CRISPRCas9 Technology and Its Application in Haematological PDF

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Br J Haematol. Author manuscript; available in PMC 2018 April 06.
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Published in final edited form as:

Br J Haematol. 2016 October ; 175(2): 208–225. doi:10.1111/bjh.14297.
CRISPR-Cas9 technology and its application in haematological

disorders
Han Zhang and Nami McCarty
The Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases
(IMM), University of Texas-Health Science Centre at Houston, Houston, TX, USA
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Summary
The recent advent of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-
CRISPR associated protein 9 (Cas9) system for precise genome editing has revolutionized
methodologies in haematology and oncology studies. CRISPR-Cas9 technology can be used to
remove and correct genes or mutations, and to introduce site-specific therapeutic genes in human
cells. Inherited haematological disorders represent ideal targets for CRISPR-Cas9-mediated gene
therapy. Correcting disease-causing mutations could alleviate disease-related symptoms in the near
future. The CRISPR-Cas9 system is also a useful tool for delineating molecular mechanisms
involving haematological malignancies. Prior to the use of CRISPR-Cas9-mediated gene
correction in humans, appropriate delivery systems with higher efficiency and specificity must be
identified, and ethical guidelines for applying the technology with controllable safety must be
established. Here, the latest applications of CRISPR-Cas9 technology in haematological disorders,
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current challenges and future directions are reviewed and discussed.
Keywords
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (Cas9)
system; haematological disorders; gene editing; haematological malignancies
Introduction
Over the past two decades, advances in molecular biology and genetics have greatly
extended our knowledge of haemaological diseases. However, the characterization of genes
of interest remains a great challenge. The optimal way to unravel the roles of particular
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genes is to manipulate their functions via gene deletion, insertion or modification. Earlier
genome editing technologies, such as homologous recombination (HR), have facilitated the
manipulation of genes by generating knockout and knock-in cells or mouse models (Thomas
et al, 1986; Mansour et al, 1988; Capecchi, 1989a, b). However HR-mediated targeting is a
less efficient, time-consuming and labour-intensive process, rendering its widespread
Correspondence: Nami McCarty, PhD, University of Texas-Health Science Centre at Houston, 1825 Pressler St., IMM-630H,
Houston, TX 77030, USA. nami.mccarty@uth.tmc.edu.
Author contributions
Han Zhang reviewed the literature, wrote the paper and made corrections. Nami McCarty designed the review, wrote the paper and
made corrections.
Zhang and McCarty Page 2
application difficult. In subsequent decades, targeting of DNA double-stranded breaks

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(DSBs) using engineered nucleases, such as zinc-finger nucleases (ZFNs) and transcription
activator-like effector nucleases (TALENs), was introduced to stimulate cellular DNA repair
pathways: non-homologous end-joining (NHEJ) and homology-directed repair (HDR)
(Rudin et al, 1989; Rouet et al, 1994; Choulika et al, 1995; Bibikova et al, 2002; Moscou &
Bogdanove, 2009). In most cases, NHEJ-mediated error-prone DNA repair generates
random insertions or deletions (indels), leading to frameshift mutations. If an exogenous
donor DNA template is available surrounding the DSB, then the DNA damage can be
repaired via an HDR pathway, which leads to precise gene modifications or gene insertions.
Although ZFNs and TALENs have resulted in more efficient and applicable site-specific
genome engineering (Joung & Sander, 2013; Urnov, 2014), the expansion of their
applications has also been hampered by complex procedures, low specificity and high cost
(Peng et al, 2015; Sanchez-Rivera & Jacks, 2015).
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The most recent development, the Clustered Regularly Interspaced Short Palindromic
Repeats (CRISPR)-CRISPR associated protein 9 (Cas9) technique, has initiated a new era of
genome editing by overcoming the limitations of these earlier methods (Cong et al, 2013;
Hsu et al, 2014; Niu et al, 2014; Konermann et al, 2015). During the past 4 years, the
CRISPR-Cas9 system has been overwhelmingly adopted as a powerful tool in biological
research, ranging from the modification of plants to the creation of animal models and the
alleviation of human diseases including haematological diseases.
Development of the CRISPR-Cas9 system

CRISPR was first described in Escherichia coli as an odd 29-nucleotide repeat sequence
with 32-nucleotide spacing by a Japanese research group in 1987, although no further
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detailed observation was reported (Ishino et al, 1987). Several years later, Francisco Mojica
discovered similar repeats in several microbial species and reported their potential use in
prokaryotes (Mojica et al, 1993, 1995). By 2002, the novel repeats were formally named as
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR); meanwhile,
scientists discovered specific CRISPR-associated (Cas) genes that were closely related to the
function of CRISPR (Jansen et al, 2002). In 2003, Mojica discovered the function of the
CRISPR system, proposing that CRISPR is an adaptive immune system that protects
microbes against specific infections (Mojica et al, 2005). Two independent groups (Bolotin
et al, 2005; Pourcel et al, 2005) reported similar conclusions, and their finding was
confirmed by experiments showing that CRISPR provides acquired immunity against viruses
in prokaryotes (Barrangou et al, 2007).
Following these discoveries, more researchers adopted CRISPR technologies into their
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research. In 2008, scientists completed the first attempt to program CRISPR in bacteria, and
for the first time they hypothesized that the target of CRISPR was not RNA, but DNA
(Brouns et al, 2008). This hypothesis was confirmed within the same year by Marraffini and
Sontheimer (2008), clarifying the mechanism by which CRISPR cleaves DNA: Cas9
nuclease guided by CRISPR RNAs (crRNAs) creates DSBs in DNA at precise positions
(Garneau et al, 2010). These findings as well as the later discovery of trans-activating

CRISPR RNA (tracrRNA) (Deltcheva et al, 2011; Jinek et al, 2012) positioned CRISPR-
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Cas9 systems on a novel stage for genome engineering.
Since the release of several milestone studies of bacteria utilizing the CRISPR-Cas9 system
(Gasiunas et al, 2012; Jinek et al, 2012), additional groups have adopted the system as an
editing tool for mammalian cells. Researchers from the Massachusetts Institute of
Technology have developed and optimized the Streptococcus pyogenes CRISPR-Cas9
system to facilitate efficient genome editing in mammalian cells (Cong et al, 2013).
Simultaneously, another group established a CRISPR-Cas9 system as an RNA-guided
editing tool for human genome engineering using a chimeric single-guide RNA (sgRNA)
instead of a tracrRNA:crRNA duplex (Mali et al, 2013a). Inspired by successful outcomes in
vitro, scientists have started to apply a CRISPR-Cas9 system in vivo. Several studies have
reported efficient genome editing in multiple organisms (e.g., zebrafish, yeast, mouse,
drosophila, pig and monkey, among others) (Jackson et al, 2005; Soda et al, 2007; Hwang et
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al, 2013; Maddalo et al, 2014; Niu et al, 2014; Xue et al, 2014; Akiyama & Gibson, 2015;
DiCarlo et al, 2015; Gantz & Bier, 2015; Redel & Prather, 2015). More interestingly, a
CRISPR-Cas9 system was applied to screen for loss-of-function using a high-throughput
screening platform in human cells (Koike-Yusa et al, 2014; Shalem et al, 2014; Wang et al,
2014; Zhou et al, 2014). These genome-scale knockout screens provide an alternative
screening system to RNA interference (RNAi), which is limited by partial depletion of target
gene levels, toxicity and transient silencing (Echeverri et al, 2006; Kaelin, 2012; Sanchez-
Rivera & Jacks, 2015).
Further modifications of the CRISPR system have been reported. Qi et al (2013)

demonstrated a catalytically inactive Cas9 protein (dCas9) lacking endonuclease activity that
can be used as a platform for RNA-guided transcription regulation; this modified system is
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called CRISPR interference (CRISPRi). Unlike RNAi-based silencing, which destructs

transcribed mRNAs, the CRISPR-dCas9 system directly blocks transcription elongation
within protein-coding regions and leads to dramatic suppression of transcription, with no
detectable off-target effects (Qi et al, 2013). The authors subsequently reported that both
repressive and activating effector domains, such as KRAB and VP64, can be fused to dCas9
to either repress (CRISPRi) or activate (CRISPRa) the transcription of target genes (Gilbert
et al, 2013). This CRISPRi/a system was further modified to control the transcription levels
of endogenous genes up to ~1000-fold, allowing researchers to determine how gene dosage
affects cellular functions of interest (Gilbert et al, 2014). Other than targeting dsDNAs,
CRISPR-Cas9 was modified to bind or cut RNA targets in vitro by providing the protospacer
adjacent motif (PAM) as part of an oligonucleotide (PAMmer). This strategy enables Cas9 to
cleave RNA targets in a programmable manner and facilitates direct transcript detection
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(O’Connell et al, 2014). The latest study further established nuclear-localized RNA-targeting
Cas9 (RCas9) as a tool to track RNAs in living cells, in which RCas9 is capable of tracking
the localization and movement of endogenous RNAs (Nelles et al, 2016). This work enables
scientists to further measure other RNA features and manipulate these processes to correct
disease-causing RNA behaviours (Fig 1).

Applications of CRISPR-Cas9 technology in haematological disorders

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Of the three types of CRISPR systems, the type II system is the simplest for genome editing
technology, requiring only Cas9 proteins and two RNAs: tracrRNA and crRNA (Brouns et
al, 2008; Wiedenheft et al, 2011; Jinek et al, 2012). The tracrRNA:crRNA duplex was
further simplified with a programmed sgRNA, which directs Cas9 endonuclease toward any
double-stranded DNA sequence of interest to trigger NHEJ or HDR-mediated genome
editing (Jinek et al, 2012; Mali et al, 2013a). The functions and mechanisms of the type II
CRISPR-Cas9 system have been extensively reviewed in detail (Hsu et al, 2014; Sanchez-
Rivera & Jacks, 2015; Lander, 2016).
Non-cancerous haematological disorders

β-thalassaemia—β-thalassaemia is one of the most common inherited blood disorders
and is characterized by mutations in the human haemoglobin beta (HBB, also known as β-
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globin) gene. Decreased HBB expression results in the accumulation of α-globin chains,
leading to ineffective erythropoiesis and thus severe anaemia (Ribeil et al, 2013; Finotti et al,
2015). One curative treatment for β-thalassaemia is allogeneic haematopoietic stem cell
transplantation (HSCT); however, this treatment is only employed for those patients with
suitable matching donors. Although extensive progress has been achieved in allogeneic
HSCT, there is always an elevated risk of suffering graft-versus-host disease (GVHD), graft
rejection or even transplant-related mortality (King & Shenoy, 2014).
Compared with allogeneic HSCT, gene therapy has been considered an alternative
therapeutic option, as several clinical trials have been successfully conducted in patients
with β-thalassaemia (Cavazzana-Calvo et al, 2010; Finotti et al, 2015). Virus-based vectors
have been widely used in gene therapy due to their ability to integrate permanently into
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genomic DNA. Nevertheless, insertional activation of protooncogenes remains a major

problem associated with vector-mediated gene therapy and can lead to secondary cancers,
such as leukaemia (Wu & Dunbar, 2011).
Similar to virus-mediated gene therapy, CRISPR-Cas9-mediated genome editing is used to

correct HBB gene mutations in patients via HDR, leading to normal erythropoiesis. In the
past two years, several research groups have successively applied CRISPR-Cas9 technology
to correct β-thalassaemia mutations in patient-derived induced pluripotent stem cells (iPSCs)
(Xie et al, 2014a; Song et al, 2015; Xu et al, 2015). Gene-corrected iPSCs can restore HBB
expression with a minimal off-target effect. In a recent study, the fibroblasts of a β-
thalassaemia patient were reprogrammed to become transgene-free naïve-state iPSCs, which
showed significantly higher targeting efficiencies in the CRISPR-Cas9 genome editing
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system compared with primed iPSCs (Yang et al, 2016).
In addition to correcting β-thalassaemia-causing mutations, other interesting attempts have

been reported in the treatment of β-thalassaemia via reactivation of fetal haemoglobin
(HbF); elevated HbF is beneficial in β-haemoglobinopathies [e.g., β-thalassaemia and
sickle-cell disease (SCD)] (Bauer et al, 2012) Unfortunately, therapy via HbF reactivation
has lagged for many years due to the unclear regulatory relationship between HbF and adult
haemoglobin (HbA) during the development of erythroid cells. This gap was not filled until

the BCL11A locus, a critical regulator of the fetal-to-adult switch and HbF silencing, was
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identified in genome-wide association studies (GWAS) of HbF (Lettre et al, 2008; Uda et al,
2008). Recently, Canver et al (2015) identified an erythroid-enhancer region within the
BCL11A gene via CRISPR-Cas9-mediated in situ saturating mutagenesis. As an ideal
therapeutic target for β-haemoglobin disorders, this region can be disrupted by individual
sgRNAs, leading to efficient HbF reactivation.
Sickle-cell disease—Along with β-thalassaemia, SCD remains a global health burden,

particularly in underdeveloped countries (Weatherall, 2010). SCD is another human genetic
disease that is caused by the single-nucleotide mutation of the HBB gene. Under
deoxygenated conditions, sickle haemoglobin polymerization within erythrocytes deforms
red blood cells (RBCs) to assume a sickle shape, clogs blood vessels and results in painful
symptoms, such as haemolysis, ischaemia, anaemia and multi-organ injury (Gewin, 2015;
Hoban et al, 2016).
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Several groups have attempted to correct the SCD mutation in patient-derived iPSCs through
ZFN-based (Zou et al, 2011) or TALENs-induced HDR (Sun & Zhao, 2014). One recent
study utilized a CRISPR-Cas9 system to target the endogenous HBB locus in human iPSCs
generated from SCD patients, resulting in higher efficiencies compared with other nucleases
(Huang et al, 2015). In addition, HbF reactivation by targeting the BCL11A gene using a
CRISPR-Cas9, as detailed above, is also a promising treatment for SCD. Alternatively,
epigenetic modifiers are beginning to emerge as potential therapeutic targets for SCD. In
murine erythroleukaemia cells, CRISPR-Cas9-mediated Ehmt2 knockout increased the
expression of embryonic Hbb genes Hbb-y (also termed Hbb-εy) and Hbb-bh1 (Hbb-βh1),
providing genetic evidence for EHMT1/2 inhibition, which induced HbF synthesis. This
approach requires further investigation as a novel therapeutic approach for the treatment of
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SCD (Renneville et al, 2015).
Fanconi anaemia—Fanconi anaemia (FA) is a genetic DNA repair-deficient human

disorder that results from mutations in FA genes. Patients with FA develop bone marrow
failure and congenital defects, and exhibit a high predisposition to malignancies such as
myelodysplastic syndrome (MDS), acute myeloid leukaemia (AML) and solid tumours
(Taniguchi & D’Andrea, 2006; Auerbach, 2009; Soulier, 2011; Kottemann &
Smogorzewska, 2013).
To overcome the limitations of HSCT-based treatment in FA patients, gene therapies have

been adopted (Bogliolo & Surralles, 2015). Somatic cells from FA patients, for example,
have been reprogrammed to generate patient-specific iPSCs; notably, the corrected FA-
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specific iPSCs generated the first disease-free haematopoietic progenitor cells (Raya et al,
2009). Subsequently, more efficient gene correction was performed by targeting FANCA
insertion into the AAVS1 safe harbour locus using ZFN (Rio et al, 2014). Recently, Osborn
et al (2015) generated a CRISPR-Cas9 system for the FANCC locus to analyse repair of the
FANCC c.456 + 4A>T point mutation in FA fibroblasts. This result indicates that CRISPR-
Cas9-mediated HDR offers a novel gene correction strategy for FA patients and can provide
a proof of principle for precise genome editing.

Diamond-Blackfan anaemia—Diamond-Blackfan anaemia (DBA) is a rare inherited

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pure erythroid aplasia that only affects RBCs with decreased erythroid progenitors in the
bone marrow. The main cause of DBA is the mutation of ribosomal protein genes, which
lead to ribosomal stress and TP53 (p53) activation (Dutt et al, 2011; Jaako et al, 2011).
In addition to bone marrow transplantation, glucocorticoid hormones as well as other

supplementary therapies have been used to improve RBC production in DBA; however,
many side effects and unresponsive cases have been reported (Vlachos et al, 2001, 2008;
Ball, 2011). Recently, one research group successfully rescued haematological defects in a
zebra-fish DBA model by inactivating tp53 (p53) in erythrocytes via a CRISPR-Cas9-based
vector system (Ablain et al, 2015). This finding exemplifies the effective use of a CRISPR-
Cas9 in vivo genetic model and its further development in model organisms other than
zebrafish.
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Thrombocytopenia—Thrombocytopenia refers to a blood disorder that involves a

decrease in thrombocytes caused by either inherited or acquired alterations (Drachman,
2004). To date, only a few cases have documented the use of CRISPR-Cas9 for the treatment
of thrombocytopenia. In one study, Zhang et al (2016a) used a CRISPR-Cas9 gene editing to
convert megakaryocyte-like cells and iPSCs from HPA-1a (human platelet alloantigen-1a) to
the HPA-1b alloantigenic epitope, which is relatively rare and difficult to obtain. This work
highlights the potential of the CRISPR-Cas9 system to produce designer platelets for
diagnostic and therapeutic purposes.
Another study focused on the thrombocytopenia caused by solid-organ xenotransplantation

(Butler et al, 2016). The authors genetically modified pigs using CRISPR-Cas9 and found
that porcine kidneys failed to consume human platelets; in contrast, porcine livers exhibited
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a significant ability to consume human platelets. This finding reveals different responses of
porcine organs to human platelets. Further genetic modification of donor pigs is needed to
prevent the development of thrombocytopenia in pig-to-primate xenotransplantation.
Haemophilia—Haemophilia refers to a group of hereditary disorders of blood coagulation

(Ratnoff & Bennett, 1973). The most common type of this disorder is haemophilia A (HA),
which is caused by a deficiency in coagulation factor VIII (FVIII). Another type,
haemophilia B (HB), is caused by coagulation factor IX (FIX) deficiency.
Several approaches have been reported to treat haemophilia using gene therapy (High,
2012). Adenovirus-associated virus (AAV) vectors expressing either human FVIII
(McIntosh et al, 2013) or human FIX (Nathwani et al, 2011) have been used in the past
decade as the first HA or HB gene therapy. Because AAV-mediated gene transfer is
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applicable only to a limited group of patients, alternative strategies have been proposed
(High, 2012). Last year, one research group developed a CRISPR-Cas9 system to revert two
inverted chromosomal regions back to the normal orientation in HA patient-derived iPSCs;
the endothelial cells differentiated from the inversion-corrected iPSCs efficiently rescued the
FVIII deficiency in an HA mouse with no off-target mutations (Park et al, 2015). More
recently, a group from China identified a novel mutation, Y371D, in the human F9 (FIX)
gene. Introduction of this mutation into mice via CRISPR-Cas9 led to a severe HB

phenotype, whereas correction of this mutation by CRISPR-Cas9-mediated in situ genome

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editing restored haemostasis in HB mice (Guan et al, 2016). These preliminary data
demonstrate the feasibility of CRISPR-Cas9 as a versatile tool in precision medicine.
Others—Similar to human blood disorders, genetic correction using a CRISPR-Cas9

system has been reported in the context of other haematological disorders. Using
myeloproliferative neoplasm patient-derived iPSCs carrying a JAK2-V617F point mutation,
Smith et al (2015) demonstrated that Cas9 could specifically target the mutant allele with
little disruption to the other allele. This result highlights the advantages of CRISPR-Cas9
technology in allele-specific genome targeting, offering a unique targeting specificity that
has not been previously achieved. Another group utilized a CRISPR-Cas9 system to create
Kdelr1 mutant mice, which had reduced expression of the T-cell receptor and increased
expression of CD44 (Siggs et al, 2015). This study indicated that KDELR1 is responsible for
T-cell lymphopenia, although the precise mechanism of T lymphopenia in Kdelr1 mutants
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has not yet been elucidated. Applications of a CRISPR-Cas9 system to non-cancerous

haematological disorders are summarized in Table I and Fig 2.
Malignant haematological disorders

Lymphoma—Despite improved responses with frontline therapies in lymphoma
(Shankland et al, 2012), relapsed and refractory cases remain a significant challenge (Brice,
2008; Gangatharan & Kuruvilla, 2013). Genome-editing technology has become a potential
therapeutic option in lymphoid malignancies, although most applications of CRISPR-Cas9
to date have been limited to bench studies.
Malina et al (2013) first established the lentiviral/retroviral-based ‘all-in-one’ CRISPR-

Cas9-mediated ex vivo editing of Trp53 in chemoresistant lymphomas in the Eµ-myc mouse
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model. Notably, the authors extended the CRISPR-Cas9 platform to positive selection
screenings (identifying those cells that ‘pass’ the selection mechanism) both in vitro and in
vivo using TP53 as a model genetic target. This work demonstrated the efficiency and
simplicity of editing unique genomic loci using a CRISPR-Cas9 system. Recently, an
exciting work unveiled the target gene for the therapeutic activity of NUTLIN3A (Valente et
al, 2016), a novel small-molecule antagonist of MDM2 that promotes TP53 activation.
Using mouse models lacking TP53 target genes, the authors demonstrated that BBC3
(PUMA) is responsible for the resistance of NUTLIN3A in lymphomas. Further CRISPR-
Cas9-mediated silencing of the BBC3 gene confirmed that BBC3 expression might predict
NUTLIN3A treatment outcomes in patients.
More recently, a study reported the application of a CRISPR-Cas9 system to disrupt CXCR4
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expression in mantle cell lymphomas (MCL), a highly aggressive subset of B-cell non-
Hodgkin lymphomas (NHLs) (Chen et al, 2016). Using lentiviral-based CRISPR-Cas9-
mediated silencing of CXCR4, the researchers found that reactive oxygen-mediated CXCR4
expression is a key signal inducing autophagy, which contributes to the survival of
bortezomib-resistant MCL cells (Chen et al, 2016). The same group also reported that
Hedgehog inhibitor LDE225 induces autophagy to promote MCL cell survival via the
upregulation of CXCR4 because LDE225 treatment failed to induce autophagy in CXCR4

knockout cells generated by CRISPR-Cas9 (Zhang et al, 2016b). To summarize, the

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CRISPR-Cas9 system generates numerous exciting opportunities to unravel the mechanisms

of drug resistance and to identify potential therapeutic targets in lymphomas.
Myeloma—Multiple myeloma (MM) is the second most frequent haematological

malignancy in the US after NHLs and is characterized by the presence of excess numbers of
malignant plasma cells in the bone marrow. Over the past several years, novel treatment
options have markedly improved the outcomes of MM patients (Richardson et al, 2007;
Raab et al, 2009; Moreau et al, 2015). However, important challenges remain that necessitate
new strategies (Moreau et al, 2015).
Wu et al (2014) first reported genome-editing in MM cells. The authors discovered that

TALENs are a very robust and efficient genome-editing tool in MM cells. Recently, Tagde et
al (2016) utilized CRISPR-Cas9 editing to silence MUC1-C, an oncogenic transmembrane
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protein, and found that MUC1-C occupies the MYC promoter and activates the MYC gene
via a β-catenin/TCF4-mediated mechanism. Further analysis using CRISPR-Cas9-mediated
microarray datasets demonstrated that MUC1-C drives MYC expression and contributes to
MM progression. Therefore, TALENs and CRISPR-Cas9 technologies may be efficient tools
for targeting specific genes, facilitating the construction of knockout reagents. In the near
future, the use of these tools will be extended to clinical treatment in MM patients.
Leukaemia—In comparison to lymphoma and MM, the CRISPR-Cas9 genome editing has
been overwhelmingly applied in leukaemia research, especially in myeloid leukaemia. Heckl
et al (2014) used the CRISPR-Cas9 system to generate mouse models of AML using
lentivirus-mediated editing of multiple genes (e.g., Runx1 and Tp53, among others) in
murine haematopoietic stem and progenitor cells (HSPC). This work highlights the
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application of CRISPR-Cas9 to generate ex vivo genome editing to delineate the complexity

of human blood cancers. Later that year, Chen et al (2014) discovered a novel tumour
suppressor gene, KMT2C (lysine methyltransferase 2C, also termed MLL3), in chromosome
7q using an ex vivo CRISPR-Cas9-mediated approach. Further experiments in a mouse
model confirmed that KMT2C is a haploinsufficient tumour suppressor in AML and its
inhibition impairs differentiation of the myeloid lineage and produces an MDS-like
syndrome in mice. Interestingly, murine AMLs with KMT2C suppression are resistant to
conventional chemotherapy. This study provides a valuable clue for the treatment of AML
patients harbouring −7/del(7q) lesions.
In the past 2 years, there have been an increasing number of publications related to CRISPR-
Cas9 technology in leukaemia research; however most studies have focused primarily on the
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delivery of gene function studies in vitro. For example, one group reported that CRISPR-
Cas9-mediated loss of EPAS1 (HIF-2α) alone had no impact on the growth of human AML
cells under hypoxic conditions (Vukovic et al, 2015). In another study, the authors combined
an RNA sequencing method and CRISPR/TALEN-based knockout techniques to identify
genes associated with drug resistance in AML (Rathe et al, 2014). In addition to gene
silencing, CRISPR-Cas9 has also been reported in the context of gene insertions and
corrections. Cooper et al (2015), using CRISPR-Cas9-induced site-mutation, found an
enhancer that is critical for normal haematopoiesis and, potentially, during myeloid

transformation. In human myeloid leukaemia K562 cells, mutation of CRISPR-Cas9-

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induced to SRSF2 revealed the mechanism of the disease-associated SRSF2 mutation in the
regulation of splicing, thereby providing a group of mis-spliced mRNA isoforms for
potential therapeutic targeting (Zhang et al, 2015a). In another recent study, CRISPR-Cas9-
mediated ASXL1 homozygous correction not only restored ASXL1 expression in chronic
myeloid leukaemia (CML) cells, but it also increased survival in mouse xenografts (Valletta
et al, 2015).
Along with many studies investigating AML, the application of a CRISPR-Cas9 system to
other types of leukaemia has been reported. In human T-cell acute lymphoblastic leukaemia
(T-ALL) cells, a CRISPR-Cas9 editing tool was used to disrupt TAL1 (Navarro et al, 2015)
or TRIB1 (TRB1) (Miyajima et al, 2015) genes to delineate their biological functions. More
recently, Dronabinol (Tetrahydrocannabinol, THC), a US Food and Drug Administration-
approved cannabinoid receptor (CNB) agonist for the treatment of chemotherapy-induced
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nausea and vomiting, was found to induce apoptosis in acute leukaemia cells, as evidenced
by the abrogation of pro-apoptotic effects of CRISPR-mediated knockout of CNB1 (CB1) or
CNB2 (CB2) following THC treatment. (Kampa-Schittenhelm et al, 2016).
Because CRISPR-Cas9 can achieve complete silencing of target genes, many research
groups have employed a CRISPR-Cas9 system in large-scale, loss-of-function screens in
blood cancer cells. One earlier study established a lentivirus-mediated library containing 73
000 sgRNAs to generate knockout collections for genome-wide screening in myeloid
leukaemia cells (Wang et al, 2014). This system provides several advancements: (i)
CRISPR-Cas9-mediated gene depletion leads to a complete loss of gene function, which
enables the targeting of non-coding regions that are conventionally considered non-
targetable by RNAi; (ii) most sgRNAs can efficiently create mutations at target sites with
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perfect discrimination between true and false positives at the significance threshold; (iii) the
screens showed minimal off-target effects. This study pioneered whole-genome analysis
using sgRNA-mediated screens in blood cancers. Recently, Vakoc and his team designed an
sgRNA library to target 192 chromatin regulatory domains (Shi et al, 2015). From this
screen, they discovered 6 known drug targets and 19 novel binding domains in murine AML
cells. This sgRNA-targeted screening required 2 weeks to unveil the 6 previously described
drug targets, an achievement that had taken researchers over half a century to identify using
conventional methods (Ricks, 2015). Applications of CRISPR-Cas9 in haematological
malignancies are summarized in Table II and Fig 2.
Secondary haematological disorders

Chronic granulomatous disease—Chronic granulomatous disease (CGD) is a rare
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genetic immunodeficiency disorder of phagocytic cells (Seger, 2008). The abnormalities of

nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits result in the lack
of an anti-pathogen oxidative burst, thereby leading to recurrent life-threatening bacterial
and fungal infections (Nienhuis, 2013). So far, only one reported study has utilized CRISPR-
Cas9 in CGD. Flynn et al (2015) corrected a single point mutation (T>G) at the end of the
first intron of the CYBB gene using CRISPR-Cas9. Mutation of this gene is responsible for
most cases of CGD. Surprisingly, the gene correction resulted in the restoration of oxidative

burst function in iPSCs derived from a CGD patient, offering a potential new gene
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therapeutic approach to CGD treatment using CRISPR-Cas9.
Human immunodeficiency virus (HIV) infection—HIV infection has been associated

with a broad range of haematological disorders (Scadden et al, 1989). Before the advent of
CRISPR-Cas9 technology, ZFN-induced disruption of the CCR5 gene, an essential HIV-1
co-receptor required for virus entry, had become a promising gene therapy and was
evaluated in clinical trials (Tebas et al, 2014). Recent advances in CRISPR-Cas9 have
further revolutionized studies of HIV-1. Since Cho et al (2013) first tested the DNA cleavage
activity of a CRISPR-Cas9 system for silencing the CCR5 gene in vitro, several studies have
reported CRISPR-Cas9-mediated disruption of CCR5 (Cradick et al, 2013; Yang et al, 2013;
Ye et al, 2014) and other co-factors, including CXCR4 (Hou et al, 2015) and XPO1
(exportin 1) (Boons et al, 2015).
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Despite much progress in combined antiviral therapy, it remains a significant challenge to

completely eradicate HIV latent infection. One strategy to achieve this goal is to remove the
proviral HIV-1 DNA from the host cell genome, and several research groups have reported
the potential of the CRISPR-Cas9 system to disrupt latent HIV-1 provirus by targeting the
long terminal repeat (LTR) region (Ebina et al, 2013; Hu et al, 2014; Zhu et al, 2015;
Kaminski et al, 2016). However, the feasibility of this approach in HIV therapy has been
questioned by the latest finding that showed viral escape from CRISPR gene-editing attack
due to the Cas9/sgRNA-derived mutations (Wang et al, 2016). Last year, investigators fused
a synergistic activation mediator (SAM) to dCas9 to target the enhancer of the LTR
promoter in HIV-1 latent cells. This target-specific compulsory reactivation led to cellular
suicide of viral proteins in HIV-1-infected cells and provided a novel therapeutic tool for the
permanent eradication of the HIV virus (Zhang et al, 2015b). More recently, another study
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identified a targeted hotspot for CRISPR activation. Among five effective sgRNA candidates
targeting different regions of the LTR, sg362F was the most effective for stimulating potent
transcriptional activation of latent HIV-1 infection in human T cells. Unlike chemical-
activating agents, CRISPR-dCas9-mediated reactivation of HIV-1 is specific in all latency
models and does not induce off-target effects (Saayman et al, 2016).
Epstein-Barr virus (EBV) infection—Wang and Quake (2014) first used CRISPR-Cas9
as a novel nuclease system for targeting key EBV-DNA in a Burkitt lymphoma (BL) cell
line. Remarkably, nearly 25% of the treated cells were completely cleared of EBV with no
detectable viral DNA, and another 50% of cells showed a substantial decrease in EBV load
relative to the untreated samples. To apply this strategy to clinical therapy, a safer and more
efficient delivery system for genome editing in patients will be required.
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Malaria—Since the CRISPR-Cas9 was successfully applied in Drosophila in vivo (Gantz &
Bier, 2015), scientists have attempted to apply this system to target either malaria parasites
(Ghorbal et al, 2014; Zhang et al, 2014) or malaria mosquitoes (Gantz & Bier, 2015;
Hammond et al, 2016). In one representative study, researchers identified a gene that is
required for female fertility in Anopheles mosquitoes and inserted it into a CRISPR-Cas9
gene-drive construct targeting female fertility loci (Hammond et al, 2016). This technique
impaired the fertility of female mosquitoes, leading to extinction of the population over

several years. In another study, parasite transmission was hindered by insertion of an anti-
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malarial gene into malaria mosquitoes using CRISPR-Cas9 (Gantz et al, 2015). This strategy
decreased the spread of malaria while maintaining the integrity of the mosquito species.
Although the same genome-editing tool was applied in these two studies, distinct endings
(extinct vs reserve species) might be achieved (Pennisi, 2015; Alphey, 2016).
Leishmaniasis—Last year, one research group from France reported the first efficient
CRISPR-Cas9-mediated genome editing in Leishmania parasites (Sollelis et al, 2015). They
incorporated the CRISPR-Cas9 system into Leishmania major by disrupting the
paraflagellar rod (PFR)-2 loci, the major constituent of the trypanosomatid-specific
filamentous network. Targeting PFR-2 genes, the authors obtained parasites in a single
round of transfection within only 1 month; importantly, no off-targets effects were detected.
Another group subsequently reported the successful implantation of the CRISPR-Cas9
technology in Leishmania donovani (L. donovani). Using this efficient system, the authors
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introduced drug selection markers and the GFP tag precisely into the L. donovani genome,
leading to improved isolation of gene disruption mutants. (Zhang & Matlashewski, 2015).
Collectively, these landmark reports have facilitated the use of CRISPR-(d)Cas9 for the
treatment of secondary haematological disorders (Table III, Fig 2).
Challenges of CRISPR-Cas9 technology

Technical limitations
CRISPR-Cas9-mediated gene modification can be achieved via transient or stable delivery
of the CRISPR components. Transient transfection in cells has been reported in several
studies (Cho et al, 2013; Cong et al, 2013; Mali et al, 2013a), although viral-based
transfection of CRISPR components is a more efficient alternative to generate stably-
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modified cells (Malina et al, 2013; Heckl et al, 2014; Chen et al, 2016; Zhang et al, 2016b).
Nevertheless, the delivery efficiency remains a significant challenge in hard-to-transfect
blood cancer cells, especially when electroporation is used as a delivery method. To
overcome these limitations, Han et al (2015) developed rapid cell mechanical deformation.
This technique generates transient holes in the membrane that enable the delivery of
biomaterials via the medium. This approach achieves high delivery efficiency in different
cell types, including lymphoma cells and embryonic stem cells, broadening the cell type
applicability of transient transfection of CRISPR plasmids. In addition to transient
transfection, viral transfection is frequently used to deliver CRISPR-Cas9 system into
haematological malignant cancer cells, although some technical issues persist, such as
relatively low infection rates.
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The critical role of target genes for cell survival is an additional challenge. For example,
MCL1, an anti-apoptotic BCL2 family member, is required for the sustained survival of
human BL cell lines because silencing of MCL1 leads to rapid cell death. In this case, the
drug-inducible CRISPR-Cas9 lentiviral system provides an option to identify the functions
of tumour-essential genes (Aubrey et al, 2015).
Due to the increased utilization of the NHEJ pathway over HDR in mammalian cells, the
low efficiency of CRISPR-Cas9-induced HDR is an additional limitation (Mali et al, 2013a;

Wang et al, 2013). Therefore, increasing the efficiency of HDR facilitates the rapid
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generation of cell lines with precisely edited genes. To resolve this issue, a recent study
identified a RAD51-stimulatory compound, RS-1, which enhances the efficiency of Cas9-
stimulated HDR by 3- to 6-fold (Pinder et al, 2015).To some extent, this discovery improves
efficiency, although greater improvement will be needed in the future.
Off-target effects
With more successful applications of CRISPR-Cas9 in mammalian cells, the issue of
specificity remains a major concern. In 2013, Nature Biotechnology simultaneously
published a series of letters regarding NHEJ-mediated off-target mismatches in human cells
(Fu et al, 2013; Hsu et al, 2013; Mali et al, 2013b; Pattanayak et al, 2013). To overcome
these hurdles, several methods have been developed to detect off-target effects. Deep
sequencing (Fu et al, 2013; Hsu et al, 2013) and whole genome sequencing (Smith et al,
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2014; Veres et al, 2014) have been considered options to identify mismatches; however, the
possibility of overlooking off-target sites and the higher cost make it difficult to perform this
step in every experiment. Alternatively, a series of online bioinformatics tools were
developed to predict off-target mutation sites (Ran et al, 2013; Fine et al, 2014; Heigwer et
al, 2014; Xie et al, 2014b). However, these computational methods failed to predict bona
fide off-target integration sites (Tsai et al, 2015). Instead, a novel method called Genome-
wide Unbiased Identification of DSBs Enabled by Sequencing (GUIDE-seq) provides an
unbiased and genome-wide method for detecting CRISPR RNA-guided DSBs in cells (Tsai
et al, 2015). In addition, other approaches, such as modified high-throughput genome-wide
translocation sequencing (HTGTS), and the integrase-defective lentiviral vector (IDLV)
assay, were developed to identify off-target effects produced by CRISPR-Cas9 (Frock et al,
2015; Wang et al, 2015). These pioneering studies have increased the technical feasibility of
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off-target detection (Koo et al, 2015; Bolukbasi et al, 2016), although ‘it is impossible to
draw broad conclusions about the specificity of engineered nucleases’, as recently reported
(Gabriel et al, 2015).
Knockdown or knockout?
Although CRISPR-Cas9 provides a tool for complete silencing, gene knockdown can still be
a valuable tool depending on the experimental objectives. Technically, shRNA-mediated
gene knockdown does not require single clone isolation, whereas CRISPR-Cas9-based gene
silencing usually involves a long duration to generate stable cell lines. Despite many
concerns about which method is more appropriate for delineating gene functions, genome-
editing technology is the apparent option for creating a true genetic null allele, introducing a
point mutation, and correcting a specific mutation (Ul Ain et al, 2015). Currently, more
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CRISPR-Cas9-based platforms have evolved to meet different demands. For example, DNA-
free CRISPR-Cas9 gene editing can be used to avoid potential integration events; the
specificity of the CRIPSR system can be enhanced using paired Cas9 nickases by targeting
two sites on opposite DNA strands; a CRISPR vector with a fluorescent marker has become
available for positive cell selection; and an inducible Cas9 expression vector has increased
experimental flexibility. These novel CRISPR-Cas9 platforms facilitate its application for
more investigative purposes and, ultimately, for therapeutic use.

Collectively, the challenges of CRISPR-Cas9 technology discussed herein only reflect those
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limitations of the studies in vitro, ex vivo or in vivo. The greatest challenge is yet to come.
Many questions remain unanswered regarding the use of this technology in humans. For
example, which delivery system is suitable for humans? How will the human immune
system respond to the administration of genome editing tools in vivo? How can off-target
effects be minimized rather than predicted or detected upon application in humans? With
these uncertainties, further applications in gene and cell therapies will require a long
duration to be fully realized (Maeder & Gersbach, 2016).
Future directions and prospects

Hereditary blood disorders, such as haemoglobin abnormalities, anaemias or coagulation
disorders, represent ideal targets for gene therapy with genome editing technology because
the partial restoration of gene functions are sufficient to reverse the symptoms. Although
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scientists are striving to unlock new possibilities of CRISPR-Cas9 in different areas, for
example, to eliminate pathogen-carrying species (e.g., malaria and Zika), to resurrect a
woolly mammoth, or to make micro-pig pets even designer babies (Reardon, 2016),
legitimate concerns are arising due to its simple and highly versatile potential to edit the
entire genome of the creatures. Last year, a team of researchers, for the first time, applied
CRISPR-Cas9 to edit the endogenous HBB genes in human embryos; however, the edited
embryos were all mosaic due to the low efficiency of HDR (Liang et al, 2015). Therefore,
exploration in human cells can also raise bioethical issues.
Any intentional or unintentional misuse of this technology may result in unexpected effects
on future generations and lead to substantial or even permanent changes in the human gene
pool. Thus, the international summit on human gene editing was held in Washington, D.C.
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last winter, where the closing statement recommended the rigorous continuation of basic and
preclinical research and emphasized that any clinical use of germline editing is irresponsible
(Olson, 2016).
In conclusion, CRISPR-Cas9 technology has sparked advancements in haematological

disorders. The scientific breakthroughs with respect to programmable nucleases will allow
us to delineate the mechanisms underlying the development of blood diseases and eventually
contribute to the alleviation of disease symptoms or the cure of certain diseases via safe and
controllable gene therapy.
Acknowledgments
This work is supported by R01 (R01CA181319, NCI) and American Society Scholar Award (RSG-11-157) given to
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NM.
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latent HIV-1 proviral DNA. Retrovirology. 2015; 12:22. [PubMed: 25808449]
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Fig 1.
Key discoveries and advances in the development of the CRISPR-Cas9 system. CRISPR,
clustered regularly interspaced short palindromic repeats; Cas, CRISPR-associated; DSBs,
DNA double-stranded breaks; crRNAs: CRISPR RNAs; tracrRNA, trans-activating CRISPR
RNA; sgRNA, single-guide RNA; dCas9, inactive Cas9; RCas9, RNA-targeting Cas9.
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Fig 2.
Applications of CRISPR-(d)Cas9 in haematological disorders. Hereditary blood disorders,
such as haemoglobin abnormalities, anaemias or coagulation disorders represent ideal
targets for CRISPR-Cas9-mediated gene therapy. Genetic controls of cells with CRISPR-
Cas9 also allow us to delineate the molecular mechanisms underlying the development of
Author Manuscript
blood diseases, and further contribute to the alleviation or cure of human diseases. CRISPR,
clustered regularly interspaced short palindromic repeats; dCas9, inactive Cas9; EBV,
Epstein–Barr virus; HIV, human immunodeficiency virus; HSPCs, haematopoietic stem and
progenitor cells; iPSCs, induced pluripotent stem cells.
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Table I
Applications of CRISPR-Cas9 technology in non-cancerous haematological disorders.
Off-target
Targeted genes Cell types Genome editing effects Outcomes/comments References
β-thalassaemia HBB Patient-derived iPSCs HDR-induced correction No Restored HBB expression and (Xie et al, 2014a)
exhibited normal karyotypes
Zhang and McCarty
HBB Patient-derived iPSCs HDR-induced correction No Restored HBB expression and (Song et al,
induced haematopoietic 2015)
differentiation
HBB Patient-derived iPSCs HDR-induced correction More Higher efficiency of CRISPR-Cas9- (Xu et al, 2015)
induced DSBs, but lower efficiency
of homologous gene targeting
compared to TALENs
HBB Patient-derived naïve iPSCs HDR-induced correction No Naïve iPSCs exhibited improved (Yang et al,
gene-correction efficiencies 2016)
BCL11A (enhancer) CD34+ HSPC-derived erythroid NHEJ-induced knockout N/A Resulted in near-complete loss of (Canver et al,
precursor cell line BCL11A expression and induction 2015)
of γ-globin and HbF protein
HBB Human embryos HDR-induced correction Apparent Mosaicism and mutations at non- (Liang et al,
target sites were apparent in the 2015)
edited embryos
Sickle-cell disease HBB Patient-derived iPSCs HDR-induced correction Less Corrected a sickle point mutation (Huang et al,
2015)
Ehmt2 Murine erythroleukaemia cells NHEJ-induced knockout N/A Increased the expression of Hbb-εy (Renneville et al,
and Hbb-βh1 embryonic globin 2015)
genes
Fanconi anaemia FANCC Patient-derived fibroblast cells HDR-induced correction No Corrected a Fanconi anaemia point (Osborn et al,
mutation 2015)
Diamond-Blackfan anaemia tp53 Zebrafish embryos NHEJ-induced knockout Maybe Rescued haematological defects in a (Ablain et al,
zebrafish DBA model 2015)

Thrombocytopenia ITGB3 Polyploid megakaryocytic cells HDR-induced knock-in No Converted from HPA-1a to HPA-1b (Zhang et al,
and iPSCs 2016a)
CMAH, GGTA1 Pigs NHEJ-induced knockout N/A Revealed different responses of (Butler et al,
porcine organs to human platelets 2016)
Haemophilia F8 Patient-derived iPSCs HDR-induced correction No Rescued the FVIII deficiency in HA (Park et al, 2015)
mice
F9 Mouse HDR-induced knock-in/correction No Identified a novel Y371D mutation (Guan et al,
in F9 gene; targeting Y371D 2016)
mutation could restore haemostasis
in HB mice
Myeloproliferative neoplasm JAK2 Patient-derived iPSCs HDR-induced correction No Specifically targeted the mutant (Smith et al,
allele 2015)
Page 26
Off-target
B-/T-lymphopenia Kdelr1 Mouse NHEJ-induced knockout N/A Reduced expression of TCR and (Siggs et al,
increased expression of CD44 in 2015)
Kdelr1 mutant mice
DBA, Diamond-Blackfan anaemia; DSBs, double strand breaks; HA, haemophilia A; HB, haemophilia B; HDR, homology-directed repair; HPA, human platelet antigen; iPSCs, induced pluripotent stem
cells; N/A, not applicable; NHEJ, non-homologous end joining; TALENs, transcription activator- like effector nucleases; TCR, T cell receptor.
Zhang and McCarty

Page 27
Table II
Applications of CRISPR-Cas9 technology in haematological malignancies.
Off–
target
Lymphoma & Myeloma Trp53 Mouse lymphoma cells NHEJ-induced knockout Limited Established the virus-based CRISPR-Cas9 (Malina et al, 2013)
Zhang and McCarty
mediated ex vivo genome editing in the

lymphoma mouse model
BBC3 (PUMA) Human cancer cell lines NHEJ-induced knockout N/A Impaired treatment responses to NUTLIN3A (Valente et al, 2016)
CXCR4 Human MCL cell lines NHEJ-induced knockout N/A Failed to induce autophagy formation upon (Chen et al, 2016;
treatment Zhang et al, 2016b)
MUC1-C Human MM cell lines NHEJ-induced knockout N/A Downregulated MYC mRNA and protein (Tagde et al, 2016)
expression
MCL1 Human BL cell lines NHEJ-induced knockout N/A Identified tumour-essential genes using inducible (Aubrey et al, 2015)
CRISPR-Cas9
Leukaemia Runx1, etc. Mouse HSPCs NHEJ-induced knockout No Generated the virus-based CRISPR-Cas9 (Heckl et al, 2014)
mediated ex vivo genome editing for modelling of
myeloid malignancies
KMT2C (MLL3) Mouse HSPCs NHEJ-induced knockout N/A KMT2C (MLL3) is a haploinsufficient tumour (Chen et al, 2014)
suppressor in AML
EPAS1 (HIF-2α) Human AML cell lines NHEJ-induced knockout N/A Loss of EPAS1 alone had no impact on human (Vukovic et al, 2015)
AML cells under hypoxic conditions
Ly6c2, etc. Murine AML cell lines NHEJ-induced knockout Little Identified genes that are associated with (Rathe et al, 2014)
cytarabine resistance in AML
Cebpa(enhancer) Murine myeloid cell line HDR-induced knock-in N/A Mutated enhancer reduced C/EBPα expression, (Cooper et al, 2015)
indicating the importance of the enhancer for
myeloid Cebpa gene regulation
SRSF2 Human CML cell line HDR-induced knock-in N/A Mutant SRSF2 misregulated hundreds of splicing (Zhang et al, 2015a)
events, thereby providing a group of mis-spliced

isoforms for potential therapeutic targeting
ASXL1 Human CML cell line HDR-induced correction No Restored ASXL1 expression in CML cells and (Valletta et al, 2015)
increased survival in mice xenografts
TAL1 Human T-ALL cell line HDR-induced knock-in N/A Induced a selective epigenetic switch (Navarro et al, 2015)
TRIB1 Human T-ALL cell line NHEJ-induced knockout N/A Loss of TRIB1 reduced interleukin-2 expression (Miyajima et al, 2015)
CNB1, CNB2 Human T-ALL, AML, CML cell NHEJ-induced knockout N/A Resulted in abrogation of pro-apoptotic effects (Kampa-Schittenhelm
lines upon THC treatment et al, 2016)
sgRNAs pool Human myeloid leukaemia cell NHEJ-induced knockout Minimal Initiated sgRNA-mediated screens for whole- (Wang et al, 2014)
lines genome analysis in human cells
sgRNAs pool Murine AML cell line NHEJ-induced knockout Low Discovered 6 known drug targets and 19 novel (Shi et al, 2015)
binding domains in murine AML cells
Page 28
AML, acute myeloid leukaemia; BL, Burkitt lymphoma; CML, chronic myeloid leukaemia; HDR, homology-directed repair; HSPCs, haematopoietic stem and progenitor cells; MCL, mantle cell
lymphoma; MM, multiple myeloma; N/A, not applicable; NHEJ, non-homologous end joining; sgRNA, single-guide RNA; T-ALL, T-cell acute lymphoblastic leukaemia; THC, Tetrahydrocannabinol
(Dronabinol).
Zhang and McCarty

Page 29
Table III
Applications of CRISPR-(d)Cas9 technology in secondary haematological disorders.
Off-target
CGD CYBB Patient-derived iPSCs HDR-induced correction 35% (21/60) Resulted in the recovery of ROS activity (Flynn et al, 2015)
Zhang and McCarty
HIV infection CCR5 HEK 293T cell line NHEJ-induced knockout No First tested the CRISPR-Cas9 system to knockout (Cho et al, 2013)
CCR5 gene in vitro
CCR5, HBB HEK 293T cell line NHEJ-induced knockout More Resulted in a wide variety of indels and point (Cradick et al, 2013)
mutations
CCR5 Human iPSCs NHEJ-induced knockout and N/A Optimized the process for genome editing in (Yang et al, 2013)
HDR-induced correction human stem cell
CCR5 Human iPSCs HDR-induced knock-in No PiggyBac technology plus CRISPR-Cas9 (Ye et al, 2014)
mediated the natural CCR5 mutation in human
iPSCs
CXCR4 Primary human CD4+ T cells NHEJ-induced knockout No Conferred cell resistance to HIV-1 infection (Hou et al, 2015)
and T cell line
XPO1 HEK 293T cell line HDR-induced knock-in N/A XPO1 mutants failed to affect viral replication (Boons et al, 2015)
upon treatment
HIV-1 (LTR region) Human 293T, Hela and T cell NHEJ-induced indels N/A Restricted transcriptionally active provirus and (Ebina et al, 2013)
lines blocked the expression of latently integrated
provirus
HIV-1 (LTR region) Human microglial, NHEJ-induced indels No Completely excised integrated proviral DNA; (Hu et al, 2014)
promonocytic and T cell lines inactivated viral gene expression and replication
in HIV latent cells
HIV-1 (LTR, pol, rev) HIV-GFP Jurkat cell line NHEJ-induced indels N/A Efficiently mutated and deactivated HIV-1 (Zhu et al, 2015)
proviral DNA in latently infected Jurkat cells
HIV-1 (LTR region) Human T cell line and NHEJ-induced indels No Excised integrated proviral DNA and eliminated (Kaminski et al, 2016)
primary T cells HIV-1 production in infected cells; reduced viral
replication in primary T cells

HIV-1 (LTR, pol, rev) SupT1 CD4+ T cell line NHEJ-induced indels Many indels HIV-1 escaped from CRISPR-Cas9-mediated (Wang et al, 2016)
suppression due to the Cas9/sgRNA-derived
mutations in the viral DNA sequence
HIV-1 (LTR region) Human microglial and T cell dCas9-induced activation No Reactivated HIV-1 latent viruses and led to (Zhang et al, 2015b)
lines suicide of the HIV-1-infectd cells
HIV-1 (LTR region) HEK 293T and human T cell dCas9-induced activation No Identified a targeted hotspot for CRISPR-dCas9 (Saayman et al, 2016)
lines activation, leading to potent transcriptional
activation of latent HIV infection
EBV infection EBV BL cell lines NHEJ-induced indels No Led to proliferation arrest and apoptosis in EBV- (Wang & Quake,
infected cells 2014)
Malaria Pysera1, etc. Parasite P. yoelii NHEJ-induced knockout No CRISPR-Cas9 can be applied to modify the (Zhang et al, 2014)
rodent malaria P. yoelii genome
Page 30
Off-target
Egfp, etc. Parasite P. falciparum NHEJ-induced knockout and No Specific gene knockouts and single-nucleotide (Ghorbal et al, 2014)
HDR-induced knock-in substitutions can be achieved in P. falciparum via
CRISPR-Cas9
AGAP007280, etc. Anopheles gambiae mosquito NHEJ-induced knockout and N/A Gene disruption conferred a recessive female- (Hammond et al,
embryos HDR-induced knock-in sterility phenotype; gene insertion at female- 2016)
fertility loci showed highly efficient gene drive
Zhang and McCarty
activity and can spread in a caged population

m2A10-m1C3 Anopheles stephensi HDR-induced knock-in No Blocked parasite development and modified (Gantz et al, 2015)
mosquito embryos mosquito population
Leishmaniasis PFR2 Parasite L. major NHEJ-induced knockout No Got null parasites in a single round of (Sollelis et al, 2015)
transfection
LdMT, etc. Parasite L. donovani NHEJ-induced knockout and N/A Identified a novel single point mutation caused by (Zhang &
HDR-induced knock-in CRISPR-Cas9 in LdMT; precisely insert a drug Matlashewski, 2015)
selection marker into genome
BL, Burkitt lymphoma; CGD, chronic granulomatous disease; EBV, Epstein–Barr virus; HDR, homology-directed repair; HEK, human embryonic kidney; HIV, human immunodeficiency virus; indels,
insertions or deletions; iPSCs, induced pluripotent stem cells; LTR, long terminal repeat; N/A, not applicable; NHEJ, non-homologous end joining; ROS, reactive oxygen species.

Page 31

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CRISPR-Cas9 technology and its application in haematological

current challenges and future directions are reviewed and discussed.

application difficult. In subsequent decades, targeting of DNA double-stranded breaks

Development of the CRISPR-Cas9 system

Br J Haematol. Author manuscript; available in PMC 2018 April 06.

Cas9 systems on a novel stage for genome engineering.

Further modifications of the CRISPR system have been reported. Qi et al (2013)

called CRISPR interference (CRISPRi). Unlike RNAi-based silencing, which destructs

Br J Haematol. Author manuscript; available in PMC 2018 April 06.

Applications of CRISPR-Cas9 technology in haematological disorders

Non-cancerous haematological disorders

genomic DNA. Nevertheless, insertional activation of protooncogenes remains a major

Similar to virus-mediated gene therapy, CRISPR-Cas9-mediated genome editing is used to

system compared with primed iPSCs (Yang et al, 2016).

In addition to correcting β-thalassaemia-causing mutations, other interesting attempts have

Br J Haematol. Author manuscript; available in PMC 2018 April 06.

Sickle-cell disease—Along with β-thalassaemia, SCD remains a global health burden,

SCD (Renneville et al, 2015).

Fanconi anaemia—Fanconi anaemia (FA) is a genetic DNA repair-deficient human

To overcome the limitations of HSCT-based treatment in FA patients, gene therapies have

Br J Haematol. Author manuscript; available in PMC 2018 April 06.

Diamond-Blackfan anaemia—Diamond-Blackfan anaemia (DBA) is a rare inherited

In addition to bone marrow transplantation, glucocorticoid hormones as well as other

Thrombocytopenia—Thrombocytopenia refers to a blood disorder that involves a

Another study focused on the thrombocytopenia caused by solid-organ xenotransplantation

Haemophilia—Haemophilia refers to a group of hereditary disorders of blood coagulation

Br J Haematol. Author manuscript; available in PMC 2018 April 06.

phenotype, whereas correction of this mutation by CRISPR-Cas9-mediated in situ genome

Others—Similar to human blood disorders, genetic correction using a CRISPR-Cas9

has not yet been elucidated. Applications of a CRISPR-Cas9 system to non-cancerous

Malignant haematological disorders

Malina et al (2013) first established the lentiviral/retroviral-based ‘all-in-one’ CRISPR-

Br J Haematol. Author manuscript; available in PMC 2018 April 06.

knockout cells generated by CRISPR-Cas9 (Zhang et al, 2016b). To summarize, the

CRISPR-Cas9 system generates numerous exciting opportunities to unravel the mechanisms

Myeloma—Multiple myeloma (MM) is the second most frequent haematological

Wu et al (2014) first reported genome-editing in MM cells. The authors discovered that

application of CRISPR-Cas9 to generate ex vivo genome editing to delineate the complexity

Br J Haematol. Author manuscript; available in PMC 2018 April 06.

transformation. In human myeloid leukaemia K562 cells, mutation of CRISPR-Cas9-

Secondary haematological disorders

genetic immunodeficiency disorder of phagocytic cells (Seger, 2008). The abnormalities of

Br J Haematol. Author manuscript; available in PMC 2018 April 06.

therapeutic approach to CGD treatment using CRISPR-Cas9.

Human immunodeficiency virus (HIV) infection—HIV infection has been associated

Despite much progress in combined antiviral therapy, it remains a significant challenge to

Br J Haematol. Author manuscript; available in PMC 2018 April 06.

Challenges of CRISPR-Cas9 technology

Br J Haematol. Author manuscript; available in PMC 2018 April 06.

Br J Haematol. Author manuscript; available in PMC 2018 April 06.

Future directions and prospects

In conclusion, CRISPR-Cas9 technology has sparked advancements in haematological

Br J Haematol. Author manuscript; available in PMC 2018 April 06.

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Br J Haematol. Author manuscript; available in PMC 2018 April 06.

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guided regulation of transcription in eukaryotes. Cell. 2013; 154:442–451. [PubMed: 23849981]

nucleases. Nature Biotechnology. 2013; 31:827–832.

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repeats in prokaryotes. Molecular Microbiology. 2002; 43:1565–1575. [PubMed: 11952905]