-1 to attach to specific cell types.

The pol gene codes for the polymerase precursor protein (Pol) that is processed into the viral

enzymes reverse transcriptase (RT) , integrase (IN) and protease (PR), which are encapsulated

within the virus particle. HIV also expresses two proteins (Tat and Rev) that regulate viral gene

expression in infected cells10, 11. The Tat protein functions in a positive feedback loop and

activates viral gene expression from the viral LTR promoter, while the Rev protein activates

export of the unspliced and single-spliced viral transcripts to the cytoplasm, thus preventing

complete splicing of the transcripts. Late in the replication cycle, most of the viral RNA is either

unspliced or singly spliced due to the action of the Rev protein, which favors the production of

the Gag, Pol and Env proteins and viral RNA genomes required for the assembly of progeny

virus.

HIV accessory proteins (vif, vpr, vpu and nef) are not absolutely required for viral infection in

vitro, but these proteins contribute to in vivo replication and pathogenesis 12, 13. For example, the

HIV Nef protein uses clathrin adaptors to evade cytotoxic T lymphocytes (CTLs) and promote

viral spread. HIV-1 Vif, Vpu, and Vpr proteins adapt cellular ubiquitin ligase adaptors to

counteract host antiviral responses.

Fig. 1. Map of the HIV-1 DNA genome. The HIV-1 proviral DNA with nine open reading frames and the 5’

and 3’ LTRs. Each LTR consists of a U5 (unique at 5’ end of HIV-1 RNA), R (repeated at 5’ and 3’ ends of

RNA) and U3 (unique at 3’ end of RNA) domains.

Apart from the protein-coding region, the HIV-1 genome contains important non-protein coding

domains. The Long Terminal Repeat (LTR) is present both at the 5’ and 3’ end of the proviral

DNA genome. Once the viral genome has been integrated into the host genome, the 5’ LTR

serves as the promoter for transcription of the HIV genome, while the 3’ LTR encodes the signal

for RNA polyadenylation, but it encodes part of the Nef protein as well 14. The upstream LTR

region contains core, enhancer and modulatory promoter elements. The sequence of the

transactivating region (TAR) that binds the viral Tat protein is located in the repeat (R) region15.

5' LTR vpr pol

rev

vpu env

tat gag vif nef

3' LTR

HIV DNA

9

Fig. 2. Mechanism of the CRISPR-Cas system in adaptive immunity. The various elements

that constitute CRISPR-Cas system are graphically depicted, including Cas genes, spacers and repeats .

CRISPR-Cas as adaptive immune system

The clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-

associated (Cas) proteins are known as adaptive immune system of bacteria and archaea that

protects against invasion by viruses and foreign plasmids 16, 17. CRISPR-Cas immune systems

proceed in three distinct steps: spacer acquisition, CRISPR RNA (crRNA) biogenesis, and target

interference (extensively reviewed in 18-22) (Fig. 2). Following invasion of the bacterial cell by

foreign genetic elements, certain Cas enzymes acquire new spacers from the exogenous nucleic

acid and install them into the CRISPR locus (step 1). These spacers are separated by repeat

sequences within a CRISPR locus. CRISPR arrays are transcribed and enzymatically maturated

through distinct pathways that are unique to each type of CRISPR system (step 2). In type II

CRISPR system, an associated trans-activating CRISPR RNA (tracrRNA) hybridizes with the

Transcription

Maturation

Repeat

Spacer

Cas genes

Spacer acquisition

crRNA biogenesis

Target interference Cas/crRNA+tracrRNA

Pre-crRNA

crRNA

Phage

10

repeats, forming an RNA duplex that is cleaved and processed by endogenous nucleases. The

mature RNAs then form a ribonucleoprotein complex with Cas proteins to mediate specific

cleavage and inactivation of homologous sequences (step 3).

Application of CRISPR-Cas systems in eukaryotic cells

The CRISPR-Cas system has been optimized for its application in mammalian cells, including

optimization of the codon usage of the Cas gene23 and the generation of a single chimeric guide

RNA (gRNA) by fusion of the crRNA and tracrRNA24. CRISPR-Cas systems can be subdivided

in two classes based on differences in the RNA-guided nuclease effectors: class 1 (type I, III, IV)

and class 2 (type II, V, VI). In class 1 systems, the effector consists of a multi-protein complex,

whereas class 2 systems rely on single-component effector proteins such as the well-

characterized Cas9 (type II) and Cpf1 proteins (type V) 25, 26. The class 2 systems are therefore

more attractive for genome editing applications.

Fig. 3. The CRISPR/Cas9 system. The widely used CRISPR-Cas9 system utilizes a gRNA, which is a

fusion between the crRNA and tracrRNA elements. Cas9 (green shape) complexes with gRNA to mediate

cleavage of target DNA sites that are complementary to the 5’ 20 nt of the gRNA and that lie next to a

PAM sequence (red line).

CRISPR-Cas systems form RNA-guided sequence-specific endonuclease complexes that bind

and cleave double stranded DNA (dsDNA)23, 24, 27. The popular Cas9 system that we used in this

thesis has been adapted from Streptococcus pyogenes (SpCas9) (Fig. 3). Cas9 nucleases can

be directed by gRNA to induce precise cleavage at endogenous genomic loci in mammalian

5’

3’

Target DNA PAM

Protospacer

gRNA

20nt

Seed region

11

cells. The 20 nucleotides (nt) guide sequence of the guide RNA (gRNA) is designed to be

complementary to the target DNA site, which also needs to contain a sequence called the

protospacer adjacent motif (PAM) immediately downstream of the target site (typically NGG for

SpCas9, see Fig. 3). DNA cleavage is executed by two distinct nuclease domains of Cas9: HNH

and RuvC. The HNH domain cleaves the complementary strand, whereas the RuvC domain

cleaves the non-complementary strand24, 28. The cleavage site is located 3 nt away from the

terminal end of the protospacer adjacent to the PAM, leaving blunt ends28. In addition, Cas9 can

be catalytically inactivated (by point mutations D10A and H840A), while maintaining its DNA-

binding specificity24, 28. This "dead Cas9" (dCas9) variant can be fused to transcriptional

activator/repressor domains to modulate gene expression in humans cells 29, 30.

SpCas9 can tolerate mismatches between the gRNA and target DNA, but this will affect the

efficiency of Cas9 cleavage23, 24, 31-33. Perfect complementarity between the gRNA and target

DNA is required in the 12 nt window adjacent to the PAM (defined as seed region34).

Mismatches are tolerated to some extent at the non-PAM end (5’ end of the guide RNA), but the

efficiency of DNA cleavage will be affected.

Targeting of HIV-1 with the CRISPR-Cas9/dCas9 systems

The CRISPR-Cas9 and dCas9 systems have been extensively employed in anti-HIV strategies

because of their high efficiency, flexibility and sequence-specificity (Fig. 4). First, CRISPR-Cas9

has been used to target nonintegrated HIV-1 DNA or the integrated provirus for cleavage 35-38.

Second, the CRISPR-Cas9 has been used to target and inactivate the cellular genes encoding

the HIV co-receptor CCR5 and/or CXCR4 39-42. Third, the CRISPR-dCas9 system can be used to

transcriptionally activate or repress the integrated HIV provirus in infected cells 43-47. In chapters 2 to 5, we focused on the first approach using CRISPR-Cas9 to target HIV. In addition, we provide an extensive review of CRISPR-Cas9 and dCas9 based approaches against HIV in

chapter 6.

DNA repair

Double-stranded breaks (DSB) in the cellular DNA occur accidentally during normal cell

metabolism or as essential intermediate during programmed recombination events. DSBs can

also be induced by exposure of cells to exogenous agents like chemotherapeutic drugs and

engineered nucleases such as ZNF, TALEN and CRISPR-Cas. DSBs threaten the genomic

integrity and can result in cell death if left unrepaired or repaired inappropriately. Two major DSB

12

repair pathways have been defined: homologous recombination (HR) and non-homologous end

joining (NHEJ) 48-51. HR refers to mechanisms in which an intact homologous donor duplex is

required to guide DNA synthesis across the DSB gap. HR can thus restore any lost sequence

information and result in accurate repair. NHEJ is a repair mechanism in which two DSB ends

are joined by direct ligation. NHEJ is recognized as having a high potential for errors introduced

around the DSB ends. The choice between these two pathways depends on the nature of the

DSB ends, the phase of the cell cycle and the presence of a homologous donor sequence.

DSBs introduced by CRISPR-Cas9 in the HIV provirus should be repaired by NHEJ as there is

no donor sequence for HR repair, which indeed has been observed 35-37.

Fig. 4. CRISPR-Cas9/dCas9 used to target HIV. The HIV particle contains two genomic RNA copies.

The Env protein exposed at the viral membrane mediates attachment to the CD4 receptor and the

CCR5/CXCR4 co-receptor of target T cells. Upon membrane fusion and virus entry, the viral RNA genome

is reverse transcribed into DNA with a complete LTR at both ends. Upon integration into the cellular

genome, this proviral DNA can be transcribed by the cellular RNA polymerase II transcription complex.

RNA transcripts are processed by the cellular capping, polyadenylation and splicing machinery and

subsequently translated. Genomic RNA dimers are packaged into new virus particles that assemble and

bud at the cellular membrane. CRISPR-Cas9 nuclease can target and cleave the dsDNA that is formed

upon reverse transcription of the viral RNA, but also the integrated proviral DNA. The integrated provirus

13

can also be targeted with CRISPR-dCas9-based transcriptional activators and repressors to either purge

virus production in these cells or to silence viral gene expression. The cellular genes encoding the CCR5

or CXCR4 co-receptor can be targeted by CRISPR-Cas9 to block HIV entry.

Scope of this thesis

In this thesis we focus on CRISPR-Cas based strategies to inhibit HIV replication and to

eradicate all HIV proviruses. In chapter 2, we employed the CRISPR-Cas9 system to target HIV. We demonstrated robust HIV-1 inhibition, but HIV could escape from this sequence-specific

attack via a new molecular mechanism: mutations introduced by the NHEJ DNA repair

machinery. We subsequently tried to prevent HIV escape by different approaches in chapter 3. First, we used the NHEJ inhibitor SCR7 to see if NHEJ-assisted escape could be prevented.

Second, the new CRISPR-Cpf1 nuclease was tested because of its property to cleave the DNA

relatively far away from the PAM, outside the seed region, which may prevent easy virus escape.

In chapter 4, a combinatorial therapy with two gRNAs was designed to prevent virus escape. The use of two gRNAs in combination improved the efficiency of virus inhibition, but viral escape

was still observed for most gRNA combinations. However, most spectacular was the observation

that two special gRNA combinations could prevent HIV escape in long-term cultures. Even more

dramatically, we described the gradual, but complete inactivation of all infectious HIV in these

infected cultures. Although these results were obtained in a relatively simple T cell culture model,

it forms an important "proof-of-concept" in the field of HIV-1 cure research. Furthermore,

although provirus excision has been proposed as the mode of action of a combinatorial

CRISPR-Cas attack, we described double HIV-1 inactivation by NHEJ-mutations in the two

targets as the major mechanism. In chapter 5 we developed a concerted attack on the viral RNA and DNA genomes with the RNA interference (RNAi) and the CRISPR-Cas9 mechanisms,

respectively. This combination generally improved the efficiency of HIV inhibition, except when

the same viral sequences were attacked at the RNA and DNA level. In that exceptional case,

early NHEJ-assisted CRISPR-Cas escape also facilitated rapid RNAi-escape, thus frustrating

the combination therapy. CRISPR-Cas9 has been used in diverse manners and by many

different groups worldwide to target HIV-1. For instance, the catalytically inactive dCas9 fused to

an activation or repressor domain was used to reactivate or repress HIV transcription,

respectively. We reviewed this booming research field in chapter 6.

14

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22. Barrangou, R. CRISPR-Cas systems and RNA-guided interference. Wiley Interdiscip Rev RNA 4,267-278 (2013).

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41. Wang, W. et al. CCR5 gene disruption via lentiviral vectors expressing Cas9 and single guidedRNA renders cells resistant to HIV-1 infection. PLoS One 9, e115987 (2014).

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43. Bialek, J.K. et al. Targeted HIV-1 Latency Reversal Using CRISPR/Cas9-Derived TranscriptionalActivator Systems. Plos One 11 (2016).

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44. Ji, H. et al. Specific Reactivation of Latent HIV-1 by dCas9-SunTag-VP64-mediated Guide RNATargeting the HIV-1 Promoter. Mol Ther 24, 508-521 (2016).

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46. Saayman, S.M. et al. Potent and Targeted Activation of Latent HIV-1 Using the CRISPR/dCas9Activator Complex. Mol Ther (2015).

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Chapter 2

CRISPR-Cas9 can inhibit HIV-1 replication but NHEJ repair facilitates virus escape

Gang Wang, Na Zhao, Ben Berkhout* and Atze T Das*

Molecular Therapy (2016); 24 (3): 522–526

18

Abstract

Several recent studies demonstrated that the CRISPR-associated endonuclease Cas9 can be

used for guide RNA (gRNA)-directed, sequence-specific cleavage of HIV proviral DNA in

infected cells. We here demonstrate profound inhibition of HIV-1 replication by harnessing T

cells with Cas9 and antiviral gRNAs. However, the virus rapidly and consistently escaped from

this inhibition. Sequencing of the HIV-1 escape variants revealed nucleotide insertions, deletions

and substitutions around the Cas9/gRNA cleavage site that are typical for DNA repair by the

NHEJ pathway. We thus demonstrate the potency of CRISPR-Cas9 as an antiviral approach,

but any therapeutic strategy should consider the viral escape implications.

Keywords: CRISPR, Cas9, HIV-1, escape, NHEJ

Introduction

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system

represents a versatile tool for genome engineering by enabling the induction of double-stranded

breaks (DSBs) at specific sites in DNA 1. Sequence specificity is due to the gRNA that directs

Cas9 to the complementary sequence present immediately upstream of a 3-nt protospacer

adjacent motif (PAM) in the target DNA. In mammalian cells, the DSBs can be repaired by the

non-homologous end-joining (NHEJ) pathway, which results in the frequent introduction of

insertions, deletions and nucleotide substitutions at the cleavage site, or by homology-directed

repair, which depends on the presence of homologous DNA sequences 1, 2.

Several studies demonstrated that the Cas9/gRNA system can be used for inhibition of human

pathogenic DNA viruses, including hepatitis B virus 3-8, Epstein-Barr virus 9, and human

papilloma virus 10. Replication of retroviruses, like HIV-1, can also be inhibited with the

Cas9/gRNA system by targeting the reverse transcribed HIV-1 DNA replication intermediate or

the proviral DNA upon integration into the cellular genome 2, 11-13. Gene therapy approaches for

the treatment of HIV-1 infected individuals have been proposed in which the Cas9 and antiviral

gRNAs are directed to HIV-1 infected cells to inactivate or delete the integrated provirus, or in

which blood stem cells are harnessed against new infections. However, Cas9/gRNA-mediated

inhibition of virus production and/or replication has been shown only in short term experiments,

19

while we know that HIV-1 can escape from most if not all types of inhibitors, including small

molecule antiviral drugs and sequence-specific attack by RNA interference (RNAi). We therefore

set out to identify viral escape strategies from Cas9/gRNA mediated inhibition.

Results and discussion

Design of gRNAs that effectively target the HIV-1 DNA genome

In silico algorithms were used to select 19 gRNAs that should target HIV-1 DNA with high

efficiency and exhibit no off-target effects on cellular DNA (Table S1). Seven gRNAs were

selected that target the long terminal repeat (LTR) region present at the 5’ and 3’ ends of the

proviral genome (Fig. 1a). Five of these (gLTR1-5) also target the accessory nef gene that

overlaps the 3’ LTR, but that is not essential for in vitro virus replication. Twelve gRNAs target

sequences that encode other viral proteins, including well-conserved domains in the essential

gag, pol and env genes and sequences of overlapping reading frames, like the tat and rev genes

(Fig. 1a). Nine selected gRNAs target sequences that are highly conserved among different HIV-

1 isolates (Shannon entropy

Figure 1. Cas9/gRNA targeting of the HIV-1 genome. (A) The HIV-1 proviral DNA with the position of

gRNAs tested in this study. (B) The efficiency of gRNAs to silence HIV-1 DNA was tested in 293T cells

transfected with plasmids expressing Cas9, gRNA and HIV-1 LAI. To quantify viral gene expression, the

viral capsid protein (CA-p24) was measured in the culture supernatant at 2 days after transfection.

Average values (± SD) of 4 experiments are shown. Statistical analysis (independent-samples t-test

analysis) demonstrated that CA-p24 expression in the presence of antiviral gRNAs differed significantly

from values measured with control gRNAs against luciferase and GFP (*, P < 0.05).

Inhibition of HIV-1 replication by the Cas9/gRNA system

SupT1 T cells were first transduced with a Cas9-expressing lentiviral vector. Stably transduced

cells were selected and subsequently transduced with a lentiviral vector expressing one of the

antiviral gRNAs. Of note, none of the selected gRNAs target the lentiviral vectors. Upon infection

of transduced cells with the HIV-1 LAI isolate, virus replication was monitored by measuring the

21

CA-p24 level in the culture supernatant. Efficient virus replication was apparent in control non-

transduced SupT1 cells and in Cas9-only transduced cells, as reflected by a rapid increase in

the CA-p24 level (Fig. 2a) and the appearance of large virus-induced syncytia and cell death

around day 10 after infection (Fig. 2b; average time of HIV-1 breakthrough replication of 4

experiments are shown). HIV-1 replication in cells transduced with Cas9 and gRNAs targeting

poorly conserved LTR sequences (gLTR1-6) was only marginally delayed (Fig. 2a and data not

shown) and breakthrough replication resulting in large syncytia was observed at 12-14 days (Fig.

2b). Replication in cells transduced with Cas9 and gLTR7, which targets the highly conserved

and essential TATA-box region of the LTR promoter, was more delayed and resulted in

breakthrough replication at 19 days. A similar split was observed when targeting protein-coding

regions. Targeting highly conserved HIV-1 sequences (gGag1, gGagPol, gPol1-4, gTatRev and

gEnv2) exhibits a more sustained antiviral effect (breakthrough replication in 20-43 days; Fig.

2b) than targeting less conserved domains (gGag2, gVpr, gEnv1 and gNef; breakthrough

replication in 11-17 days; Fig. 2b). Surprisingly, despite their potency to suppress virus

production (Fig. 1b), some of the gRNAs inhibited virus replication only briefly and none

prevented breakthrough virus replication. Moreover, the time required for breakthrough

replication did not correlate with the potency of inhibiting HIV-1 production in 293T cells (Fig.

S1).

The breakthrough viruses could represent viral escape variants that are no longer suppressed

by the Cas9/gRNA system. Interestingly, the time required for breakthrough virus replication was

longer for target sequences that are more conserved (Fig. 2c: inverse correlation between the

day of breakthrough replication and the Shannon entropy). Along these lines, the early escape

observed for the gRNAs targeting non-conserved domains could be explained by the many

escape options that are available to the virus, whereas the relatively late escape observed for

gRNAs targeting conserved domains could be due to the fewer escape options because

important sequences are targeted. Nevertheless, the poor inhibition and very swift viral escape

observed for some of the gRNAs is remarkable, as the evolutionary process underlying viral

escape, that is the generation of sequence variation and subsequent outgrowth of variants with

improved fitness, usually takes several weeks or even months, e.g. for RNAi inhibitors tested in

the same experimental system 14.

22

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gGag2 gGag1gVprSupT1gLTR5gLTR1 gLTR7

gPol3

0102030405060

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TR

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TR

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ag1

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)

A

B

C

23

Figure 2. HIV-1 replication in Cas9 and gRNA expressing cells. (A-B) SupT1 cells stably transduced

with Cas9 and gRNA expressing lentiviral vectors were infected with HIV-1 LAI. Virus replication was

monitored by measuring the CA-p24 level in the culture supernatant (A) and by scoring the formation of

virus-induced syncytia (B). The day at which massive syncytia were observed, which reflects breakthrough

virus replication, is indicated. Average values of 4 experiments (± SD) are shown. SupT1, control non-

transduced cells. SupT1-Cas9, cells transduced only with the Cas9 expressing vector. (C) Correlation

between the level of inhibition (day of breakthrough replication; as shown in panel b) and the conservation

of target sequence amongst different HIV-1 isolates (Shannon entropy as shown in Table S1) . The

Pearson’s correlation coefficient was calculated: r = -0.58.

NHEJ-induced mutations around the Cas9 cleavage site cause rapid HIV-1 escape

We first tested whether the breakthrough viruses were indeed resistant to the specific

Cas9/gRNA set by passage onto fresh matching Cas9/gRNA SupT1 cells and control non-

transduced cells. The breakthrough viruses replicated with similar efficiency on both cell lines

(Fig. S2), which confirmed the escape phenotype. Both cell lines were also infected with wild-

type HIV-1 LAI, showing the selective replication block in restricted Cas9/gRNA cells.

We next sequenced the gRNA-target region of breakthrough viruses in multiple independent

cultures. Strikingly, we observed mutations in the target for all escape viruses (Fig. 3 and Fig.

S3). The viruses that escaped rapidly from gRNAs targeting non-conserved LTR domains

(gLTR1-6) frequently acquired deletions (1 to 31 nt in size; in 20 cultures) and insertions (1 to 3

nt; in 6 cultures), and a single culture acquired a point mutation in the target. In contrast, the

gLTR7 resistant viruses that evolved more slowly had acquired substitutions (1 or 2 nt; in 3

cultures, once in combination with a 1-nt deletion) and 1-nt insertions (3 cultures). The gLTR7

target includes the TATA box, which could explain why large deletions are not tolerated. The

occurrence of single nucleotide substitutions at critical target positions confirms the exquisite

sequence specificity of Cas9/gRNA action.

This trend of a differential mutational spectrum between conserved and less conserved targets

was confirmed for the gRNAs that target protein coding regions. We predominantly observed

nucleotide substitutions (1 or 2 nt) in conserved essential genes. Insertions were restricted to the

size of 3-nt, such that a codon is added but the open reading frame is not disrupted. A fair

percentage of the acquired substitutions represent silent codon changes, again suggesting

pressure on the virus to maintain the coding potential. In contrast, nucleotide deletions and

insertions that shift the open reading frames were frequently observed when targeting the less

conserved vpr and nef genes that are not required for HIV-1 replication on SupT1 cells.

24

Figure 3. HIV-1 escapes from Cas9/gRNA inhibition through mutations in the target region. The gRNA target region in breakthrough viruses obtained in 2 to 6 independent HIV-1 cultures on the different

SupT1-Cas9-gRNA cells was sequenced. For every gRNA, the wild-type HIV-1 nucleotide sequence is

shown on top. Codons are boxed in grey if applicable, with the translated amino acid sequence on the

right hand side. The PAM sequence is boxed and the arrowhead indicates the Cas9 cleavage site at

position -3. Nucleotide and amino acid substitutions, insertions and deletions (Δ) are indicated. Data for all

tested gRNAs are shown in Fig. S3.

The position of all observed mutations was plotted relative to the gRNA target (position -1 to -20;

Fig. 4) and 3-nt PAM (position 1 to 3) and we indicated the position of the expected DSBs.

Except for a single point mutation in the PAM region, all mutations cluster around the Cas9

cleavage site at position -3 15-17, suggesting that the escape mutations were generated in the

process of HIV-1 inhibition. More specifically, we propose that Cas9/gRNA inhibits by DNA

cleavage, but subsequent repair by the NHEJ pathway will generate the mutations that provide

viral resistance. The coupled Cas9 cleavage and NHEJ repair explains the immediate HIV-1

escape when non-critical sequences are targeted.

gLTR1 G A T T G G C A G A A C T A C A C A C C A G G G C C A G G G G T C A G A T A T C C A D W Q N Y T P G P G V1 2 3

· · · · ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ · · · · · · · · · · · · · · · · · · A G C · · · · · · · · · · · · · · · · · · · · · · · ·

· * · · · · * · · · · · · S · · · · ·

gLTR5 G A C A G C C G C C T A G C A T T T C A T C A C G T G G C C C G A G A G C T G C A T S R L A F H H V A R E L H1 2 3 4 5 6

· · · · · · · · · · · · · · · G · · · · · · · · · · · · · · · · · · · · · · · · · ·· · · · ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ · · · · · · · · · · · · · · ·· · · · · · · · · · · · · · G · · · · · · · · · · · · · · · · · · · · · · · · · · · ·· · · · · · · · · · · · · · · · ∆ ∆ ∆ ∆ ∆ · · · · · · · · · · · · · · · · · · · · ·· · · · · · · · · · · · · · ∆ ∆ ∆ ∆ · · · · · · · · · · · · · · · · · · · · · · · ·· · · · · · · · · · · · · · C · ∆ ∆ · · · · · · · · · · · · · · · · · · · · · · · ·

· · · · V · · · · · · · ·· P R A A S G V L Q E L L· · · · I S S R G P R A A· · · · S R G P R A A S G· · · · I P W P E R L H P· · · · S S R G P R A A S

gLTR7 G G C G A G C C C T C A G A T G C T G C A T A T A A G C A G C T G C T T T T T G C C T G T1 2 3 4 5 6

· · · · · · · · · · · T · · ∆ · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·· · · · · · · · · · · · C · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·· · · · · · · · · · · · C · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·· · · · · · · · · · · · C · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·· · · · · · · · · · · · A · G · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·· · · · · · · · · · G · C · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

gGag1 G T T A A A A G A G A C C A T C A A T G A G G M L K E T I N E E1 2 3 4 5

· · · · · · · · · · · · · · · · · C C · · · ·· · · · · · · · · · · · · · · · C · · · · · ·· · · · · · · · · · · · · · · · C · · · · · ·· · · · · · · · · · · · · · · · · · · · · A ·· · · · · · · · · · · · · · · A · · · · · · ·

· · · · · · T · ·· · · · · · H · ·· · · · · · H · ·· · · · · · · · ·· · · · · · · · ·

gGag2 G C T A C C A T A A T G A T G C A A A G A G G A T I M M Q R G1 2 3 4

· · · · · · · · · · · · · · · G C · · · · · ·· · · · · · · · · · · · · · A · · · · · · · ·· · · · · · · · · · · · · · · · · G G · · · ·· · · · · · · · · · · · · · · · T T T C · · · · · ·

· · · · · A · ·· · · · I · · ·· · · · · · G ·· · · · · L S · ·

gPol3 G C A T G G G T A C C A G C A C A C A A A G G A W V P A H K G1 2

· · · · · · · · · · · · · · C · · · · · · · ·· · · · · · · · · · · · · · · · · T · · · · ·

· · · · · · · ·· · · · · · · ·

gVpr A G A A T A G G C G T T A C T C A A C A G A G G A G A G C A A G A A A T R I G V T Q Q R R A R N1 2 3 4 5 6

· · · · · · · · · · · · · · · · · · G A C · · · · · · · · · · · · · · · · · · · · · · · · · A ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ · · · · · · · · · · · · · · · · · · · · · · · · · · · · G ∆ · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ · · · · · · · · · · · · · · · · · · · T C C T C · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ∆ ∆ ∆ ∆ ∆ ∆ ∆ · · · · · · · · · · · · · · · · · ·

· · · · · · D · · · · · ·· · E ∆ ∆ ∆ ∆ ∆ ∆ · · ·· · · · · · G G E Q E M· · · · · H ∆ ∆ ∆ ∆ ∆ G· · · · · · S S R G E Q E· · · · R G E Q E M E P

25

Figure 4. HIV-1 escape mutations cluster around the Cas9 cleavage site. The position of all observed

nucleotide deletions, insertions and substitutions (as shown in Fig. 3 and Figure S3) was plotted relative to

the gRNA target sequence (position -1 to -20) and PAM (position 1 to 3). The arrowhead indicates the

Cas9 cleavage site at position -3.

Insertions and deletions form the hallmark of NHEJ action, but such genome changes are not

acceptable in critical HIV-1 sequences, which explains the frequent observation of nucleotide

substitutions in conserved targets. Alternatively, these mutations could have been generated

during the error-prone reverse transcription process during viral replication or by cellular

APOBEC activity, as documented in more standard virus evolution scenarios 18. Regular HIV-1

evolution is dominated by transitions with G-to-A as the predominant mutation. For example, in a

similar virus evolution study with RNAi antivirals 19, 80% of the acquired mutations were

transitions (91 of 113 substitutions; Table S2) and 46 G-to-A changes were scored. A completely

different pattern was observed in this Cas9/gRNA study: only 44% transitions (27 of 62

mutations) and the A-to-C transversion was the most frequent mutation. Together with the

clustering of the mutations around the Cas9 cleavage site and their rapid appearance, these

findings strongly suggest the involvement of the Cas9-NHEJ pathway in the generation of most

and perhaps all escape mutations. On the other hand, we cannot formally exclude that regular

HIV-1 evolution contributed to virus escape, e.g. by creating the G-to-A mutation in the PAM.

0

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deletions

insertions

substitutions

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frequ

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frequ

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26

Implications for antiviral strategies

Taken together, we demonstrate that HIV-1 can be targeted effectively by the Cas9/gRNA

system, but that the coupled NHEJ repair process creates viral escape variants. This results in

immediate escape when non-essential viral sequences are targeted. When conserved protein-

coding HIV-1 sequences are targeted, viral escape can be significantly delayed and the level of

inhibition is comparable to that observed for some antiviral shRNAs targeting conserved HIV-1

domains. Combinations of such potent shRNAs provide durable inhibition of virus replication 20-

22. The CRISPR/Cas9 antiviral strategy may similarly provide a sustained therapeutic effect

when gRNAs targeting highly conserved HIV-1 sequences are applied in a combinatorial mode.

Since Cas9 cleaves the DNA at position -3 and most escape mutations cluster around this

position, this subdomain of the target sequence should be particularly conserved to reduce the

viral escape options. The coupled Cas9-NHEJ cleavage-repair action may also suggest an

alternative anti-viral strategy where mutations are introduced to weaken the fitness of the

persisting virus. We observed rapid mutation-mediated escape when non-essential sequences

were attacked, but even these mutations will likely reduce the viral replicative fitness in vivo,

suggesting the potency of such an attenuation strategy. Alternatively, cellular genes that are

essential for HIV-1 replication, like the genes encoding the CCR5 coreceptor, could be targeted

by Cas/gRNAs. Moreover, these CRISPR/Cas9 strategies could be combined with other antiviral

approaches, either regular antiviral drugs or gene therapy strategies.

Materials and methods

Plasmids

The lentiviral vector LentiCas9-Blast (Addgene plasmid # 52962) containing the human codon-

optimized S. pyogenes Cas9-expression cassette and LentiGuide-Puro (Addgene plasmid #

52963) with gRNA expression cassette were gifts from Feng Zhang 23. Oligonucleotides

encoding the gRNAs were ligated into the BsmB1 site of the LentiGuide-Puro vector. Control

gRNAs targeting the firefly luciferase and EGFP gene 13 were included (Table S3). The plasmid

pLAI encodes the HIV-1 subtype B isolate LAI 24.

Cell culture and transfection

Human embryonic kidney 293T cells and SupT1 T cells were cultured as described previously 25.

293T cells was transfected with 200 ng pLAI, 500 ng LentiCas9-Blast plasmid and 500 ng

LentiGuide-Puro plasmid by calcium phosphate precipitation.

27

Lentiviral vector production and transduction

The lentiviral vector was produced and titrated as previously described 20. Briefly, the vector was

produced by transfection of 293T cells with the lentiviral vector plasmid and packaging plasmids

pSYNGP, pRSV-rev, and pVSV-g with Lipofectamine 2000 (Invitrogen). After transfection, the

medium was replaced with OptiMEM (Invitrogen) and the cells cultured for 48 hr. The lentiviral

vector containing supernatant was filtered (0.45 μm), aliquoted and stored at −80 °C. SupT1

cells (2 × 105 cells in 1 ml culture medium) were transduced with an equal amount of LentiCas9-

Blast (Cas9) virus particles (based on CA-p24) and cultured with 1 ng/ml blasticidin for one week

to select transduced cells. The cells were subsequently transduced with an equal amount of the

different LentiGuide-Puro (gRNA) virus particles and cultured with 1 ng/ml puromycin to select

for dually transduced cells.

HIV-1 infection and evolution

The HIV-1 LAI stock was produced by transfection of 293T cells with the pLAI molecular clone.

Virus production was measured by CA-p24 enzyme-linked immunosorbent assay 26. SupT1 T

cells (2 × 105 cells in 1 ml culture medium) were infected with an equal amount of HIV-1 LAI

virus corresponding to 1 ng CA-p24. Cells were passaged twice a week. Virus spread was

monitored by measuring the CA-p24 production in the culture supernatant and scoring the

formation of syncytia every 3 or 4 days. At the peak of infection, when massive syncytia were

observed, cell-free virus was passaged to fresh, matching transduced cells. When syncytia were

apparent in the newly infected cells, cellular DNA containing the integrated provirus was isolated

for sequencing analysis of the gRNA target region (PCR and sequencing primers listed in Table

S4), as previously described 27.

28

Acknowledgements

G. Wang is recipient of a fellowship of the China Scholarship Council (CSC). We thank E.

Herrera Carrillo, C. Cristella and Y. Zheng for assistance.

Competing interests

The authors declare no competing interests.

29

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Cas9 RNA-guided endonuclease. Nat. Biotechnol. 31, 230-232 (2013).18. Smyth, R.P., Davenport, M.P. & Mak, J. The origin of genetic diversity in HIV-1. Virus Res. 169,

415-429 (2012).19. von Eije, K.J., ter Brake, O. & Berkhout, B. Human immunodeficiency virus type 1 escape is

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31

Supplementary material

Figure S1. The capacity of Cas9/gRNA to inhibit HIV-1 replication does not correlate with the efficiency of silencing viral gene expression.

Figure S2. Efficient replication of HIV-1 escape variants on SupT1-Cas9/gRNA expressing cells.

Figure S3. Sequence of the gRNA target region in breakthrough viruses obtained in 2 to 6 independent cultures of HIV-1 on different SupT1-Cas9-gRNA cells.

Table S1. Selected gRNAs targeting HIV-1.

Table S2. Nucleotide substitution patterns observed in HIV-1 escape variants.

Table S3. Target sequence of control gRNAs.

Table S4. Primers used for sequencing of gRNA target regions in HIV-1.

32

Figure S1. The capacity of Cas9/gRNA to inhibit HIV-1 replication does not correlate

with the efficiency of silencing viral gene expression. For every tested gRNA, the time

required for HIV-1 breakthrough replication in SupT1-Cas9/gRNA cells (as shown in Fig.

2b) was plotted against the CA-p24 level produced in 293T cells transfected with plasmids

expressing Cas9, gRNA and HIV-1 (as shown Fig. 1b; the average CA-p24 level produced

with the control gRNAs [gLuc, gGFP1, gGFP2] was set at 100%). No correlation was

apparent (Pearson’s correlation coefficient r = 0.036).

33

Figure S2. Efficient replication of HIV-1 escape variants on SupT1-Cas9/gRNA expressing

cells. SupT1 cells stably transduced with Cas9 and gRNA expressing lentiviral vectors and control

(non-transduced) SupT1 cells were infected with wild-type HIV-1 LAI (HIV panels) and the

breakthrough viruses obtained upon culturing of HIV-1 LAI in the corresponding Cas9/gRNA

expressing SupT1 cells (HIV-gRNA panels). Virus replication was monitored by measuring the

CA-p24 level in the culture supernatant.

0.1

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SupT1-gGagPol

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0.1

1

10

100

1000

10000

0 3 6 9

HIV-gLTR7

SupT1-gLTR7

SupT1

0.1

1

10

100

1000

10000

0 3 6 9

HIV

SupT1-gLTR6

SupT1

0.1

1

10

100

1000

10000

0 3 6 9

HIV

SupT1-gLTR7

SupT1

0.1

1

10

100

1000

10000

0 3 6 9

HIV-gPol4

SupT1-gPol4

SupT1

0.1

1

10

100

1000

10000

0 3 6 9

HIV-gTatRev

SupT1-gTatRev

SupT1

0.1

1

10

100

1000

10000

0 3 6 9

HIV-gEnv2

SupT1-gEnv2

SupT1

0.1

1

10

100

1000

0 3 6 9

HIV

SupT1-gPol4

SupT1

0.1

1

10

100

1000

10000

0 3 6 9

HIV

SupT1-gTatRev

SupT1

0.1

1

10

100

1000

10000

0 3 6 9

HIV

SupT1-gEnv2

SupT1

0.1

1

10

100

1000

10000

0 3 6 9

HIV-gPol2

SupT1-gPol2

SupT1

0.1

1

10

100

1000

10000

0 3 6 9

HIV

SupT1-gPol2

SupT1

CA

-p2

4 (

ng

/ml)

CA

-p2

4 (

ng

/ml)

CA

-p2

4 (

ng

/ml)

CA

-p2

4 (

ng

/ml)

days days days days

34

a

gLTR1 G A T T G G C A G A A C T A C A C A C C A G G G C C A G G G G T C A G A T A T C C A C D W Q N Y T P G P G V

1

2

3

· · · · ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ · · · · · · · · · · · · · · · · · · · · · · ·

· · · · · · · · · · · ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ·

· · · · · · · · · · · · · · · · · · A G C · · · · · · · · · · · · · · · · · · · · · · · · ·

· *

· · · · *

· · · · · · S · · · · ·

gLTR2 G T C A G A T A T C C A C T G A C C T T T G G A T G G T G C T A C A A G C T A G T A C V R Y P L T F G W C Y K L V

1

2

3

4

5

· · ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ · · · · · · · · · · · · · · · · · ·

· ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ · · · · · · · · · · · · · · · · · · ·

· · · · · · · · · · · · · · · · · ∆ ∆ ∆ ∆ · · · · · · · · · · · · · · · · · · · · · ·

· · · · · · · · · · · · · · · · · · · · · · ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ·

· · · · · ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ · · · · ·

W C Y K L V P V E P D K V E

W C Y K L V P V E P D K V E

· · · · · · D G A T S *

· · ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ·

gLTR3 G A G A G A G A A G T G T T A G A G T G G A G G T T T G A C A G C E R E V L E W R F D S

1

2

3

4

5

6

· · · · · · · · · · · · · · · · · · C · · · · · · · · · · · · · · ·

· · C · · · · · · · · · · · · · ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ · · · · · ·

· · · · · · · · · · · · · · · · ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ T · · · · ·

· · · · · · · · · · · · · · · · ∆ ∆ ∆ ∆ ∆ ∆ ∆ · · · · · · · · · ·

· · · · · · · · · · · · · · · · · · G G · · · · · · · · · · · · · · ·

· · · · · · · · · · · · · · · · · · ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ·

· · · · · · M E V *

· · · · · G Q P P S I

· · · · · V Q P P S I

· · · · · · G G G L T

· · · · · · P P S I S

gLTR4 A G C C G C C T A G C A T T T C A T C A C G T G G C C C G A G A G C T G C A T S R L A F H H V A R E L H

1

2

3

· · · · · · · · · · · · · · · · · · · C · · · · · · · · · · · · · · · · · · · ·

· · · · · · ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ · ·

· · · · · · · · · · · · · · · · · · · C C · · · · · · · · · · · · · · · · · · · ·

· · · · · · P R G P R A A

· · · R S T S R T A D I E

· · · · · · P T W P E S C

gLTR5 A G C C G C C T A G C A T T T C A T C A C G T G G C C C G A G A G C T G C A T S R L A F H H V A R E L H

1

2

3

4

5

6

· · · · · · · · · · · · G · · · · · · · · · · · · · · · · · · · · · · · · · ·

· ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ · · · · · · · · · · · · · · ·

· · · · · · · · · · · G · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

· · · · · · · · · · · · · ∆ ∆ ∆ ∆ ∆ · · · · · · · · · · · · · · · · · · · · ·

· · · · · · · · · · · ∆ ∆ ∆ ∆ · · · · · · · · · · · · · · · · · · · · · · · ·

· · · · · · · · · · · C · ∆ ∆ · · · · · · · · · · · · · · · · · · · · · · · ·

· · · · V · · · · · · · ·

· P R A A S G V L Q E L L

· · · · I S S R G P R A A

· · · · S R G P R A A S G

· · · · I P W P E R L H P

· · · · S S R G P R A A S

gLTR6 G A C A T C G A G C T T G C T A C A A G G G A C T T T C C G C T G G G G A C T T T C C A G G G

1

2

3

4

· · · · · · · · · · · · · · · · · · · · ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ · · · · · · · · · · · · ·

· · · · · · · · · · · · · · · · · G ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ · · ·

· · · · · · · · · · · · · · · · · · · · · · · · · · · · ∆ · · · · · · · · · · · · · · · · · ·

· ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ · · · · · · · · · · · · · · · · ·

gLTR7 G G C G A G C C C T C A G A T G C T G C A T A T A A G C A G C T G C T T T T T G C C T G T

1

2

3

4

5

6

· · · · · · · · · · · T · · ∆ · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

· · · · · · · · · · · · C · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

· · · · · · · · · · · · C · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

· · · · · · · · · · · · C · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

· · · · · · · · · · · · A · G · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

· · · · · · · · · · G · C · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

gGag1 G T T A A A A G A G A C C A T C A A T G A G G M L K E T I N E E

1

2

3

4

5

· · · · · · · · · · · · · · · · · C C · · · ·

· · · · · · · · · · · · · · · · C · · · · · ·

· · · · · · · · · · · · · · · · C · · · · · ·

· · · · · · · · · · · · · · · · · · · · · A ·

· · · · · · · · · · · · · · · A · · · · · · ·

· · · · · · T · ·

· · · · · · H · ·

· · · · · · H · ·

· · · · · · · · ·

· · · · · · · · ·

gGag2 G C T A C C A T A A T G A T G C A A A G A G G A T I M M Q R G

1

2

3

4

· · · · · · · · · · · · · · · G C · · · · · ·

· · · · · · · · · · · · · · A · · · · · · · ·

· · · · · · · · · · · · · · · · · G G · · · ·

· · · · · · · · · · · · · · · · T T T C · · · · · ·

· · · · · A · ·

· · · · I · · ·

· · · · · · G ·

· · · · · L S · ·

Figure S3. (a-b) Sequence of the gRNA target region in breakthrough viruses obtained in 2

to 6 independent cultures of HIV-1 on different SupT1-Cas9-gRNA cells. For every gRNA, the

wild-type HIV-1 nucleotide sequence is shown on top. If applicable, protein codon triplets are

boxed in grey and the translated amino acid sequence is shown on the right. The PAM sequence

is boxed and the arrowhead indicates the Cas9 cleavage site at position -3. Nucleotide and amino

acid substitutions, insertions and deletions (∆) are indicated.

35

b

gGagPol C C C T C A G A T C A C T C T T T G G C A A C P S D H S L A T F P Q I T L W Q R

1

2

3

4

· · · · · · A · · · · · · · · · · · · · · · ·

· · · · · · A G · · · · · · · · · · · · · · ·

· · · · · · A C · · · · · · · · · · · · · · ·

· · · · · · A C · · · · · · · · · · · · · · ·

· · N · · · · ·

· · S · · · · ·

· · T · · · · ·

· · T · · · · ·

· · · · · · · · ·

· · · V · · · · ·

· · · L · · · · ·

· · · · · · · · ·

gPol1 G T A C C A G T A A A A T T A A A G C C A G G V P V K L K P G

1

2

3

4

· · · · · · · · · · · · · · · C · · · · · · ·

· · · · · · · · · · · · · · · · G · · · · · ·

· · · · · · · · · · · · · · · G C · · · · · ·

· · · · · · · · · · · · · · · · G · · · · · ·

· · · · · Q · ·

· · · · · R · ·

· · · · · A · ·

· · · · · R · ·

gPol2 G G G C A A G T C A G A T T T A C C C A G G G W A S Q I Y P G

1

2

3

4

· · · · · · · · · · · · · · · · · T · · · · ·

· · · · · · · · · · · · · · · · T · · · · · ·

· · · · · · · · · · · · · · · · · A · · · · ·

· · · · · · · · · · · · · · · T · · · · · · ·

· · · · · · S ·

· · · · · · · ·

· · · · · · T ·

· · · · · F · ·

gPol3 G C A T G G G T A C C A G C A C A C A A A G G A W V P A H K G

1

2

· · · · · · · · · · · · · · C · · · · · · · ·

· · · · · · · · · · · · · · · · · T · · · · ·

· · · · · · · ·

· · · · · · · ·

gPol4 G A T T G G G G G G T A C A G T G C A G G G G G I G G Y S A G E

1

2

· · · · · · · · · · · · · · · C C · · · · · ·

· · · · · · · · · · · · · G C C C · · · · · ·

· · · · · · P · ·

· · · · · A P · ·

gVpr G A A T A G G C G T T A C T C A A C A G A G G A G A G C A A G A A A T R I G V T Q Q R R A R N

1

2

3

4

5

6

· · · · · · · · · · · · · · · · · G A C · · · · · · · · · · · · · · · · · ·

· · · · · · A ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ · · · · · · · · · ·

· · · · · · · · · · · · · · · · · G ∆ · · · · · · · · · · · · · · · ·

· · · · · · · · · · · · · · · · ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ∆ ·

· · · · · · · · · · · · · · · · · T C C T C · · · · · · · · · · · · · · · · · ·

· · · · · · · · · · ∆ ∆ ∆ ∆ ∆ ∆ ∆ · · · · · · · · · · · · · · · · · ·

· · · · · · D · · · · · ·

· · E ∆ ∆ ∆ ∆ ∆ ∆ ∆ · · N

· · · · · · G G E Q E M

· · · · · H ∆ ∆ ∆ ∆ ∆ G

· · · · · · S S R G E Q E

· · · · R G E Q E M E P

gTatRev C C T A T G G C A G G A A G A A G C G G A G A S Y G R K K R R M A G R S G D

1

2

3

4

5

· · · · · · · A C G · · · · · · · · · · · · · · · ·

· · · · · · · A C G · · · · · · · · · · · · · · · ·

· · · · · · · G C · · · · · · · · · · · · · ·

· · · · · · · G · · · · · · · · · · · · · · ·

· · · · · · · A C G · · · · · · · · · · · · · · · ·

· · · · R · · · ·

· · · · R · · · ·

· · · · · · · ·

· · · · · · · ·

· · · · R · · · ·

· D · · · · · ·

· D · · · · · ·

· G · · · · ·

· G · · · · ·

· D · · · · · ·

gEnv1 G T A A C A T T A G T A G A G C A A A A T G G C N I S R A K W

1

2

3

4

· · · · · · · · · · · · · · C · T · · · · · ·

· · · · · · · · · · · · · · A T G G · · · · ·

· · · · · · · · · · · · A · · · · · · · · · ·

· · · · · · · · · · · · · · T T C C · · · · ·

· · · · · P · ·

· · · · · M E ·

· · · · K · · ·

· · · · · F Q ·

gEnv2 G G A G C A G C A G G A A G C A C T A T G G G G A A G S T M G

1

2

3

· · · · · · · · · · · · · · · · G G · · · · ·

· · · · · · · · · · · · · · · · · G · · · · ·

· · · · · · · · · · · · · · · · · G · · · · ·

· · · · · I · ·

· · · · · · · ·

· · · · · · · ·

gNef G C T A T A A G A T G G G T G G C A A G T G G M G G K W

1

2

3

· · · · · · · · · · · · · ∆ ∆ · · · · · · · ·

· · · · · · · · · · · · · ∆ · · · · · · · · ·

· · · · · · · · · · · · · · · · G G G G · · · · · ·

· · · V V

· · A S G

· · · G · ·

Gag

Pol

Tat

Rev

Figure S3 continued

36

Name position in HIV-1 LAI DNA Target sequence + PAMa Orientation On-target activity (%)

b Efficiency

c Conservation

d

gLTR1 79-101, 9211-9233 ATTGGCAGAACTACACACCAGGG sense 78 0.77 0.20

gLTR2 112-134, 9244-9266 GATATCCACTGACCTTTGGATGG sense 67 0.49 0.24

gLTR3 241-263, 9373-9395 AGAGAGAAGTGTTAGAGTGGAGG sense 51 0.46 0.41

gLTR4 272-294, 9404-9426 CCGCCTAGCATTTCATCACGTGG sense 80 0.89 0.58

gLTR5 275-297, 9407-9429 CCTAGCATTTCATCACGTGGCCC anti-sense 74 0.35 0.49

gLTR6e 343-365, 9475-9497 GCTACAAGGGACTTTCCGCTGGG sense 88 0.46 0.52

gLTR7 413-435, 9544-9566 CCCTCAGATGCTGCATATAAGCA anti-sense 60 0.16 0.09

gGag1 1389-1411 GTTAAAAGAGACCATCAATGAGG sense 64 0.65 0.15

gGag2 1909-1931 GCTACCATAATGATGCAAAGAGG sense 66 0.87 0.42

gGagPol 2288-2310 CCCTCAGATCACTCTTTGGCAAC anti-sense 66 0.31 0.07

gPol1 2607-2629 GTACCAGTAAAATTAAAGCCAGG sense 61 0.81 0.11

gPol2 3382-3404 GGGCAAGTCAGATTTACCCAGGG sense 74 0.87 0.17

gPol3 4185-4207 GCATGGGTACCAGCACACAAAGG sense 64 0.83 0.13

gPol4 4835-4857 GATTGGGGGGTACAGTGCAGGGG sense 61 0.26 0.08

gVpr 5833-5855 GAATAGGCGTTACTCAACAGAGG sense 86 0.56 0.44

gTatRev 6002-6024 CCTATGGCAGGAAGAAGCGGAGA anti-sense 54 0.16 0.11

gEnv1 7263-7285 GTAACATTAGTAGAGCAAAATGG sense 53 0.76 0.55

gEnv2 7841-7863 GGAGCAGCAGGAAGCACTATGGG sense 69 0.25 0.07

gNef 8836-8858 GCTATAAGATGGGTGGCAAGTGG sense 72 0.47 0.30

a PAM sequence in bold and underlined.

b The on-target activity, computed as 100% minus a weighted sum of off-target hit-scores in the human genome

(hg19), was calculated using the CRISPR design web tool from crispr.mit.edu 1, accessed December 3, 2014.

c The gRNA design web tool from www.broadinstitute.org/rnai/public/ 2 was used to calculate the gRNA activity. This

score can vary from 0 to 1, with 1 being the most effective.

d The Shannon entropy was calculated to estimate the variation in the gRNA target sequence amongst the HIV-1

isolates (group M) described in the HIV database 2014 (hiv.lanl.gov; only the complete viral sequences were included).

The entropy can vary from 0 to 1.5, with an invariant sequence having a score of 0.

e This gRNA was previously tested by Ebina et al. 3

Table S1. Selected gRNAs targeting HIV-1

37

Table S2. Nucleotide substitution patterns observed in HIV-1 escape variants.

Nucleotide substitutions observed in HIV-1 variants that escaped from inhibition mediated

by Cas9/gRNA (this study) and RNAi (as published in reference 4) are shown.

mutation Cas9/gRNA RNAi

A→T 4 11

A→C 13 2

A→G 9 28

T→A 0 0

T→C 3 7

T→G 4 0

C→A 2 5

C→T 6 10

C→G 6 1

G→A 9 46

G→T 1 3

G→C 5 0

gRNA Target Sequence

gLuc GCTGTTTCTGAGGAGCCTTCAGG

gGFP1 GGGCGAGGAGCTGTTCACCGGGG

gGFP2 GAGCTGGACGGCGACGTAAACGG

Table S3. Target sequence of control gRNAs. The gRNAs targeting the GFP

gene have been described previously in reference 5.

38

Table S4. Primers used for sequencing of gRNA target regions in HIV-1

sense primer antisense primer

gLTR1-7 CAG CAT CTC GAG ACC TGG AAA AAC AT GCC ACC TGA CGT CTA AGA AAC CAT T

gGag1 CAT ATC ACC TAG AAC TTT AAA TGC AGT TTT ATA GAA CCG GTC TAC ATA

gGag2 GCA GGA ACT ACT AGT ACC CTT CA CCT GAA GCT CTC TTC TGG TG

gGagPol TCA GAG CAG ACC AGA GCC AAC AG CCA ATC TGA GTC AAC AGA TTT CTT CC

gPol1 GGA GCA GAT GAT ACA GTA TTA GA GAC CTA CAC CTG TCA ACA TAA T

gPol2 AAA TCC ATA CAA TAC TCC AG CTG CCA GTT CTA GCT CTG CTT C

gPol3 ATA GTA ACA GAC TCA CAA TAT GCA AGG TTA AAA TCA CTA GCC ATT GCT CTC C

gPol4 CCC TAC AAT CCC CAA AGT CAA AAT CAT CAC CTG CCA TCT GTT TTC C

gVpr ATA TCA AGC AGG ACA TAA CAA GG CTA TGA TTA CTA TGG ACC ACA CA

gTatRev ATA TCA AGC AGG ACA TAA CAA GG CTA TGA TTA CTA TGG ACC ACA CA

gEnv1 GTA CAA GAC CCA ACA ACA ATA CAA G TTA CAG TAG AAA AAT TCC CCT CCA CAA

gEnv2 GCA CCC ACC AAG GCA AAG AGA AGA GTG G CAA CCC CAA ATC CCC AGG AGC TGT TGA TCC

gNef GCA GTA GCT GAG GGG ACA GAT AGG UGU GCU UCU AGC CAG GCA C

39

Supplementary References

1. Hsu, PD, Scott, DA, Weinstein, JA, Ran, FA, Konermann, S, Agarwala, V, et al. (2013).

DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 31: 827-832.

2. Doench, JG, Hartenian, E, Graham, DB, Tothova, Z, Hegde, M, Smith, I, et al. (2014).

Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation. Nat

Biotechnol 32: 1262-1267.

3. Ebina, H, Misawa, N, Kanemura, Y, and Koyanagi, Y (2013). Harnessing the CRISPR/Cas9

system to disrupt latent HIV-1 provirus. Sci Rep 3: 2510.

4. von Eije, KJ, ter Brake, O, and Berkhout, B (2008). Human immunodeficiency virus type 1

escape is restricted when conserved genome sequences are targeted by RNA interference.

J Virol 82: 2895-2903.

5. Liao, HK, Gu, Y, Diaz, A, Marlett, J, Takahashi, Y, Li, M, et al. (2015). Use of the

CRISPR/Cas9 system as an intracellular defense against HIV-1 infection in human cells.

Nat Commun 6: 6413.

40

Chapter 3

Strategies to prevent NHEJ-mediated HIV-1 escape from CRISPR-Cas9 attack

41

Introduction

Several studies demonstrated that targeting of HIV-1 with the CRISPR-Cas9 system can

effectively inhibit virus replication and inactivate the integrated provirus1-4. However, we and

others recently described that HIV can rapidly escape from such CRISPR-Cas9 inhibition

through the selection of mutations in the Cas9-gRNA target sequence 5, 6. These mutations were

introduced during the error-prone non-homologous end joining (NHEJ) repair of the Cas9-

induced double-stranded breaks (DSBs) in the target DNA. We investigated two approaches to

prevent this NHEJ-assisted escape route: direct inhibition of this NHEJ activity by drugs or the

application of an alternative CRISPR system, CRISPR-Cpf1.

1. Inhibition of NHEJ activity

We demonstrated in chapter 2 that the NHEJ DNA repair mechanism facilitates HIV-1 mutation and escape from CRISPR-Cas attack. We therefore tested whether an NHEJ inhibitor could

block this viral escape route. The small molecule inhibitor SCR7 was reported to inhibit the

NHEJ mechanism and to impede cancer progression 7. It was subsequently used to increase the

efficiency of precise genome editing by homologous recombination (HR) through inhibiting the

NHEJ pathway8, 9. We studied the effect of SCR7 in long-term virus inhibition experiments.

SupT1 cells stably expressing Cas9 and a gRNA targeting either the LTR region (gLTR6) or the

Vpr coding region (gVpr) were infected with HIV-1 and cultured in the absence or presence of

SCR7. In control cells not expressing any gRNA, the virus replicated efficiently both in the

absence and presence of SCR7, resulting in the rapid appearance of large virus-induced

syncytia and massive cell death around day 10 (Fig. 1). As previously presented 6, HIV-1

replicated less efficiently in cells protected with gLTR6 or gVpr, but breakthrough replication due

to virus escape was nevertheless observed at around day 14. However, similar inhibition and

relatively fast rapid breakthrough replication was observed when the infected gLTR6/gVpr-cells

were cultured in the presence of 1 or 10 µM SCR7. In contrast, a significant delay in

breakthrough replication was measured at 100 μM SCR7 (around 31 and 28 days for gLTR6 and

gVpr expressing SupT1 cells respectively), which indicates that inhibition of the NHEJ activity

can indeed reduce HIV escape (Fig. 1). However, the SupT1 cells showed significant cytotoxic

effects at this high SCR7 dose, which is in agreement with an earlier report 7. These results may

suggest that NHEJ-inhibition is a successful strategy for preventing HIV-1 escape, although we

cannot exclude that virus replication is unspecifically hindered by the cytotoxic effects of the drug.

Thus, it may be worthwhile to test additional NHEJ-inhibitors that are less toxic or that are more

active at lower, non-toxic concentrations.

42

Fig. 1. SCR7 reduces HIV escape from CRISPR-Cas9 inhibition. SupT1 cells stably expressing Cas9

and gLTR6 or gVpr (produced as previously described 6 and control SupT1 cells were infected with 0.1 ng

HIV-1 LAI virus. The cells wells cultured with 0, 1, 10 or 100 µM SCR7 (Xcess Biosciences, USA,

dissolved in DMSO). The day at which massive syncytia were observed, reflecting breakthrough

replication, was scored as described previously 6. Average values (±SEM) of at least three experiments

are shown.

2. Targeting HIV-1 with CRISPR-Cpf1

CRISPR-Cas9 from Streptococcus pyogenes, which is used in most virus inhibition studies,

cleaves the target DNA 3 bp upstream of the PAM site in a region that is critical for gRNA

recognition. Subsequent NHEJ repair will introduce mutations at or around this position that

frequently result in mutated target sites that are no longer recognized and cleaved by CRISPR-

Cas9. To prevent such resistance, one would ideally like to use a Cas9-like nuclease that

cleaves outside the gRNA recognition sequence. Unfortunately, such systems are not yet

available. Alternatively, one could use the CRISPR-Cpf1 system from the Lachnospiraceae or

Acidaminococcus.10 Cpf1 is guided by a single crRNA molecule, thus without a tracrRNA, to a 24

nt target sequence. Cpf1-crRNA cleaves the target DNA at 18/23 bp downstream of the PAM in

a region that is less important for crRNA binding10. Target sites with mutations introduced at this

more distal position may still be recognized and cleaved by Cpf1 and thus will not lead to

resistance and virus escape. We therefore tested the capacity of CRISPR-Cpf1 to inhibit HIV-1

replication and to prevent virus escape. We designed 7 crRNAs that targeted both conserved

and non-conserved sequences in HIV-1 DNA (Fig. 2a).

0

5

10

15

20

25

30

35

40

45

gLTR6 gVpr SupT1

Brea

kthr

ough

repl

icat

ion

(day

)

DMSO

100μM SCR7

43

a

b

Fig. 2. Cpf1/crRNA targeting of the HIV-1 genome. (a) The HIV-1 proviral DNA with the position of crRNAs tested in this study. (b) The efficiency of CRISPR-Cpf1 to silence HIV-1 DNA was tested in 293T

cells transfected with plasmids expressing HIV-1 LAI, Cpf1, and crRNAs. crLuc is a crRNA targeting the luciferase gene. crEmpty is a crRNA scaffold without targeting sequence. Methods: The Cpf1 gene

(Addgene plasmid #69982, a gift from Feng Zhang 10) and crRNAs scaffold10 were cloned into the

lentiviral vector (Addgene plasmid # 52962 and # 52963 respectively, a gift from Feng Zhang)11. 293T

cells were transfected in a 24-well plate with 500 ng of the lentiviral vector plasmids expressing Cpf1 and

crRNAs and 300 ng HIV-1 LAI plasmid, as previously described 6. To quantify viral gene expression, the

CA-p24 level was measured in the culture supernatant at 2 days after transfection. Average values (±SEM)

of four experiments are shown.

We first tested the capacity of the CRISPR-Cpf1 system to inactivate HIV-1 DNA. For this, 293T

cells were transfected with the HIV-1 encoding plasmid pLAI and plasmids expressing Cpf1 and

the crRNAs. Unfortunately, we observed a rather poor efficiency of crRNAs/Cpf1 in inactivating

the HIV-1 DNA plasmid and reducing viral gene expression (Fig. 2b) compared to the knock-

down observed with the regular Cas9 system in chapter 2. These results indicate that Cpf1

gag

v pr

v if

uva-dare (digital academic repository) crispr-cas based … · and transcription activator-like...

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