CRISPR /Cas9 Based Genome Editing for Treating Genetic
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Preface
The Nobel Prize in Chemistry 2020 was awarded jointly to Emmanuelle Charpentier
and Jennifer A. Doudna who have discovered the CRISPR/Cas9 genetic scissors. The
CRISPR/Cas 9 genome editing system is a highly versatile and easy-to-use technology
that has revolutionized both basic and applied research in many disciplines ranging
from medicine to agriculture, democratizing genome editing in laboratories around
the world. In medicine, CRISPR/Cas9 has provided a powerful tool for research
and holds an enormous potential for the treatment of genetic disorders and non-
inherited diseases. Accordingly, this book offers an updated in-depth review on the
implementation status of CRISPR/Cas9 technology in research and clinical settings.
Moreover, this book brings to readers a selection of CRISPR-based gene editing
platforms, as well as perspectives from professionals in non-medical fields pertinent
to the use of CRISPR-Cas9. Each of the chapters in CRISPR-/Cas9 Based Genome
Editing for Treating Genetic Disorders and Diseases has been selected to provide
a full understanding of the various (bioethical, legal, technical) challenges related
to the applicability of CRISPR-Cas9 in human cells. The book sets a benchmark
for the involvement of CRISPR-Cas9 system in bioethics and regulatory issues,
neuroscience research, gene therapy, epigenome editing, cancer research, telomere
length, genome mapping and virus infection control.
I sincerely wish to thank the reviewers, Prof. Hallam Stevens, Prof. Zdenka
Ellederova, Prof. Bernhard Schmierer, Prof. You Lu, Dr. Manh T. Tran, Dr. Marta
Martinez-Lage, Prof. Jonathan Ploski, and Prof. Hiroshi Yamaguchi, whose work
and insightful comments have enriched the quality of this book.
Luis María Vaschetto
Contents
Preface iii
1. The CRISPR/Cas9 Genome-editing System: Principles and Applications 1
Cecilia Pop-Bica, Andreea Nutu, Roxana Cojocneanu, Sergiu Chira
and Ioana Berindan-Neagoe
2. Recent Applications of CRISPR-Cas9 in Genome Mapping
and Sequencing 23
Dharma Varapula, Lahari Uppuluri and Ming Xiao
3. Gene Editing and Genetic Disorders: Ethical and Legal Concerns 46
Vicente Bellver and Federico de Montalvo
4. Political, Regulatory and Ethical Considerations of the CRISPR/Cas
Genome Editing Technology 71
Eduardo Rodriguez Yunta and Luis María Vaschetto
5. CRISPR/Cas and Gene Therapy: An Overview 85
Luis María Vaschetto
6. CRISPR and Cancer: From Bench to Clinic 90
Muhammad Usama Tariq and Amina Chaudhry
7. The Advances of the CRISPR/Cas9 Technology for Correcting
Genetic Disorders: Using Sarcoma as a Model System 116
Pichaya Thanindratarn, Dylan C. Dean, Francis J. Hornicek
and Zhenfeng Duan
8. CRISPR/Cas9 Technology as a Strategy Against Viral Infections 132
Huafeng Lin, Haizhen Wang, Jun He, Xiangwen Peng, Aimin Deng,
Lei Shi, Lei Ye and Hanyue Gong
9. CRISPR/Cas9-based Genome and Epigenome Editing in
Neuroscience Research 158
Nereo Kalebic and Wieland B. Huttner
10. Non-viral Alternatives to Delivery of CRISPR-Cas Gene
Editing Components 183
Kate Senger and Benjamin Haley
vi Contents
11. CRISPR Medicine: Advance, Progress and Challenges 224
Guillermo Ureña-Bailén, Justin S. Antony, Yujuan Hou, Janani Raju,
Andres Lamsfus-Calle, Alberto Daniel-Moreno, Rupert Handgretinger
and Markus Mezger
Subject Index 260
CHAPTER
1
The CRISPR/Cas9 Genome-editing
System: Principles and Applications
Cecilia Pop-Bica1, Andreea Nutu1, Roxana Cojocneanu1, Sergiu Chira1 and
Ioana Berindan-Neagoe1,2*
1 Research Center for Functional Genomics, Biomedicine and Translational Medicine,
“Iuliu-Hatieganu” University of Medicine and Pharmacy, Cluj-Napoca, Romania
2 Department of Functional Genomics and Experimental Pathology, The Oncology Institute
“Prof. Dr. Ion Chiricuţă”, Cluj-Napoca, Romania
Introduction
CRISPR/Cas9 represents an adjustable and widespread genome-editing tool
adapted from the bacterial immune system, which is presently used in basic and
applied biomedical research. Genetic alterations are frequently the cause of genetic
diseases. Even though recent technological advances eased the discovery of disease-
associated gene alterations, general therapeutic options are usually designed to
treat the symptoms, not to restore the altered genetic sequences responsible for the
disease phenotype. In this context, gene therapies appeared to support the concept of
restoring the genetic mutations in order to prevent or treat the disorders caused by
these alterations (Mirgayazova et al. 2020). In what concerns the CRISPR/Cas system
(clustered regularly interspaced short palindromic repeats-CRISPR associated), the
fundamental principle includes genome editing and the regulation of physiological
phenomena of various organisms and cells. This system was first discovered
in bacteria, where it functions as an adaptive “immune” strategy used to destroy
foreign genetic material or develop resistance to phage infections (Wright et al. 2016,
Yadav et al. 2021). The CRISPR/Cas systems is characterized by a sequence of about
20-50 bp organized as direct repeats, isolated by spacers of comparable length, and
tailed by an AT-rich “leader” region (Jansen et al. 2002, Kunin et al. 2007). This
system has two main components – a guide RNA (gRNA), comprised of a CRISPR
RNA (crRNA) and trans-activating crRNA (tracrRNA) (Figure 1 and Figure 2),
which acts as a guide RNA for the complementary DNA sequence, and the Cas
proteins (encoded by cas genes), which are involved in the degradation of the target
DNA/RNA sequence (Strich and Chertow 2019). The principle of the complementary
2 CRISPR-/Cas9 Based Genome Editing
binding of the crRNA prompted the researchers to use this system to design sequences
that would bind to and cut the sequences of interest in different pathologies (Jinek et
al. 2012). Furthermore, this technology allowed researchers to identify the function
of a gene through the introduction of genetic alterations (Shalem et al. 2015). The
reduced time-frame needed to perform CRISPR/Cas9 gene editing, together with the
reported efficiency of this system in terms of reduced off-target effects, makes this a
superior technique compared to others based on ZFNs (Zinc Finger Nucleases) and
TALENs (Transcriptor Activator-Like Effector Nucleases), in which the efficiency is
exclusively based on the nucleases affinity and specificity (Qu et al. 2013).
The bacterial origin of CRISPR/Cas
The discovery of CRISPR/Cas nucleases system
The coexistence of prokaryotes and viruses generated various defense mechanisms
such as restriction modifications, toxin-antitoxin systems, and, in the later years,
CRISPR/Cas systems were discovered (Labrie et al. 2010). The discovery of
CRISPR as a component of the prokaryotic “immune” system, and the repurposing
of this system as a genome editing tool, determined a broad use of this technology
in molecular biology applications, making it one of the most used technologies in
active research in biology (van Soolingen et al. 1993, Bolotin et al. 2005, van der
Oost et al. 2009, Mali et al. 2013a, Cho et al. 2013) (Figure 1). Even though the first
CRISPRs were observed decades ago in bacteria (Ishino et al. 1987), and subsequent
studies revealed the presence of CRISPRs in archaea (Mojica et al. 1993), it was only
in the early 2000s that researchers discovered the sequence similarities between the
viruses, bacteriophages, and plasmids and the spacer regions in CRISPR, managing
to uncover the defense function of CRISPR (Mojica et al. 2005, Bolotin et al.
2005). Independent parallel studies revealed a set of genes associated with CRISPR,
consequently named cas (CRISPR-associated), and, in 2008, Marakova et al.
suggested the existence of a CRISPR/Cas complex that acts as an acquired immune
system to protect the bacterial cell against invading phages or other exogenous
genetic material (Jansen et al. 2002, Makarova et al. 2006).
Components of the CRISPR/Cas system
CRISPR/Cas system ensures the immunity of the bacteria in three steps that require
target recognition and cleavage (Barrangou and Marraffini 2014, Sorek et al. 2013).
The first step is the adaptation, which allows the copy and paste of the foreign nucleic
acids into the ‘spacers’ of the CRISPR arrays, thus providing acquired resistance
against the invading phage (Barrangou et al. 2007). The next step includes the
biogenesis of the crRNA (expression stage), in which the small interfering RNAs
are generated through transcription and further processing. In the interference stage,
the gRNA heads the Cas enzymes to cleave the DNA (Marraffini and Sontheimer
2008). The CRISPR-Cas system can be classified into two main categories according
to the effectors: first, where all functionalities in the effector complexes are carried
out using a protein, and second, the multi-unit effector complexes (Shmakov et al.
2017). These two classes are further divided into six types of CRISPR-Cas systems
The CRISPR/Cas9 Genome-editing System: Principles and Applications 3
Figure 1. Timeline progression and development of CRISPR and a brief description of
fundamental discoveries. In 1987, during a study focused on alkaline phosphatase loci in E.
coli, the first observation of invariably repeated sequences flanking specific sequences, later
described as CRISPR arrays (in 2002 (Jansen et al. 2002)), was made by Ishino et al. (Ishino
et al. 1987). Later, CRISPR sequences were discovered in other microorganisms (Mojica et al.
2000), and in 2005 foreign sequences (spacers) from phages and plasmids were identified in
CRISPR (Bolotin et al. 2005, Haft et al. 2005). The bacterial “immune” function (Marraffini
and Sontheimer 2008) of CRISPR, and the crRNAs that serve as gRNAs for DNA targeting
(Brouns et al. 2008) are discoveries made in the following years. Between 2009-2011,
CRISPR/Cas system to cleave RNA molecules (Hale et al. 2009), the specific DSB DNA
cleavage (Garneau et al. 2010), and tracrRNA-crRNA structure associated with Cas9 were
first described (Deltcheva et al. 2011). In 2012, another major breakthrough was made, as
two separate studies indicated that CRISPR system operates as RNA-guided endonucleases
(Gasiunas et al. 2012), respectively, that the Cas9 are reprogrammable and this system could
potentially serve as a tool for genome-editing (Jinek et al. 2012). Another breakthrough is
represented by the transfer of this technology from prokaryotes to eukaryotic cells in 2013,
and in 2014 the first sgRNA libraries were used for genome-wide screening using Cas9 (Cong
et al. 2013, Mali et al. 2013a, Wang et al. 2014, Shalem et al. 2015). Recent studies proved
the efficiency of CRISPR/Cas systems in cancer biology (Sanchez-Rivera and Jacks 2015),
and embryonic stem cells systems (Jin and Li 2016). In 2018, the first gene editing of HIV-1
co-receptors using CRISPR was made (Allen et al. 2018). In 2019, CRISPR/Cas9 was used to
control genetic inheritance in mice (You et al. 2019), and in 2020, it was the first time when
CRISPR was used as a genetic scissor directly administered into a patient (Ledford 2020).
4 CRISPR-/Cas9 Based Genome Editing
according to the presence of the signature genes. In type I systems, the signature
protein is Cas3, in which the cleavage of the external DNA is carried out by the
nuclease and helicase domains, and the recognition of the target sequence is made
by the multi-protein-crRNA complex Cascade. In type II systems, the unique protein
required for the interference is Cas9 (signature protein). In type III systems, the
signature gene is cas10, a gene encoding a multidomain protein (Cas10) that is
assembled into an interference complex needed for the identification and cleavage
of the target sequence. Type IV systems are usually not linked to a CRISPR array.
The effector complex is represented by Csf1 (csf1 being considered the signature
gene) and the other two proteins encoded by cas genes (Makarova and Koonin
2015). Type V systems comprise a unique Cas9-like nuclease, which can be Cpf1,
C2c1, or C2c3 according to the subtype of the CRISPR/Cas type V system (Shmakov
et al. 2017, Zetsche et al. 2015). In type VI systems, the protein C2c2 with two
HEPN RNase domains is the signature protein (Shmakov et al. 2017). All these
systems are classified as either Class1 systems (Type I, III, IV) as they have a multi-
subunit effector or as Class 2 systems (Type II, V, VI) as they are characterized by
a single-subunit effector (Shmakov et al. 2017, Makarova and Koonin 2015). Table
1 illustrates a brief classification and characterization of the CSISPR/Cas systems.
Class 2 systems are seen as a more straight-forward device for genetic editing, thus
being intensively used by the research community (Adli 2018, Wang et al. 2016).
Table 1. Overview of classification and characteristics of CRISPR/Cas systems (Shmakov
et al. 2017, Pyzocha and Chen 2018, Makarova et al. 2020, Perez Rojo et al. 2018,
Tamulaitis et al. 2017)
Class System Target for Effector Nuclease domain
type cleavage
I I DNA Cas proteins – Cas3, HD fused to Cas3
Cas5-Cas8, Cas10, and
III DNA/RNA HD fused to Cas10
Cas11
IV DNA Unknown
II II DNA Cas9 RuvC and HNH
V DNA Cas12a/b/c RuvC and Nuc
VI RNA Cas13a/b/c HEPN domains
HD – Histidine-aspartate domain; HEPN – Higher eukaryotes and prokaryotes nucleotide
binding domain
CRISPR technology originated as an adaptive prokaryotic “immune” system
that ensures protection against exogenic nucleic acids, and is now utilized as a precise
genetic/genomic tool in molecular biology (Barrangou et al. 2007, Haapaniemi
et al. 2018). The defense response generated by the CRISPR-Cas system consists
of three points, which are as follows: adaptation, expression and maturation, and
interference. In the adaptation step, a complex of Cas proteins is responsible for
the identification of the target sequence in the DNA (protospacer-adjacent motif –
PAM), binding, and the generation of double-strand breaks in this sequence. This
target DNA releases a short sequence that will be acquired by the CRISPR array as a
The CRISPR/Cas9 Genome-editing System: Principles and Applications 5
‘spacer’. The expression step consists of the transcription of the CRISPR array into
a precursor RNA that is further processed by the Cas proteins and other accessory
factors into smaller entities of RNA – CRISPR RNAs (crRNAs) that, together with
the Cas proteins, form the Cas-crRNA complex. The last step – the interference –
includes the action of the Cas-crRNA complex to recognize and cleave the invading
nucleic acid (Amitai and Sorek 2016, Makarova et al. 2006).
Depending on the type of CRISPR-Cas system, there are particular stages of
expression and interference. In the type I system, the Cas complex acts by cleaving
at the junction of the ssRNA (single-stranded RNA) and the dsRNA (double-stranded
RNA) formed by the hairpin loops (Makarova and Koonin 2015). In type II CRISPR-
Cas systems, the double break in the target DNA is induced by the Cas9 protein
(guided by the tracrRNA:crRNA complex). Specifically, each DNA strand is cleaved
by two different Cas9 endonuclease domains (Jinek et al. 2012). In type III systems,
there are two main nucleases – one sequence-specific ribonuclease to degrade the
ssRNA, and one deoxyribonuclease to target and degrade the ssDNA (Tamulaitis et
al. 2017). The mechanism is based on the DNA sequence recognition, and does not
require hairpin loops (Janik et al. 2020, Garneau et al. 2010, Gasiunas et al. 2012).
Features of the CRISPR/Cas9 for genome engineering
Cas9 is specific for the Type II CRISPR locus. Given the variety of the cas genes,
this type II CRISPR locus comprises three subtypes (IIA, IIB, IIC). Cas9, cas1, and
cas2 together with the CRISPR array and the tracrRNA are the main component
of type II CRISPR loci (Makarova et al. 2011). The three genes are shared by all
three subtypes of type II CRISPR loci, whereas type IIA has an additional csn2 gene
and type IIB an additional cas4 gene (Chylinski et al. 2013, Makarova et al. 2011).
Nonetheless, this classification is not relevant for the structural diversity of the Cas9
proteins. These nucleases are characterized by sequence homology and variable
lengths (Hsu et al. 2014). Despite of this variation in length (usually associated with
the REC domain), Cas9 proteins are characterized by a structure consisting of two
nuclease domains – RuvC (responsible for the cleavage of the non-target strand)
and HNH (responsible for the cleavage of the target strand), REC domains, and the
bridge helix (Makarova et al. 2011, Fonfara et al. 2014). The preference for this
type II CRISPR/Cas system in applications for genome editing relies on the fact
that there are few specifications that few specifications need to be fulfilled to ensure
a programmable DNA targeting, such as the presence of Cas9 endonuclease and
the existence of a guide RNA (gRNA) that can be customized (Jinek et al. 2012).
The DNA cleavage depends on two factors: the crRNA that targets the protospacer,
and the PAM, a sequence located downstream of the protospacer in the non-target
strand (tracrRNA) (Garneau et al. 2010). CRISPR/Cas9 systems are now used as
efficient gene/genome-editing tools in different organisms, including human cells,
facilitating the multiplex genome engineering by editing multiple sites at the same
time by using multiple gRNAs (Mali et al. 2013b, Wang et al. 2013, Cong et al.
2013). In addition, in mammalian genomes, it was shown that the adaptation of Cas9
into a nickase facilitates homology-directed repair (HDR) (Mali et al. 2013a, Ran et
al. 2013). Nowadays, there are a few RNA-guided Cas9 isolated from Streptococcus
6 CRISPR-/Cas9 Based Genome Editing
pyogenes, Streptococcus thermophilus, and Neisseria meningitides converted and
used as gene-editing tools (Charpentier and Doudna 2013, Horvath and Barrangou
2013, Hou et al. 2013, van der Oost 2013).
Molecular mechanism of CRISPR/Cas9 for
genome editing
The use of CRISPR-Cas9 system in gene editing is based on the development of guide
RNA (gRNA) sequences, which, in turn, compel for a unit fusion molecule – such as
single-guide RNA (sgRNA) – or a specific crRNA with the trans-activating CRISPR
RNA (tracrRNA) required (Yadav et al. 2021). Specifically, to target and cleave a
double-stranded DNA (dsDNA) sequence, this system is based on the formation of
the complex of sgRNA and the Cas protein (Figure 2). CRISPR/Cas9 employs Cas9
endonuclease to recognize and cleave the DNA strands using one of the two domains
(RuvC and HNH) responsible for the cleavage of the target and non-target DNA
strands (Garneau et al. 2010, Gasiunas et al. 2012, Jinek et al. 2012). During this
stage, the tracrRNA – a small noncoding RNA – directs the maturation of crRNAs
by pairing with the repeat sequence and forming a dual-RNA hybrid structure meant
to guide the Cas9 at the target sequence (usually a 20 nucleotide sequence) adjacent
to the PAM (Deltcheva et al. 2011, Gasiunas et al. 2012, Jinek et al. 2012). Jinek et
al. (2012) showed that by combining the tracrRNA and the crRNA into a chimeric
sgRNA would simplify the system and maintain Cas9 function for DNA cleavage
at a specific target. Therefore, the modification of the spacer sequence within the
crRNA would result in a simplified CRISPR-Cas system that can be engineered to
target the desired DNA sequence and induce double-strand breaks (Jinek et al. 2012).
The generated double-strand breaks (DSB) are then restored either by error-prone
nonhomologous end joining (NHEJ) – this repair generates small random indels at
the DSB site – or by high-fidelity homology directed repair (HDR) – this type of
repair is based on the use of a homologous template that produces precise genome
modification at the cleavage site (Lieber 2010, San Filippo et al. 2008) (Figure
2). The insertion of frameshift mutations leading to the impairment of the protein
function makes NHEJ less reliable and, therefore, makes its use restricted. On the
other hand, HDR is the mechanism preferred in the CRISPR technology as the long
stretches of homologous sequences used to repair the DNA lesions do not cause
mutations (Deng et al. 2013).
Mechanisms of targeted modifications
The adaptation of the CRISPR/Cas9 for the editing of several eukaryotic models
implies the exploration of this system to generate DSB that would facilitate the
insertion of mutations at specific locations in the genome. Therefore, to achieve
loss-of-function mutations, a DSB should be targeted in a constitutively spliced
coding exon (Shalem et al. 2015). Repair of the DSB through NHEJ can result
in the introduction of indels and the generation of frameshift mutations and
premature stop codons, producing the nonsense-mediated decay of the transcript.
The insertion of indels that introduce frameshift mutations can also result in non
The CRISPR/Cas9 Genome-editing System: Principles and Applications 7
Figure 2. Mechanism of action in CRISPR/Cas9 genome-editing system (after Chira et al.
(Chira et al. 2017)). The discovery of the original CRISPR/Cas9 system in bacteria facilitated
its transformation into a genome-editing tool. The tracr-RNA-crRNA forming a dual-RNA
hybrid structure serving as gRNA can be inserted into the target cell, directed towards the
nucleus. Then the cas9 gene is transcribed and exported outside the nucleus and translated into
protein (Cas9). The interaction between the gRNA and the functional Cas9 protein generates
a ribonucleic-protein effector complex able to target the gDNA (genomic DNA) at a specific
locus, nearby to the PAM sequence. DSBs are then introduced, and the restoration of these
brakes can be performed either through NHEJ (which increases the possibility of introducing
indel mutations), or via HDR (which requires the presence of a donor DNA).
functional proteins, not necessarily nonsense-mediated decay. In this respect, the
preferred exons for sequence targeting using CRISPR/Cas9 are the early exons due
to the high probability of introducing premature stop codons if indels or frameshift
mutations are inserted (Doench et al. 2014). Various studies focused on the use of
RNA-mediated programmed Cas9 on genome-scale knockout in mammalian cell
cultures highlighted the fact that this approach offers promising phenotypic effects,
high reagent consistency, and significant validation rate of the knockout efficiency
(Shalem et al. 2015, Wang et al. 2014, Koike-Yusa et al. 2014, Zhou et al. 2014).
In addition to the knockout function for genes, deactivated Cas9 (dCas9) or other
complexes using Cas9 or sgRNAs fused with transcriptional repressors, activators,
or recruitment domains are used to regulate gene expression at a specific site. This
approach provides the possibility of gene-editing without introducing irreversible
changes in the DNA sequence. For this purpose, CRISPRi and CRISPRa are dCas9
systems employed for the activation and inhibition of transcription at a specific
target site (Larson et al. 2013, Bikard et al. 2013). Oi et al. (2013) developed a
CRISPRi system to induce the repression of gene expression in HEK293 human
8 CRISPR-/Cas9 Based Genome Editing
cells. Their system uses dCas9 and can be employed for effective gene modulation
(repression/activation) (Qi et al. 2021). Another study demonstrated that the binding
of dCas9 at the promoter region inhibits transcription, most likely by preventing
RNA polymerase from binding. Moreover, by engineering this system to direct
dCas9 binding at open reading frames (ORF), transcription elongation is obstructed.
Another highlight of this study was the fusion of one RNA polymerase subunit with
dCas9, thus transforming it into a transcription activator. This strategy is efficient
in enhancing the transcription of underexpressed genes (Bikard et al. 2013, Dove
and Hochschild 1998). On the other hand, gain-of-function screens are restricted to
cDNA overexpression libraries (Yang et al. 2011). Given the partial coverage of these
libraries due to the impediments of cloning large cDNA constructs, the deficiency in
covering the complexity of transcript isoforms, these gain-of-function screens are
limited compared to the loss-of-function screens. Various transcriptional activators
(VP64, p65) are fused with dCas9 to ease gain-of-function screens (Konermann et al.
2013, Maeder et al. 2013, Perez-Pinera et al. 2013).
CRISPR/Cas9 applications
Recent years showed an increased interest in using CRISPR/Cas9 systems in
basic research studies, biotechnological applications, and for the development
of new therapeutic strategies for complex diseases (Komor et al. 2017). Current
developments include functional gene screening (Shalem et al. 2015), transcriptional
studies (Konermann et al. 2015), generation of cellular and animal models (Wang et al.
2013, 2016), genome fluorescence imaging (Chen et al. 2013), and the development
of sequence-specific anti-microbials/anti-virals (Bikard et al. 2014) (Figure 3).
Figure 3. General overview of CRISPR applications.
The CRISPR/Cas9 Genome-editing System: Principles and Applications 9
CRISPR/Cas9 as a tool for biomarkers discovery
Molecular biomarkers offer valuable information regarding the biological/molecular
characteristics of a patient that can be reflected in the progression of the disease, in the
outcome of the patients, or can predict the efficiency of a targeted therapy. CRISPR/
Cas systems can be used for the identification of these biomarkers by evaluating the
genetic/epigenetic alterations that occur in the tumor tissue (Matsuoka and Yashiro
2018, Zamborsky et al. 2019).
In a study by Xiao et al. (2018), CD44 (Cluster of Differentiation 44)
was identified as a prognostic biomarker, its overexpression predicting a poor
overall survival. The researchers used CRISPR/Cas9 system to knockout CD44
in drug-resistant osteosarcoma cell lines, followed by a decrease in the levels of
P-glycoproteins. This resulted in an increased intracellular absorption of doxorubicin
in the cells. These results reveal that using CRISPR/Cas9, CD44 was highlighted as
being involved in the response to chemotherapy in osteosarcoma cell lines (Xiao et
al. 2018). Mutations in the WNT signaling pathway induce its constitutive activation
and are usually associated with drug resistance (Duchartre et al. 2016). Using
CRISPR/Cas9 technology to create xenograft mouse models (tumors derived from
HCT116 cells in nude mice) with TIAM1(T-cell lymphoma invasion and metastasis
1) knocked-down in colorectal tumors, Izumi et al. (2019) observed an increased
sensitivity of these cells in the treatment with 5′ FU (5′ fluorouracil) and reduced
tumor size and mass measured in comparison with the controls. These results
indicated a direct correlation between upregulation of TIAM1 and drug resistance,
making this gene a potential therapeutic target that can be used in colorectal patients
in the attempt to overcome drug resistance (Malliri et al. 2006, Izumi et al. 2019). In
lung cancer studies, the knockdown of YES1 gene using CRISPR/Cas9 technology
was correlated with reduced metastasis rate and diminished tumor burden, indicating
YES1 as a prognostic biomarker for lung cancer patients (Garmendia et al. 2019).
CRISPR/Cas9 is also effective in editing non-coding molecules, such as
microRNAs (miRNAs). These molecules modulate a vast number of molecular
pathways by targeting a variable number of genes, acting either as tumor suppressors
or as oncogenes, based on their downregulation or upregulation encountered in
cancer progression (Berindan-Neagoe et al. 2014). Therefore, in a study published
by Zhou et al. (2017), the authors demonstrated that upregulation of miR-3188 in
hepatocellular carcinoma is associated with an unfavorable outcome for the patients.
Using CRISPR/Cas9 to knockout the expression of this microRNA in hepatocellular
carcinoma cell lines by aiming the sequences next to the Drosha processing sites
harbored in the miR-3188 secondary loop, the authors observed that underexpression
of miR-3188 suppressed malignant characteristics. These results indicated miR-3188
as a potential biomarker for early diagnosis and therapeutic target for the treatment
of hepatocellular carcinoma (Zhou et al. 2017).
In epigenetic studies, CRISPR/Cas9 was used to mediate the DNA methylation
of DERARE (Distal Element Multiple Retinoic Acid Response Element), which
inhibited the expression of HOXB and diminished leukemogenesis. This was possible
by using the epigenetic editing tool developed by Vojta et al. (2016), in which the
group constructed a dCas9-DNMT3A (deactivated Cas9-DNA methyltransferase
