Review

CRISPR/Cas9: Transcending
the Reality of Genome Editing
Sergiu Chira,1 Diana Gulei,2 Amin Hajitou,3 Alina-Andreea Zimta,1 Pierre Cordelier,4
and Ioana Berindan-Neagoe1,2,5
1Research Center for Functional Genomics, Biomedicine, and Translational Medicine, “Iuliu Hatieganu” University of Medicine and Pharmacy, Cluj-Napoca, Cluj 400377,
Romania; 2MedFuture Research Center for Advanced Medicine, “Iuliu Hatieganu” University of Medicine and Pharmacy, Cluj-Napoca, Cluj 400377, Romania;
3Cancer Phage Therapy Group, Division of Brain Sciences, Imperial College London, London SW7 2AZ, UK; 4Cancer Research Center of Toulouse, Université Fédérale
Toulouse Midi-Pyrénéées, Université Toulouse III Paul Sabatier, INSERM, 31100 Toulouse, France; 5Department of Functional Genomics and Experimental Pathology,
The Oncology Institute “Prof. Dr. Ion Chiricuta,” Cluj-Napoca, Cluj 400015, Romania

With the expansion of the microbiology field of research, a new effector nucleases (TALENs), in terms of simplicity of design and
genome editing tool arises from the biology of bacteria that versatility1–3 (Table 1). By 2013, the ability of the CRISPR/Cas9 sys-
holds the promise of achieving precise modifications in the tem to engineer mammalian cell genomes has been experimentally
genome with a simplicity and versatility that surpasses previous validated, and the crystal structure of the Cas9 effector complex
genome editing methods. This new technique, commonly was resolved in 2014.4 Among the three CRISPR/Cas systems
named CRISPR/Cas9, led to a rapid expansion of the (I–III) identified in both bacteria and archaea, the type II system
biomedical field; more specifically, cancer characterization from Streptococcus thermophilus or Streptococcus pyogenes is the
and modeling have benefitted greatly from the genome editing most versatile for genome engineering purposes.5
capabilities of CRISPR/Cas9. In this paper, we briefly summa-
rize recent improvements in CRISPR/Cas9 design meant to The CRISPR/Cas9 type II system consists of the Cas9 nuclease and a
overcome the limitations that have arisen from the nuclease ac- single guide RNA (sgRNA or gRNA), which is a fusion of a CRISPR
tivity of Cas9 and the influence of this technology in cancer RNA (crRNA) and a trans-activating crRNA (tracrRNA) that binds
research. In addition, we present challenges that might impede Cas9 nuclease and directs it to a target sequence based on a comple-
the clinical applicability of CRISPR/Cas9 for cancer therapy mentary base-pairing rule. The target sequence must be adjacent
and highlight future directions for designing CRISPR/Cas9 de- to a protospacer-adjacent motif (PAM) consisting of a canonical
livery systems that might prove useful for cancer therapeutics. NGG or NAG sequence. At the recognition site, a double-strand
break (DSB) is generated that can be repaired by non-homologous
The increasing burden of cancer in the human population represents end joining (NHEJ), resulting in small insertions or deletions (indels)
a major concern for our society, and finding alternative treatments usually associated with loss of function (knockdown/knockout) (Fig-
that are safe as well as efficient has become a major goal for re- ure 1). In the presence of an exogenous donor DNA, by a homology-
searchers around the world. For the past decades, we have witnessed directed recombination (HDR) mechanism, precise modifications
an effervescence of technologies that explore DNA structure and can be achieved at the targeted site, resulting in gain of function
function, and our understanding of cancer has expanded to an extent (knockin).6
that enables characterization of this disease at a deeper molecular
level. The progression of basic research to reach clinical applications However, Cas9 can tolerate, to a certain extent, mismatches between
necessitates reliable pre-clinical models of cancer in which therapeu- the sgRNA and the target sequence in the genome, resulting in off-
tic strategies and agents can be evaluated for efficacy or efficiency. target effects, as some previous studies have shown.7,8 These undesir-
able effects of the CRISPR/Cas9 system might impede the use of this
Introducing targeted modifications in the genome for functional genome editing technology for clinical applications; therefore, a great
studies and cancer modeling or, moreover, for therapeutic purposes,
requires highly efficient systems that are able to alter the existing
DNA pattern with great precision. Nucleases of bacterial origin repre- http://dx.doi.org/10.1016/j.omtn.2017.04.001.
sent powerful tools that have been widely used for genome engineer- Correspondence: Sergiu Chira, Research Center for Functional Genomics,
Biomedicine, and Translational Medicine, “Iuliu Hatieganu” University of Medi-
ing purposes in an attempt to studying gene function and, ultimately, cine and Pharmacy, 23 Gheorge Marinescu Street, 8 Victor Babes Street, Cluj-
to implement new therapeutic strategies. Napoca, Cluj 400377, Romania.
E-mail: [email protected]
Correspondence: Pierre Cordelier, Cancer Research Center of Toulouse, Université
Among the existing nucleases with genome editing capabilities,
Fédérale Toulouse Midi-Pyrénéées, Université Toulouse III Paul Sabatier, INSERM,
the CRISPR system surpasses other nuclease-based systems, such 31100 Toulouse, France.
as zinc-finger nucleases (ZFNs) and transcription activator-like E-mail: [email protected]

Molecular Therapy: Nucleic Acids Vol. 7 June 2017 ª 2017 The Author(s). 211
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
www.moleculartherapy.org

Review

Table 1. Comparison of ZFNs, TALENs, and CRISPR/Cas9 Nuclease Systems for Genome Editing

Genome Editing Tool ZFNs TALENs CRISPR/Cas9

Features ZF-FokI nuclease protein fusion TALE-FokI nuclease protein fusion Cas9 nuclease and sgRNA
RNA-DNA interaction, site selection restricted by
protein-DNA interaction, capable of targeting protein-DNA interaction, capable of targeting
Target site identification the NGG motif, which occurs statistically every
virtually any site virtually any site
8 nt in a random DNA sequence
DSBs directed by the FokI domain, repaired DSBs directed by the FokI domain, repaired DSBs directed by Cas9, repaired through NHEJ
Genome altering
through NHEJ or HDR through NHEJ or HDR or HDR
+ ++ +++
Design custom design based on the target sequence, custom design based on the target sequence, labor very simple design by altering the crRNA
labor intensive and time-consuming intensive, less time-consuming than ZFN sequence of the sgRNA
Efficiency ++ ++ +++
Biallelic targeting ++ ++ +++
Off-target effects ++ +++ +
low compared with ZFN and TALEN as a result of
specific more specific than ZFN allowed mismatches by the Cas9 nuclease between
sgRNA and the DNA target sequence
+ + +++
Multiplexing yes, capable of targeting multiple sites
rarely used rarely used
simultaneously
+++ ++ +
Size and delivery the ZFN monomer is significantly smaller TALEN monomers are situated in the middle massive Cas9 protein encoded by a 4.2-kb
than Cas9 of Cas9 and ZFN nucleases in terms of size sequence

deal of effort has been made to improve the efficiency and specificity to Cas9 nuclease, thereby reducing the off-target effects the CRISPR
of CRISPR/Cas9. system exhibits under normal conditions. Placing Cas9 under the con-
trol of a tetracycline-responsive element (TRE) promoter confers the
In this review, we summarize recent improvements made in CRISPR/ possibility to achieve a conditional expression in the presence of tetra-
Cas9 design and flexibility and the applications of this genome editing cycline/doxycycline (Tet/Dox).12,13 However, even in the off state,
technology for cancer research. At the end of the manuscript, we discuss when Tet/Dox are absent, Cas9 expression still exhibits “leakiness,”
challenges and future perspectives of CRISPR/Cas9 in cancer therapy. as recently reported.14 Placing the sgRNA under control of a hybrid
Tet-responsive promoter proved to be a more advantageous alternative
Minimizing the Off-Target Effects of CRISPR/Cas9 to obtain a tighter control of the off-target cleavage in a Dox-dependent
Optimizing the sgRNA design represents one approach for reducing manner.14 Regardless whether Cas9 or sgRNA expression is regulated
the off-target effects of CRISPR/Cas9. It has been shown that the base in a Dox-dependent manner, such inducible systems require additional
composition of the 50 sequence of the sgRNA can have a profound trans-acting factors and promoters to be incorporated in the construc-
effect on the efficiency of CRISPR/Cas9,9 and large-scale studies tion of the delivery system, which, in the case of adeno-associated virus
of sgRNA libraries led to design algorithms for maximizing the on- (AAV) vectors, is quite problematic because of their limited cloning ca-
target activity and reducing off-target effects.10 Several online tools pacity.15 Therefore, inducible CRISPR/Cas9 systems that use minimal
are now available to assist researchers in designing sgRNAs with a genetic elements to achieve conditional expression of Cas9 might prove
higher specificity for a desired genomic locus (Table 2). to be useful in designing delivery vectors for therapeutic applications.

The activity of Cas9 nuclease is another prime factor causing undesir- In one study, researchers rendered Cas9 nuclease activity dependent
able off-target effects of the CRISPR/Cas9 system; specifically, on 4-hydroxytamoxifen (4-HT) by fusing a hormone-binding
elevated levels of Cas9 has been associated with unspecific cleavage.11 domain of the estrogen receptor (ERT2) to Cas9.16 This fusion en-
Therefore, controlling the activity of Cas9 nuclease would lead to a sures sequestration of Cas9 nuclease to the cytoplasm in the absence
significant reduction of unwanted “side effects.” In this regard, several of 4-HT, consequently limiting the activity of the enzyme. A nuclear
approaches that use chemical or physical agents to achieve condi- import is seen within the first 6 hr after induction with 4-HT, and
tional expression of Cas9 have been under investigation by re- optimal nuclease activity occurs at a time interval of 4–8 hr at
searchers and are illustrated in Figure 2. 1 mM 4-HT; therefore, the off-target effects are reduced to a mini-
mum. In a similar manner, Davis et al.17 used the self-splicing prop-
Turning the activity of Cas9 on and off confers to the researcher a erties of inteins to render Cas9 nuclease active in the presence of
means for a temporal control, limiting the exposure time of the genome 4-HT. In this report, the ERT2 domain is fused to intein to render

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Table 2. Online sgRNA Design Tools

Platform Platform Link Reference

8
CRISPR Design http://crispr.mit.edu/
87
E-CRISP http://www.e-crisp.org/E-CRISP/
http://www.multicrispr.net/basic_ 88
CRISPR MultiTargeter
input.html
http://portals.broadinstitute.org/gpp/ 10,89
sgRNA Designer: CRISPRko
public/analysis-tools/sgrna-design
90
Off-Spoter https://cm.jefferson.edu/Off-Spotter/
http://crispr.cos.uni-heidelberg.de/ 91
CCTop
index.html
92,93
CHOPCHOP http://chopchop.cbu.uib.no/index.php

Other models have used the propriety of light-inducible heterodime-

rization proteins for the modulation of Cas9 activity. These “light ap-
proaches” use a catalytically inactive form of Cas919 or a split Cas9
variant that is fused to light-responsive proteins.20 Upon stimulation
with blue light, Cas9 becomes active either by transitioning from an
inactive form19 or by reconstitution of the whole active Cas9 nuclease
from the C domain and N domain of the enzyme.20 Although such
systems are to some extent reversible and adjustable for regulating
Cas9 activity, they are limited to in vitro applications, under which
blue light is relatively easy to apply. For in vivo applications, stimu-
lating Cas9 activity with blue light requires more invasive and dedi-
Figure 1. CRISPR/Cas9 Mechanism of Action cated equipment to achieve an efficient effect, a fact that might limit
The original bacterial CRISPR/Cas9 design has been translated into an engineered
the use of such systems in clinical contexts.
instrument for genome editing purposes and is capable of introducing specific
modifications in the target cell. In this regard, the vector comprising the crRNA and
tracrRNA that together constitute the RNA molecule for Cas9 guidance (gRNA) is A recent study elegantly used a self-restricted CRISPR/Cas9 system to
introduced in the desired cell, where it passes the cytoplasmic milieu toward the achieve control of Cas9 nuclease to minimize off-target effects.21 In
nucleus. After delivery to the nucleus, the Cas9 gene encoded by the experimental addition to a specific targeting sgRNA, the authors co-expressed an
vector is transcribed and exported into the cytoplasm for translation of Cas9 additional sgRNA that targets Cas9 itself. Using this approach, they
nuclease. After synthesis of the active protein, the gRNA, transcribed by its own were able to limit the expression of Cas9 to several days, even in
promoter, interacts with the Cas9 nuclease, resulting in the ribonucleic-protein
the context of an integrative lentiviral vector. Furthermore, it has
effector complex that is internalized back into the nucleus. The cleavage of the
double-stranded genomic DNA takes place in a guided manner, where the crRNA
been shown that altering the energetic state of the Cas9-sgRNA-target
sequence of gRNA directs Cas9 toward the specific locus, based on sequence RNA by substituting four positive amino acid residues to neutral ones
complementarity, which is positioned adjacent to the PAM. When cleaved, the con- in the DNA binding loop of Cas9 results in a “high-fidelity” Cas9
tinuity of the host DNA can be restored through NHEJ, where the hanging ends join nuclease that displays no detectable undesired genomic alterations.22
together, creating small indels, or through HDR in the presence of a donor DNA.
As reviewed above, limiting undesired off-target effects is not an easy
it sensitive to 4-HT. Upon induction, intein becomes active and is task, requiring additional genes and inducers, modifications to Cas9,
effectively spliced from an inactive variant of Cas9, in which intein or even invasive approaches to obtain spatiotemporal control of
was inserted, leading to an active form of Cas9. However, this CRISPR/Cas9 so that this technology can move forward to therapeu-
4-HT-inducible system is not reversible compared with another sys- tic applications in a clinical context. In regard to “simple is beautiful,”
tem proposed by Liu et al.;16 therefore, it does not offer the advantage we definitely need an easy and, at the same time, reliable system that
of an on/off switch for more adjustable control of Cas9 activity. It is will give us the possibility to engineer our “faulty” genomes to a
well worth mentioning that simple split Cas9-intein systems have healthy state and, furthermore, to an improved state of well-being.
been developed in which an active form of Cas9 is reconstituted
post-translationally from a Cas9-C domain and a Cas9-N domain af- Multiplexing CRISPR/Cas9 Editing Capabilities
ter splicing of fused intein.18 These two inducible CRISPR/Cas9 sys- Despite the fact that we currently might have a simple CRISPR/
tems use minimal genetic elements to achieve conditional expression Cas9 system that is, to some extent, safe and efficient in targeting a
of Cas9, which might prove their usefulness in designing delivery vec- specific genome site, there are still many other obstacles to overcome
tors for therapeutic applications. to successfully implement this strategy in clinical therapy. In some

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Figure 2. Strategies for Regulating Cas9 Nuclease Activity

Increased levels of Cas9 can lead to unspecific cleavage, which causes hazardous effects in the target cell, resulting in off-target effects (red 50 in Figure 1). Different strategies
have been implemented to minimize and control the activity of Cas9. (A) Introduction of a Tet-controlled promoter that allows monitoring of Cas9 expression through an on/off
system dependent on Tet/Dox. (B) Fusion of Cas9 with an estrogen receptor domain (ERT2) that enables the supervision of Cas9 activity through 4-HT presence/absence.
(C) Control of the enzymatic activity via intein and its splicing properties. The N-terminal and C-terminal domains of Cas9, each containing a fused intein domain, are joined
together by a splicing event, and, upon expression of gRNA, the intein is auto-excised, and gRNA forms with Cas9 an active complex. (D) Holding of Cas9 activity through
fusion with light-responsive elements, which allows Cas9 performance only after stimulation with blue light. (E) A self-restricted CRISPR/Cas9 system that contains, in the
engineered vector, a gRNA that targets the Cas9 gene itself, resulting in an auto-regulated loop. CRY2, cryptochrome circadian clock 2; VP64, viral protein 64 transactivation
domain; CIBN, N-terminal domain of CIB1; P, promoter.

instances, targeting a single site in the genome is not sufficient to for targeting multiple sites and gene networks. To date, researchers
achieve a full therapeutic effect because some diseases, like cancer, have developed two strategies for this approach. As mentioned above,
have a multigenic basis and require targeting whole gene networks one approach uses multiple sgRNA expression cassettes made of an
that sustain the pathological state of the cell. Therefore, such an individual RNA polymerase III promoter, sgRNA, and a transcription
approach would require the use of multiple sgRNAs able to target terminator, all imbedded in the same construct.23,24 This means that
multiple genomic loci, adding an extra level of complexity for the construct would express multiple sgRNA transcripts able to target
designing CRISPR/Cas9 systems. multiple genomic loci. In the second approach, multiple sgRNAs are
released from one single transcript, produced from either one RNA
Traditionally, this would mean that one must have at least two sgRNA polymerase III promoter25 or one RNA polymerase II promoter.26
expression cassettes on the same construct or placed on two different Such a polycistronic transcript offers the possibility of encoding
constructs. Co-transfection of two vectors limits the efficiency of the additional exogenous factors to sgRNAs in the case of RNA polymer-
CRISPR/Cas9 system in vitro and, furthermore, in vivo. In addition, ase II-driven promoters, although efficient processing of the tran-
having multiple sgRNA expression cassettes on the same construct script for releasing active sgRNAs and polyadenylated transcripts
poses a limit of physical constrains in the number of sgRNA cassettes for nuclear export is rather complex in nature. First, sgRNA must
because of their limited cloning capacity, as seen for AAV vectors.15 be released from the primary transcript and, therefore, requires the
addition of flanking sequences that are recognized by exogenous
Indeed, multiplexing CRISPR/Cas9 editing capabilities would offer factors that must be provided in trans, such as Cys4 protein from
several advantages over a conventional single sgRNA-based system Pseudomonas aeruginosa.26 Second, the remaining transcript must

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be properly processed as a translational active molecule. Additionally, The extensiveness of the library is also a critical factor, where there
a shortcoming of this system is that Csy4 has cytotoxic effects at is the possibility to target the whole genome with the use of a single,
high concentrations,26 a fact that might hinder the clinical scenario. large “pooled” library of sgRNAs or to target specific subsets of
Moreover, this number of supplemental sequences significantly ham- genes that are thought to be interconnected, known as sub-pooled
pers the efficiency of the targeting and editing capabilities of the libraries.32,33 Moreover, the permanent nature of the genetic modifica-
CRISPR/Cas9 system because of the addition of extra variables. tions mediated by CRISPR/Cas9 technology permits the evaluation of
the modified cells for extended periods of time, where there is the pos-
Exploiting the endogenous RNA processing factors would be sibility that a certain phenotype will manifest only after several cell
preferred, and simplistic approaches for multiplexing CRISPR/Cas9 divisions.
have already been reported. In one study, the endogenous tRNA pro-
cessing machinery was used for processing an sgRNA-tRNA polycis- The first step when performing a screening investigation is viral
tronic transcript to release multiple active sgRNAs.25 This approach is administration of the selected sgRNAs in cells that consistently ex-
a powerful and reliable system for multiplexing CRISPR/Cas9 because presses Cas9 enzyme or in combination with the Cas9-encoded
it takes advantage of the abundance of endogenous cellular RNAases sequence under the construct. After selection of the transfected
without heterologous expression of additional and potential cytotoxic cells, further selective agents can be added that will enrich the resis-
factors that might interfere with normal cell function. This interfer- tant phenotype population. The final step consists of sequencing
ence with normal cell function could also make the evaluation of arrays that can reveal the difference between the experimental
results rather difficult and unreliable. and control groups in terms of enriched sgRNAs and depleted
ones, which can be seen in Figure 3. When these data are overlap-
Having a system that can easily and efficiently target gene networks, ped with the original sgRNA library and screened for the target
multiplexing CRISPR/Cas9 offers the advantage of deleting genes genes, statistical analysis can reveal the key genes within the pheno-
or even inserting new genes in the genomes of choice. This provides type of concern.31,34.
a toolbox that opens new horizons that researchers could only dream
of, with an ease never previously attained. The feasibility of CRISPR/ Shalem et al.35 and Wang et al.36 pioneered the genome-wide
Cas9 systems for knockin in reporter genes in different genomic sites screening of cells using CRISPR/Cas9 libraries and presented the ad-
has already been reported for different cell types, such as mouse vantages of this method in contrast to small interfering RNA.
haploid embryonic stem cells27 and human embryonic pluripotent Although their approaches were quite different, where Shalem
stem cells.28 The authors used an HDR approach in which the re- et al.35 transfected cells with a vector containing both sgRNAs and
porter cassette was flanked by two homologous arms on the region a Cas9-expressing sequence, and Wang et al.36 administered viral par-
of interest in the genome, and two sgRNAs were used for guiding ticles containing only sgRNAs molecules in cells that already stably
Cas9 nuclease to the targeted sites. Upon cleavage, the reporter expressed Cas9 enzyme, the results proved encouraging for both
cassette is inserted in the genome by homologous recombination groups. Thus, even though this approach for the identification
with an efficiency that varies depending on the targeted site.28 Using of entire genes set functions in living cells is still in its infancy,
this approach, studies have shown that such a procedure permits the CRISPR/Cas9 libraries hold significant advantages over the preceding
generation of mice harboring reporter genes up to 5 kb29 or a condi- technologies (e.g., small interfering RNA [siRNA libraries]), allowing
tional allele30 with ease, surpassing traditional approaches in terms of the generation of a permanent “mutated” phenotype that can be stud-
simplicity, time length, and cost-effectiveness. The only requirement ied across several cell division steps under different experimental
is the presence in the donor plasmid of two flanking homologous conditions.
arms that may vary in length from 500 bp27 to 900 bp29 and two
sgRNAs sequences able to direct Cas9 toward the targeted genomic The Big Step of CRISPR/Cas9 in Cancer Research
fragment. In the absence of a donor DNA, large genomic deletions Cancer is one of the most investigated fields in medicine because of
up to 65 kb can be easily generated,29 making CRISPR/Cas9 a its high prevalence in the human population, the complexity and
preferred system for obtaining knockout mouse models for transla- heterogeneity of the disease, which makes current treatments rather
tional research. unspecific, raising the death toll of the affected individuals.37–39 Un-
derstanding the molecular mechanisms that underline the prolifera-
CRISPR/Cas9 Libraries tive process is a prerequisite for the development of reliable
Taking advantage of the CRISPR/Cas9 system’s ability to target almost experimental models in which the altered cellular pathways can be
any genetic loci within a target cell, researchers have created entire reproduced and novel targeted therapies can be implemented. Hence,
libraries of sgRNAs that are directed toward specific genes to examine it did not take long until the genome editing capabilities of CRISPR/
the meaning of a “custom-made” phenotype in different experimental Cas9 proved its utility in cancer research and development of thera-
setups.31 By introducing into cells sgRNA libraries via delivery vectors peutic strategies. Such applicability ranges from functional validation
that are most often lentiviral particles, there is the possibility to of genes implicated in cancer development and progression to cancer
repress, activate, or even knock out different target genes at once to modeling and therapeutic concepts, as described in Table 3. There-
elucidate their function within a pathologic or homeostatic context. fore, CRISPR/Cas9 has spread to every aspect of cancer research in

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Figure 3. Generation and Quality Control of sgRNA Libraries for CRISPR/Cas9-Mediated Phenotypic Screening
The first step illustrates the synthesis of sgRNA libraries consisting of a heterogeneous population of sequences approximately 100 bases in length with a reduced number of
mutations. Quality control is significantly facilitated when a barcode of a known sequence is incorporated into each sgRNA and analyzed at the final stages through next-
generation sequencing (NGS). After detachment of oligonucleotides from the solid support, the targeted sequences are amplified through PCR and then cloned in lentiviral
vectors that contain a selection marker for antibiotic resistance (e.g., puromycin) and also an enrichment sequence that can be detected with the help of fluorescence-
activated cell sorting (FACS) (e.g., GFP). Every sgRNA is under the activity of an U6 promoter that will facilitate its expression. The entire complex is packed in viruses that are
further used for the transduction of the target cells. The first selection consists of the capacity of cells to survive in an antibiotic-enriched medium because of their integrated
gene for puromycin or any other antibiotic that is used in the experiment. The second selection consists of the enrichment of the transduced cell population through GFP
selection by FACS. The remaining cells that contain the viral construct are then collected for DNA extraction and analyzed through NGS, which detects individual sgRNAs
because of the inserted barcode.

a very short period of time, making it a versatile technology that could lack of an immune response might interfere with the results obtained
leave an important footprint with regard to redefining the meaning in preclinical studies, a crucial step for evaluating the safety profile of
of cancer biology and its treatment. From small-scale studies40–42 to novel therapeutic agents.
high-throughput screens of gene function,43–45 CRISPR/Cas9 has
brought new insights into cancer progression and metastasis and A few studies published recently on in vitro46–48 and in vivo49,50 can-
led to the identification of new potential therapeutic targets. Cancer cer models have paved the way for CRISPR/Cas9 in therapeutic appli-
modeling has an opportunity with CRISPR/Cas9 for both in vitro cations for the oncology niche. In this regard, cancer immunotherapy
and in vivo models because the traditional Cre/LoxP recombination gained special attention by reprograming T cells through disruption
technology for animal models has shortcomings in terms of labor, of the programmed death-1 receptor (PD-1) over conventional
time length, and costs for producing stable in vivo lines of cancer PD-1 antibody therapy.48 Because the PD-1 ligand is a negative regu-
models. In addition, this might bypass tumor xenograft models, lator of T cell activity and expressed on dendritic cells as well as in
where the risk of immune incompatibility can interfere with the pro- some tumors, knocking down PD-1 with CRISPR/Cas9 could repro-
duction of reliable in vivo models. The use of immunocompromised gram T cell activity toward PD-1 ligand-expressing tumors. This
animals requires special conditions for maintenance of the lines, and approach is already in phase I clinical trials for castration-resistant

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Table 3. List of CRISPR/Cas9 Applications in Cancer Research Organized by In Vitro and In Vivo Studies of Gene Function, Cancer Modeling, and Therapy

Cell Line/Organism Delivery System Cancer Type Targeted Gene Local/Systemic Reference
Gene Function Studies In Vitro
45
A375 melanoma cell line (human) LV melanoma SAM library
43
A375 melanoma cell line (human) LV melanoma GeCKO library
44
Non-small-cell lung cancer cell line LV non-small-cell lung cancer GeCKO library
42
MDA-MB-231, MCF-7 (human) LV triple-negative breast cancer Shcbp1
T24, J82, 5637, SW-780 bladder 63
plasmid transfection bladder cancer p21, E-cadherin, hBax
cancer cell lines (human)
Cd74-Ros1
64
HEK293T (human) plasmid transfection non-small-cell lung cancer Eml4-Alk
Ki5b-Ret
65
HEK293 (human) plasmid transfection non-small-cell lung cancer Met
66
Dld-1 (human) plasmid transfection colon cancer Pkc
B16-F10, Ret melanoma cell lines 67
plasmid transfection melanoma Id1, Id3
(mouse)
RV/plasmid 68
BT-474, SKBR-3, MCF-7 (human) breast cancer Her2
transfection
In Vivo
ex vivo 69
Mouse LV prostate cancer TGFBRII
DU145 cells (human)
Nanog1 ex vivo 40
Mouse LV prostate cancer
Nanoggp8 DU145 cells (human)
ex vivo 41
Mouse LV triple-negative breast cancer Cripto-1
JygMC(A) (mouse)
ex vivo 70
Mouse LV triple-negative breast cancer Ctbp1
MDA-Mb-231 (human)
ex vivo 71
Mouse plasmid transfection cervical cancer HPV E6, E7
SiHa, C33-A (human)
ex vivo 72
Mouse LV Burkitt lymphoma Mcl-1, TP53
HSC (mouse)
ex vivo 73
Mouse plasmid transduction acute myeloid leukemia Mll3
HSC (mouse)
Cancer Modeling In Vitro
74
Myoblast cells (mouse) LV alveolar rhabdomyosarcoma Pax3, Foxo1
HEK293A, hMSCs, PBMCs, plasmid Ewing sarcoma, acute myeloid 75
Ewsr1, Fli1, Runx1, Eto
HL-60 (human) transduction/EP leukemia
In Vivo
76
Mouse LV non-small-cell lung cancer Eml4-Alk local
77
Mouse LV non-small-cell lung cancer Nkx2.1, Pten, Apc local
78
Mouse LV/Ad pancreatic ductal adenocarcinoma Lkb1 local
79
Mouse Ad non-small-cell lung cancer Eml4-Alk local
80
Mouse AAV lung adenocarcinoma TP53, Lkb1, Kras local
hepatocellular carcinoma 81
Mouse HI multiple systemic
intrahepatic cholangiocarcinoma
82
Mouse HI hepatocellular carcinoma Pten, TP53 systemic
83
Mouse EP glioblastoma Pten, Apc, Nf1 local
(Continued on next page)

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Table 3 Continued

Cell Line/Organism Delivery System Cancer Type Targeted Gene Local/Systemic Reference
83
Mouse PEI/EP medulloblastoma Ptch1 local
84
Mouse EP pancreatic cancer multiple local
ex vivo 85
Mouse LV acute myeloid leukemia Tet2, Dnmt3a, Nf1, Ezh2
HSCs (mouse)

Apc, TP53, Kras, Smad4, ex vivo 86

Mouse LV colorectal cancer
PIK3CA intestinal stem cells (human)
Therapeutic Concepts In Vitro
Osteosarcoma cell lines (KHOS), 46
EP osteosarcoma Cdk11
u-20 s (human)
47
Primary T cells (human) EP tumor immunotherapy Cxcr4, PD-1
48
Primary T cells (human) EP tumor immunotherapy PD-1
In Vivo
49
Mouse LV pancreatic ductal adenocarcinoma p57 local
prostate cancer ex vivo 50
Mouse LV/EP TCR, B2M, PD-1
leukemia CAR-T cells (human)
RV, retrovirus; Ad, adenovirus; HI, hydrodynamic injection; EP, electroporation; PEI, polyethylenimine; HSC, hematopoietic stem cell; hMSC, human mesenchymal stem cells; PBMC,
peripheral blood mononuclear cells; CAR, chimeric antigen receptor

prostate cancer, muscle-invasive bladder cancer, metastatic non- cells within our body, and gene therapy emerged as a vison for treat-
small-cell lung cancer, and metastatic renal cell carcinoma (https:// ment of human diseases that would be safe and specific.
clinicaltrials.gov).
Most of the biotherapies are based on oncolytic vectors that selectively
Challenges and Future Perspectives of CRISPR/Cas9 for Cancer replicate and destroy cancer cells, releasing tumor-specific antigens,
Therapy resulting in a second immune-humoral response against distant
The simplicity that resides in the genome editing capabilities of metastatic tumors.53 However, these therapies are limited to local
CRISPR/Cas9 attracted the attention of many researchers around administration, a fact that imposes several limitations, such as tumor
the world, as confirmed by the overwhelming numbers of studies localization, that require invasive approaches that are not feasible in
that have been published on the subject for the past 2 years. This certain cancer types. For the time being, CRISPR/Cas9 has been
newly characterized RNA-guided Cas9 nuclease system opened the investigated as a potential therapeutic strategy in ex vivo preclinical
era of “molecular surgery,” and, as reviewed above, cancer research setups for cancer immunotherapy in both hematological and solid tu-
has and will continue to benefit from this new genome editing tech- mors by targeting the PD-1 gene in T cells (Table 3), reprograming
nology. In particular, modeling oncogenesis in mice using CRISPR/ these cells of the immune system to recognize and attack cancer cells.
Cas9 surpassed previous genome editing tools in terms of time length To date, there are four clinical trials under investigation based on the
and cost effectiveness without the need of multiplying colonies strategy of targeting PD-1 by CRISPR/Cas9 in four different carci-
of mice. nomas (https://clinicaltrials.gov). Both the preclinical and clinical
studies are using lentiviruses (LVs) as delivery systems in T cells of
Functional studies of multiple genes in cancer cell lines with CRISPR/ the CRISPR/Cas9 components. For ex vivo therapeutic setups, trans-
Cas9 (Table 3), especially large-scale studies with sgRNAs libraries duction of primary T cells from cancer patients, characterization,
(Table 3),43–45 again stressed the complexity of this insidious killer and expansion in the laboratory of the transduced cells are quite
that makes cancer particularly difficult to treat. The multigenic and labor-intensive and expensive. Although lentiviruses have been re-
multi-mutated status gives cancer an unique heterogeneity character- ported to be efficient for tumor regression on in vivo models, with
istic; this heterogeneity is even seen in different individuals affected CRISPR/Cas949 or other experimental setups, like utilizing antago-
by the same cancer type,51,52 and this truly represents the major mirs,54 a valuable lesson has been learned in terms of the tumorigenic
challenge for clinical therapy. Although surgery and chemotherapy/ potential of integrating vectors in clinical trials.55
radiation therapy are currently in clinical use for the treatment and
palliative care of cancer patients, the lifespan, because of tumor Having a technology that gives us the possibility to knock out or
relapse, is quite short, and the quality of life because of adverse effects knock in single or multiple genes with an ease that surpasses other
of therapy is rather poor. All of this gave researchers the impulse to genome editing tools is clearly a major achievement. Furthermore,
find alternative treatments that are efficient for targeting the diseased researchers have engineered Cas9 nuclease into an RNA-guided

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Review

transcriptional activator by fusing the VP64 transcription activation vectors? Perhaps, but we cannot envision such a scenario at this
domain to a catalytically inactive Cas9 and linking a MS2-p65- time because more preclinical studies must be implemented to
HSF1 activation complex to sgRNA via a hairpin aptamer.45 test its clinical significance and evaluate the pharmacokinetic
Rendering sgRNA from 20 bp to 11–15 bp, an RNA-guided transcrip- properties of phage-derived nanoparticles. However, we must
tional activation complex, Cas9-MS2-p65-HSF1, was obtained with keep in mind that the multigenic and heterogeneous nature of can-
an active Cas9.56 This system offers the possibility of simultaneous cer is definitively the major challenge for an efficient therapy. Even
orthogonal activation of gene expression with gene knockout by “clas- with a systemic delivery vector that can efficiently deliver CRISPR/
sical” Cas9-sgRNA system in a single cell population. The specificity Cas9 to cancer cells without side effects, there is no guarantee that
of such an approach can be further enhanced by substituting the a full therapeutic effect will be achieved. A thoughtful understand-
Streptococcus pyogenes Cas9 (spCas9) with the Streptococcus aureus ing of the proliferative process and connections between different
Cas9 (saCas9); this allows for recognition of the PAM of the cellular pathways in cancer cells is mandatory for developing effi-
consensus sequence NNGRR(T), which is more complex than the cient therapeutics, especially considering that each type of cancer
NGG spCas9 PAM.57 SaCas9 also has a significantly smaller size has its own genomic and phenotypic profiles. Whether CRISPR/
than spCas9, a particularity that might be useful when physical con- Cas9 technology will make a difference in cancer therapeutics is
strains are imposed by the type of vector that is used as the delivery hard to tell, but it is definitely worth the effort. And the knowledge
vehicle. However, such a complex PAM reduces the number of that will arise from this process of “trial and error” will pave the
genomic loci that can be targeted. way for CRISPR/Cas9 in our daily medical practice and, if not
for cancer, then perhaps for other diseases that are threatening
Despite the numerous efforts that have been made to improve the our own existence and well-being.
specificity and efficiency of CRISPR/Cas9, a major hurdle experienced
with other gene therapy approaches lies before us. Delivery into the CONFLICTS OF INTEREST
body and targeting the diseased cells without side effects is not some- The authors declare no conflict of interest.
thing new, and each type of delivery vector has its own advantages
and limitations.58 Therefore, a safe and efficient delivery vector for ACKNOWLEDGMENTS
systemic administration of CRISPR/Cas9 would be highly desirable. We would like to thank Mihail Buse for English proofreading
An emerging class of gene delivery vectors is derived from an M13 of the manuscript. This work was supported by national grants
filamentous bacteriophage in which a tumor-targeting ligand is ex- Nr.128,014-PN-II-PT-PCCA-2013-4-2166 (GenCanD) and POC
pressed on the bacteriophage capsid.59,60 Because bacteriophages do Nr.35/01.09.2016, ID 37_796 (CANTEMIR).
not have natural receptors on human cells, attaching a ligand like
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