www.impactjournals.com/oncotarget/ Oncotarget, Vol. 7, No.

32

Review
CRISPR/Cas9 therapeutics: a cure for cancer and other genetic
diseases
Faheem Ahmed Khan1, Nuruliarizki Shinta Pandupuspitasari1, Huang Chun-Jie1,
Zhou Ao1, Muhammad Jamal2, Ali Zohaib3, Farhan Ahmed Khan4, Muthia Raihana
Hakim5 and Zhang ShuJun1
1
Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Education Ministry of China, Huazhong
Agricultural University, Wuhan, People’s Republic of China
2
State Key Laboratory of Agriculture Microbiology, Huazhong Agricultural University, Wuhan, People’s Republic of China
3
Key Laboratory of Special Pathogens and Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese
Academy of Sciences, Wuhan, China
4
Department of Cardiovascular Medicine, Zulfiqar Ali Bhutto Medical University, Pakistan Institute of Medical Sciences,
Islamabad, Pakistan
5
Tongji Medical College, Huazhong University of Science And Technology, Wuhan, China
Correspondence to: Zhang ShuJun, email: sjxiaozhang@mail.hzau.edu.cn
Keywords: CRISPR/Cas9, genetic diseases, cancer, genome editing, next generation sequencing
Received: March 12, 2016 Accepted: May 19, 2016 Published: May 26, 2016

Abstract
Cancer is caused by a series of alterations in genome and epigenome mostly
resulting in activation of oncogenes or inactivation of cancer suppressor genes.
Genetic engineering has become pivotal in the treatment of cancer and other genetic
diseases, especially the formerly-niche use of clustered regularly interspaced short
palindromic repeats (CRISPR) associated with Cas9. In defining its superior use, we
have followed the recent advances that have been made in producing CRISPR/Cas9
as a therapy of choice. We also provide important genetic mutations where CRISPRs
can be repurposed to create adaptive immunity to fight carcinomas and edit genetic
mutations causing it. Meanwhile, challenges to CRISPR technology are also discussed
with emphasis on ability of pathogens to evolve against CRISPRs. We follow the recent
developments on the function of CRISPRs with different carriers which can efficiently
deliver it to target cells; furthermore, analogous technologies are also discussed
along CRISPRs, including zinc-finger nuclease (ZFN) and transcription activator-like
effector nucleases (TALENs). Moreover, progress in clinical applications of CRISPR
therapeutics is reviewed; in effect, patients can have lower morbidity and/or mortality
from the therapeutic method with least possible side-effects.

Introduction much lower cost. Cancers are characterized by DNA and

RNA alterations including mutations, gene duplications
In recent years, available therapies for cancers have and changes in messenger RNAs. The integrative approach
been evolving to the betterment of prognosis in patients. to utilize genomic and transcriptomic advances can unveil
Chemotherapy, radiotherapy and surgery are used in the complete picture of individual genome. This approach
combination to reduce the cancerous cells to remission, is also being used in clinical setting to make critical
that increases lifespan to a maximum of five years. decisions regarding patient treatment [2].
However, harmful side effects and toxicity increases the Cancer exsists in multiple complex forms making
mortality whilst it significantly reduces the quality of it difficult to prevent and/or treat. It is of ought most
life [1]. The understanding of cancer biology is of key importance to study etiology, pathogenesis, prognosis
importance to develop novel anti-cancer therapies. The and its phenotypes to develop new therapies and improve
present day advances in sequencing technology have the existing treatments. Mutations are among the leading
helped to explore the cancer genome more efficiently with causes of cancers. To date approximately 140 genes with

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 deleterious mutations are reported. This further complicate (Figure 1) [9]. This natural adaptive immunity of bacteria
the ability to develop appropriate effective therapeutics and archaea can be redesigned to achieve desired genome
[3]. The multiple steps in cancer development provides editing and more importantly repurpose it as a therapy
ample time for therapeutic strategies to work against its against long awaiting cancerous and genetic disorders.
appearance. The initiation of cancer begins with DNA Epigenetic changes defines the environment for
mutation but several contributing factors arise from the cancer development. The mutations in tissue development
epigenome of the individual which needs novel approaches genes stimulate cancer development. Previously Khan
in maintaining the homeostasis. Recent advances and colleagues discussed SUMOylation as one of the
in sequencing technologies have helped sequence a epigenetic events causing cancer that might be exploited
diversified variety of cancer neoplasms, providing novel in novel therapeutic strategies to cure cancers [7]. The
insights into cancer prevention and therapy. present review is an attempt to see the applicability of
The human genome is composed of two haploid sets CRISPR/Cas9 system in cancer and genetic disease
of 23 chromosomes, each containing approximately six therapies. Furthermore, several genetic mutations and
billion nucleotides. Approximately 20,000 genes exist in suitability to CRISPR/Cas9 system is explored to provide
each set of chromosome. These genes are transcribed into researchers to focus on the translation of laboratory
messenger RNAs (mRNA), ribosomal RNA (rRNA) and research to clinics.
transfer RNA (tRNA) constituting the whole transcriptome
of an individual [4, 5]. Other species of RNAs termed as Next generation sequencing,
non coding RNAs (ncRNAs) which includes micro-RNAs mutations, genetic diseases and
(miRNAs) that does not encode proteins but may activate
or inhibit the gene expression [6]. miRNAs have been tumorigenic genes
reported to stimulate several genes and are evolving as
important players in therapeutic strategies against several The understanding of cancer has been revolutionized
diseases [7]. The completion of Human Genome Project in by the present day next generation sequencing
2003 established foundations for precision medicine based technologies (NGS). The NGS provides the identification
on sequencing technologies continues its journey from of specific mutations relevant to cancers and other genetic
RNAi, ZFNs and TALENs and now it steps into a unique diseases at genomic level that can be edited by genome
CRISPR/Cas9 genome editing tool. The present day editing technologies the ZFNs, TALENs and CRISPRs
understanding of gene functions and mutations owing to or the combination of them. The NGS technologies
omics results in establishment of various molecular tools include whole genome sequencing, whole exome
to diagnose risk factors for various diseases having genetic sequencing, RNA sequencing, reduced representation
components enlisting Diabetes, Alzheimer’s, Huntington, bisulfite sequencing, and chromatin immunoprecipitation
Duchenne muscular dystrophy, Inborn blindness and sequencing each of which is employed for specific
Rheumatoid arthritis. The CRISPR/Cas9 technology has objectives reviewed in Yadav et al., 2015 [10]. In cancers
presently been shown to correct the mutations causing often the whole exome sequencing is performed to get
those diseases and has a potential to be developed as a specific mutations at the cellular levels.
promising therapy at genetic level to protect patients at A substantial quantity of recent researches identifies
risk. mutations in onco-genes that causes cancer. The well
Previously, several therapies to treat cancer were known onco-genes are p53, AKT1 (v-akt murine thymoma
introduced but none sustained for long time. Major causes viral oncogene), BRCA1 (breast cancer in females and
of failure include the development of the self-resistance prostate cancer in males), BRCA2 (breast cancer in females
and the deleterious side effects. Previously DNA domain and prostate cancer in males), ALK (anaplastic lymphoma
binding proteins zinc fingers nucleases (ZFNs) and receptor tyrosine kinase), BRAF (B-Raf proto-oncogene,
transcription activator-like effector nucleases (TALENs) serine/threonine kinase), EGFR (epidermal growth factor
were employed to treat cancers but their efficiency was receptor ), KRAS (Kirsten rat sarcoma viral oncogene),
limited due to their inability to effectively target the MET (proto-oncogene, receptor tyrosine kinase), NRAS
epigenetic changes that occur during carcinogenesis [8]. (neuroblastoma RAS viral (v-ras) oncogene homolog),
Recently a more versatile genome editing technology, RET (ret proto-oncogene), ROS1 (ROS proto-oncogene 1,
clustered regularly interspaced short palindromic receptor tyrosine kinase), Bcl11A (B-cell CLL/lymphoma
sequences (CRISPRs) associated with HNH domain 11A), Bcl11B (B-cell CLL/lymphoma 11B) and HER2/
protein Cas9 shows promise towards reliable long term neu (erb-b2 receptor tyrosine kinase2). It is a necessity
cancer therapy. CRISPR/Cas9 is an adaptive immune to understand the normal signaling pathways as well as
system in bacteria and archaea against phage invasion in dysfunctional signaling mediated by gene mutations.
natural environment. Bacteria evolve this system through Some of the mutations in genome causing cancers and
capturing DNA sequences and used it as a memory to be other genetic diseases are listed in Table1.
identified as enemy and destroy it on its attack in future Several studies in past proposed therapies that might
be useful in treating cancers. Among those therapies the

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 Table 1: Cancers, genes, mutations, and CRISPRs editing ability
Mutations that can be corrected with CRISPR/Cas9 Gene References
Cancers/ Genetic diseases CRISPR/Cas9* targets
Lung exon 19 deletion and L858R EGFR [11]
G309A, D769H, D769Y, V777L, P780ins, HER2/Neu,
Breast V842I, and R896C and BRCA [12]
BRCA1/2 mutations

Thyroid C228T and C250T TERT promoter [13]

HBB
β- Thalesemia IVS2-654 (C > T) [14]

Huntington p.(Gln302) and p.(Tyr539Cys) RNF216 [15]

Limb girdle muscular c.5713C>T; p.R1905X, and missense Dysferlin, and alpha- [16]
dystrophies types 2B and 2D c.229C>T; p.R77C sarcoglycan
Alzheimer's H214N, R220P Presenilin 1 [17]
*These are only few of the representative mutations causing cancers or other genetic diseases

nuclease guided therapies carries the potential to correct have been associated with Rasonco-genes (Hras, Kras,
the mutations and dysfunction in a homeostatic epigenetic Nras). Recently mutations in Ras genes were shown to
environment that causes cancers. dysfunction the wild type allele and hence generating
The correlation of chronic inflammation is well proto-oncogenes that suppresses the carcinogenesis [20].
defined in cancer acceleration but its cellular and The findings of To and colleagues is of high importance as
molecular mechanisms remain unknown. A recent study in it is possible to produce desired mutations in Ras genes in
this regards found that KrasG12D an onco-gene that induces patients at risk of lung and other cancers. The generation
expression of IL-17 receptors on pancreatic intraepithelial of mouse cancer models become efficient with CRISPR/
neoplasia (PanIN) and also synergistically employs Cas9 technology. Several laboratories have reported
TH17 and IL-17+/gdT Cells stimulate the expression of useful results in the progress towards cancer cure such as
PanIN epithelial gene expression hence providing insight the NANOG and NANOGP8 involvement in malignant
into the pancreatic neoplasia [18]. Lung cancer that potential of prostate cancer [21] which can be corrected
accounts for 1.6 million deaths worldwide in 2012 [19] with CRISPR/Cas9 or in combination with TALENs or

Figure 1: The CRISPR/Cas9 mechanism.

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 either ZFNs. pathways. The development of RNAi technology in the
Apart from cancers there are several other genetic early 90’s and its application in mammalian cells to unveil
diseases including Huntington, Alzheimer’s, Diabetes, the molecular functions of genes gave rise to the era of
Sickle cell anemia which are caused by mutations in reverse genetics. Since the discovery of RNAi technology
relevant genes. Notably many of these mutations are more efficient tools naming zinc finger nucleases (ZFNs),
now known with the help of NGS technologies. The TALENs and CRISPRs [23, 24] are developed which can
developments in the genome editing technologies have the perform the genome wide screens efficiently and have
potential to precisely correct those mutations and revert the recently been employed to correct several environmentally
defect to its original form at DNA level. The programmed induced mutations and inborn genetic defects.
nucleases ZFNs and TALENs were used previously to The modifiable ability of genome editing nucleases
correct these deleterious mutations, however, the success to make specific double stranded DNA breaks (DSBs)
of the technology fall well short of expectations. which are primarily repaired by naturally present non-
homologous end joining (NHEJ) DNA repair pathway
Genome Editing Tools that is prone to frame-shift mutations resulting in gene
disruption [25, 26]. This condition can be precisely
The interpretation of gene expression, its corrected by providing a template along with nucleases
stimulatory or suppressive role in biological pathways that will follow a homologous repair (HR) pathway to
and its interaction with disease phenotypes remains the mend DSB. It has been previously reported that a nuclease
core aim of classical genetics and today’s age molecular induced DSB near a disease mutation can significantly
biology [22]. The design of any therapeutic technology enhance HR pathway [27]. Hence it is very much possible
at molecular level that can cure diseases should have to correct the mutated gene by providing a template of
the ability to precisely correct malfunctioned cells and wild type gene thus greatly enhances its applications in
biomedical research.

Figure 2: Methods for delivery of Cas9-sgRNA complex to cell A. Microinjection based delivery of Cas9-sgRNA B. viral vector
(AAV) based delivery C. Lipofection D. Cell penetrating peptides (CPP) based delivery of Cas9-sgRNA complex into mammalian cells
have shown successful genome editing with high efficiency.

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 CRISPRs were discovered firstly in Escherichia been worked out to keep the CRISPRs/Cas9 on target and
coli where Ishino and colleagues observed unusual is extensively reviewed recently [25].
repeat structures in 3’ flanking region of iap gene [28]
with several later observations of similar structures in Altering Epigenome with CRISPRs/
other bacterial species, and named CRISPR by Jansen Cas9
and colleagues [29]. It was later known that the CRISPR
loci captured spacer sequences from the invading viruses Epigenetic modifications consisting of DNA
and used them as a memory to provide bacterial host and methytlation and histone modifications provides an
or archaea to develop an adaptive immunity through the essential environment for stimulating gene expression
Cas proteins which makes a double stranded DNA breaks that defines their cell proliferation and differentiation
(DSBs). Based on the phylogeny of Cas gene, crRNA activity [35, 36]. Histone proteins responsible for
biogenesis and mechanism of nucleic acid cleavage (DNA packaging the whole DNA in eukaryotic cells undergo
and RNA), more than 13 different CRISPR-Cas systems several epigenetic modification including ubiquitination,
have been recognized which is classified into three phosphorylation, SUMOylation, and acetylation. All
major groups (I, II and III) and at least 12 subtype (A-F) those are of reversible nature and are under the control
respectively [30, 31]. The recent studies on Cas proteins of epigenetic modification enzymes [37, 38]. These
demonstrate its utility in initial identification and excision epigenetic covalent modifications of histones are of high
of attacking viral DNA genomes [32]. The RNA guided importance in repression or activation of gene expression
DNA breaks is elucidated by understanding the crystal [39]. Apart from histone modifications, the structure of
structure of SpCas9 and constructing the truncated Cas9 chromatin is also defined by DNA methylation. The first
mutant that facilitates its in vivo therapeutic application ever epigenetic modification identified is carried out by
by providing a mechanism of its packaging in size restraint DNA methyltransferase enzymes (DNMTs) [8], that
viral vectors [33]. The SpCas9 crystal structure opened provides environment to prevent binding of transcription
several avenues for its practical applications and scientists factors and/ or bring repressive protein complexes to DNA
around the globe start working it out in their labs for large [40]. DNA methylation is more stable in comparison with
functional screens of their libraries [34]. The problem of post translational modifications of histone but can still
CRISPRs/Cas9 off-target effects are widely questioned for be demethylated by active and passive mechanisms, and
its clinical application and hence several strategies have

Figure 3: CRISPR/Cas9 can be redesigned to cure mutations causing cancers and genetic diseases.

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 is responsible for the normal development and cellular alter genome sequences, hence providing an opportunity
differentiation [41]. These days several non coding RNA to correct disease causing mutations, but requires potent
(ncRNA) species for example micro RNA (miRNA), short delivery methods to cells. The Cas9 can be delivered
interfering RNA (siRNA) are found to inhibit or activate to cells as mRNA, however the mRNA is unstable and
genes that are involved in the epigenetic regulation of is not suitable for long term gene therapy purposes,
critical biological processes of growth and development however the alterations to genome remains and widely
[42]. miRNAs are implicated in several disease conditions used in model organisms including mouse, zebrafish,
and can be used to target specific gene expression for Drosophila and C. elegans [46, 47]. The expression of
its up and/or down regulation as required [7]. Recently, Cas9 and gRNA complex in cultured mammalian cells
CRISPR/Cas9 has been successfully employed to edit is mostly done physically by delivering non-replicating
genetic switches [9], and many of the miRNAs that are plasmids expressing these cassettes. These methods
involved in cancer progression and development can be involve electroporation [48], microinjection [49, 50]
specifically targeted with CRISPR/Cas9 genome editing lipofection [51, 52] nucleofection [51-53]. The main
system. disadvantage of using plasmid is the random integration
The recent advances in genome editing technologies of plasmid or its part in both off-target and on-target sites
especially CRISPR/Cas9 marvelous outcomes have leading to insertion inactivation of genes. To address
given strong hope to deal with deadly genetic and these shortcomings, several efforts were put in to deliver
cancerous diseases. The suppression of gene expression Cas9 protein in conjugation with cell penetrating proteins
was demonstrated when dCas9 (double mutant) fused (CPPs) complexed with guide RNA (gRNA) that forms
with a repressor Krüppel-associated box KRAB, but its nanoparticles with positive charge showed efficient
genome wide specificity and heterochromatin specificity disruption of genes [54]. An enhanced delivery vehicle
was not known until reports of its binding HS2 enhancer, inspired from DNA nanoclews and DNA nanoparticles
having distinct role in expressing many globin genes. The is recently been reported having the capability of
observation of highly specific H3K9 trimethylation and simultaneously delivering the Cas9 protein and sgRNA
limited chromatin accessibility of enhancer and promoter to human cell nuclei and disrupt genes efficiently while
suggesting individual enhancers can be successfully maintaining cell viability [55]. Viral vectors such as
modified to control epigenome changes [43]. Adeno-associated virus (AAV), lenti-virus are broadly
Epigenetic drugs are in extensive use with reliable used as gene delivering tool due to efficient introduction of
efficacy but have some strong side-effects that need an an exogenous DNA fragment into genome by robust HDR.
alternative platform to specifically modify the epigenome Although low pathogenicity and oncogenic risk makes
for treating cancers. Epigenetic drugs have been AAV a suitable choice, yet large size of SpCas9 (4.2 kb
implicated in severe off-target effects that trigger several in length, ~1,400 amino acids) limit AAV transducing
genes including p53, Akt, cMyc and cause dysfunction abilities as it allows only 4.7 kb fragment to be transduced.
in several biological pathways including metabolic This problem has been resolved by a smaller size ´SaCas9´
and immunity pathways. Hilton and colleagues have (3.3 kb). AAV mediated delivery of this Cas9 variant
recently demonstrated that a fused nuclease-null dCas9 shows the same efficiencies as the natural SpCas9 on the
with catalytic core of human acetyltransferase p300 same genomic loci with no off-target mutation [56].
cotransfected with multiple gRNAs in HEK297T cells to Mostly for gene therapy ex vivo treatment of cells is
target IL1RN, MYOD and OCT4 endogenous promoters performed which is later transferred to body and the most
resulted in highly specific gene expression through stable way of transferring Cas9 and gRNA in this regard
acetylation of H3 lysine27 (H3K27) by its promoters is with the use of non-viral DNA plasmids [46]. It is of
and enhancers, thus provides robust tool for gene paramount importance to develop tools and methods to
modifications [45]. As the major portion of human cancers efficiently and specifically stimulating or inhibiting gene
is because of the loss in global methylation patterns or expression to achieve desired results for cancer therapy.
hypermethylation of specific loci, the present progress and The strategy to control cancer disease can be done in
further findings of epigenome editing tools will be of key two ways either by removing tumor tissue or to control
interest in curtailing cancers. transgene expression by the tumor-specific promoter
such as telomerase promoters which are often involved in
Carriers of CRISPR/Cas9 to cells cancers. The in vivo delivery of nucleases is addressed by
Zuris and colleagues, they conclusively show that proteins
There are difficulties in uniform and sustained delivery by cationic lipid is a viable approach for genome
transportation of CRISPR system to cells, that needs editing and is capable of carrying Cre recombinase, TALE,
to be addressed. The specialized methods developed CRISPR/Cas9: gRNA and Cas9 transcription factor
to deliver Cas9 and gRNA to target site within a cell or activators [57] (Figure 2).
tissue claims to aid CRISPR specificity. The nuclease The delivery of ribonucleoprotein (RNP) (Cas
genome editing technologies effectively and efficiently protein and gRNA) to cells induced site specific mutation

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 of up to 79% and reduced off-target cleavage associated successfully corrected β-catenin gene mutation frequently
with plasmid transfection at off-target sites that differ involved in cancer with CRISPR [62].
by one or two nucleotides from on-target sites [58, 59]. Burkitt’s lymphoma is a cancer caused by mutations
The efficiency of delivering RNPs via electroporation in cMyc gene and almost every patient suffering from
transformation is reported two times higher as compared it, has Epstein-Barr virus (EBV) infection. Recently
to plasmid mediated transformation. Through RGEN CRISPR/Cas9 system is used against EBV that reduced
RNP delivery, two endogenous genes (dpy-3 and unc-1) the viral load and tumor proliferation [63]. Furthermore,
in C.elegans have been heritably knocked down without recent studies demonstrates successful editing of tumor
indel formation [60]. This method has some advantages as suppressor gene Trp53 in Arf−/−Eμ-Myc lymphomas
this complex provides a control Cas9 amount to the cell [64], the over-expression of Myc gene is responsible for
followed by rapid degradation. For therapeutic application several kind of lymphoma cancers [7]. Subsequent studies
the invention of safe and improved delivery method of shows Mll3 as another important tumor suppressor gene
Cas9-sgRNA into the cell or organisms is urgently needed. disruption via CRISPRs ex vivo in acute myeloid leukemia
Improvement of existent method and invention of new [65]. The successful application of the genome editing tool
method as a cargo such as use of nano particles might TALENs in one year old girl patient of leukemia provides
enable efficient and specific genome editing [58, 61]. the grounds for the use of more efficient CRISPRs
application in clinics.
CRISPRs Path to clinics Apart from cancers, CRISPRs are also used
to correct several genetic diseases. An inherited eye
The rapid development of the genome editing disease Retinitis pigmentosa that causes breakdown of
technologies need an adequate attention towards photoreceptor cells resulting in gradual loss of vision
improving pre-clinical and clinical assays to assess the is been recently edited successfully in iPSC for RPGR
toxicity, off-target effects, and other possible side effects. gene, which in health state is responsible for production
Several attempts have been made in using CRISPRs to of proteins involved in normal vision, to give new
correct the mutated genes. One of such study was carried hope to patients blinded with Retinitis pigmentosa
out in mice cancer model with mutated Pten and p53 [66]. Efforts are put in Editas pharmaceuticals to make
genes. The mice were transfected with a vector carrying CRISPR therapeutics against children genetic disease
designated CRISPR through a tail vein to achieve 20% Leber congenital amaurosis, that causes blindness by
of hepatocytes to transform through blood that also editing the defected gene with CRISPR in eye cells and

Figure 4: The two DNA repair pathways. NHEJ is naturally favored while HDR pathway has therapeutic application in correcting
several mutated genes.

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 plans to launch it by the end of the year 2017. Another The editing frequency achieved by NHEJ ranges from
recessive X-linked disease the Duchenne muscular 2% to 25%, which cause high efficiency deletion of the
dystrophy (DMD), is primarily caused by a frame-shift intervening sequence [52]. HDR pathway uses donor DNA
mutation in dystrophin protein which is essential for as a template to repair the DSB in a ‘copy-and-paste’-
proper functioning of muscles, and is very much suitable type using homologous recombination mechanism. By
for genome editing inspite of its very large size of 79 providing an appropriately designed donor DNA (≥400bp
exons. It does not require whole of the gene to express in case of plasmid and 25-65 bp in case of single stranded
and with little changes to sequence which causes disease oligodeoxynucleotides) [72], precise small or large
can bring improvements in muscle functioning. Thus an modifications can be made to the genome (HR-mediated
exon skipping technology can be applied that is proved genome editing). The genome editing ability of CRISPR-
successful in mouse model of Duchenne muscular Cas9 using HDR pathway is not fully developed. As the
dystrophy [67-69]. The exon skipping technology with NHEJ pathway is erroneous causing InDels (insertion-
CRISPR/Cas9 opens the door to treat several other deletion) formation at the cleaved site, may lead to frame
diseases such as Ataxia telangiectasia, congenital disorder shift mutation resulting in a malfunction proteins or non-
of Glycosylation, and Niemann-Pick disease type C sense mediated decays of transcripts ultimately causing
caused by errors in splicing. A well renowned experiment gene disruption [73]. The rate of specificity increase with
in SUN-YATSEN university China in human embryos to increase in HDR mediated repair of DSB induced by
treat Thalasemia causing gene in human embryos shows CRISPR Cas9. Hence the use of HDR is more favored.
only a few embryos out of 80 received a corrected form However, NHEJ competes with HDR to rectify the DSBs.
of gene copy (Figure 3). These examples of successful The use of Scr7 inhibitor antagonistic of DNA ligase IV
clinical application with CRISPR defines bright future (a principal enzyme involved in NHEJ repair pathway)
of the technology, but have still to work out several increased the genome editing efficiency up to 19 fold by
preclinical and clinical assays to determine the side effects preventing NHEJ [74]. It has been shown that cell cycle
on patients health, the immunogenic responses to vector synchronization of the nuclease in G2 increases HDR
carriers, and possible drawbacks on over all genome as a efficiency while reducing unwanted NHEJ events [75].
result off-targets. The studies in mouse models confirmed Similarly substituting the normal Cas9 for Cas9 nickase
the tumor suppressor activity of SWI/SNF subunits. A (Cas9n) activates HDR with low off-target potentials [49].
recent study demonstrates that mutations in EZH2 affects However the efficiency of recombination is low (1 in 106-
the tumor suppressor activity of SWI/SNF subunits and it 109 cells), limiting the large-scale applications of HDR in
also suggests that inhibitors of EZH2 in developmental gene-targeting assay [72]. The specificity and accuracy of
phase will also not fully control the oncogenic activity of the gene editing process following site-specific genomic
EZH2. Hence carefully designed CRISPR/Cas9 system breaking by RGENs depends upon the nature of donor
can be utilized to correct the mutations that can help regain DNA [76]. If the foreign DNA have a homology more
the tumor suppressor activity of SWI/SNF subunits [70]. than 400bp with the target, it will lead to more efficient
introduction of precise nucleotide substitutions or
Challenges in CRISPR/Cas9 deletions, endogenous gene labeling, as well as targeted
clinical therapeutics transgene [77]. Increasing the length of homology arms of
the homology-directed repair template facilitates targeting
There are some obstacles that limits the commercial efficiency, while increasing the length of the DNA insert
therapeutic application of CRISPR. The gene editing reduced it [59].
ability of Cas9 is of paramount importance. The dsDNA The specificity of the CRISPRs is also under
break by Cas9 is followed by two natural pathways question and many clinical laboratories are concerned
present in the cell; Non-homologous end joining (NHEJ) about its off-target effects and the ways that can minimize
and homology directed repair (HDR). NHEJ is naturally those off-targets and develop clinical assays to measure.
favored pathway for gene correction in nature but is Several advances are made in delivering nucleases to
error prone and causes undesired mutations hence is not destination cells ex vivo and in vivo but there is still need
suitable for the application of CRISPR as therapeutic to improve the delivery systems for realizing the dream
agents. HDR on the other hand is accurate and error free of CRISPRs therapeutics. Besides the specificity, key to
but is not naturally favored pathway for DSBs correction, success is the isolation of mutant cells (having DNA of
hence requires means to make HDR to be favored over interest) from a diverse population of cells.
NHEJ in natural environment to efficiently translate The evolution of host-pathogen interaction is a
CRISPRs benefits to clinics (Figure 4). NHEJ is mostly continuous process. The natural development of CRISPRs
dominating during G1, S and G2 phase while HDR in in bacteria and archaea as adaptive immune responses
late S and G2 phase [71]. These two pathways have been took hundreds of years to fight the invasion of pathogens.
manipulated by researchers for genome editing using Recently some groups identified anti-CRISPRs proteins in
CRISPR in mammalian cell for the first time [52, 53]. viruses that can destroy the bacterial CRISPRs with all its

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 memory records of invading viruses and hence exposing 94.
it to the larger threat of attacking viruses. Such findings 7. Khan FA, Pandupuspitasari NS, Huang CJ, Hao X, Zhang
stress the need to further develop more accurate clinical S. SUMOylation: A link to future therapeutics. Curr Issues
assays of longer efficacy for the introduced therapeutic MolBiol. 2015; 18: 49-56.
CRISPRs against evolutionary pressure asserted by 8. Sachdeva M, Sachdeva N, Pal M, Gupta N, Khan IA,
pathogens. Majumdar M, Tiwari A. CRISPR/Cas9: molecular tool
for gene therapy to target genome and epigenome in the
Conclusions treatment of lung cancer. Cancer gene therapy. 2015; 22:
509-517 doi:10.1038/cgt.2015.54.
There is much buzz around genome editing 9. Hsu PD, Lander ES, Zhang F. Development and
technologies specially CRISPRs to be used against several applications of CRISPR-Cas9 for genome engineering. Cell.
life threatening diseases at the molecular level. The phrase 2014; 157: 1262-1278.
“Nip the evil in the bud” may rightly be used for CRISPRs 10. Yadav SS, Li J, Lavery HJ, Yadav KK, Tewari AK.
therapeutics but it is critical to develop all the requisite Next-generation sequencing technology in prostate
clinical tests for its efficacy, safety and specificity before cancer diagnosis, prognosis, and personalized
its use in clinics. The short journey of CRISPRs till now treatment. UrolOncol. 2015; 33: 267-13 doi: 10.1016/j.
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medical cure against deadly diseases.
11. Ramalingam SS, O’Byrne K, Boyer M, Mok T, Jänne
PA, Zhang H, Liang J, Taylor I, Sbar EI, Paz-Ares L.
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mutated advanced nonsmall-cell lung cancer (NSCLC):
Financial assistance from the natural Science pooled subset analyses from two randomized trials. Ann
Foundation of China (3157236, 31272427) are greatly Oncol. 2016; 27: 423-9 doi: 10.1093/annonc/mdv593.
appreciated. the constructive discussions of laboratory
12. Bose R, Kavuri SM, Searleman AC, Shen W, Shen D,
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Koboldt DC, Monsey J, Goel N, Aronson AB, Li S, Ma CX,
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