CRISPR/Cas9-Mediated Genome Editing of Epigenetic Factors for Cancer Therapy

Abstract

Advances in engineered recombinant nuclease have provided facile and reliable methods for genome editing. Especially with the development of the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein-9 nuclease) system, the discovery of various versions of Cas9 proteins and delivery carriers, it is now practicable to introduce desired mutations into the genome, to correct disease-related mutations, and to activate or suppress genes of interest. Epigenetic regulators are often disturbed in cancer cells and are essential for the transformation of normal to cancerous cells. Tumor-related epigenetic alterations or epigenetic factor mutations play a major part during the various steps of carcinogenesis and affect a variety of cancer-related genes and a wide range of cancerous phenotypes. Therefore, epigenetic regulatory enzymes might be candidate targets for cancer therapy. In this review, we discuss prospects of CRISPR/Cas9-based genome editing in targeting epigenetics for cancer gene therapy.

Introduction

Classical epigenetics is defined as a heritable molecular mechanism that changes gene expression patterns without alterations in DNA sequence. This mechanism involves multiple DNA and histone covalent modifications, which constitute an essential platform for the actions of chromatin-remodeling and gene expression regulators.^1,2 Epigenetics has become an important topic in cancer research, and accumulating evidence has shown that malignant transformation of normal cells and tumor maintenance require extensive epigenomic reconfigurations.^2
–4 DNA methyltransferases (DNMTs) and various histone modification enzymes, such as NSD2, EZH2, and LSD1, are dysregulated in cancerous cells, causing gene expression reprogramming at a genome-wide scale.^5

–11 Multiple epigenetic factors, including histones themselves, were found to be recurrently mutated in multiple types of human cancer, including glioblastoma, hematological malignancies, chondrosarcoma, and osteosarcoma, which directly linked dysregulated epigenetic control with carcinogenesis.^12

–15 Further, emerging evidence indicates that these epigenetic abnormalities are also essential for tumor maintenance.^16,17 On the basis of this evidence, targeting epigenetic regulatory enzymes might be an important strategy for cancer therapy.^18,19

Rapid advances in engineered nuclease-mediated genome editing have provided efficient and convenient tools for gene therapy. To date, three major nucleases—zinc finger nucleases (ZFNs),²⁰ transcription activator-like effector nucleases (TALENs),²¹ and the clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein-9 (Cas9) nucleases²²—have been developed to induce site-specific genome modifications. Compared with ZFNs and TALENs, CRISPR/Cas9 is much more easily reprogrammed to new targets and has significantly higher amounts of targetable sites.^22,23 Another advantage of CRISPR/Cas9 is the ability to manipulate multiple genes simultaneously. These advantages of the CRISPR/Cas9 system have substantially improved our ability to make desired changes in the genome, which provides a promising tool for targeted gene therapy.²⁴ Since the pioneering works showed that the bacteria type II CRISPR system is reprogrammable,^25

–29 numerous efforts have been focusing on exploring the modes of action of the CRISPR/Cas9 system and developing new versions of Cas9 proteins and carriers. On the basis of these efforts, a list of CRISPR/Cas9-based toolkits is already available to introduce desired mutations into the genome, to correct disease-related mutations, and to activate or suppress the expression of certain genes.^30

–35 These exciting advances are beginning to show remarkable promise in clinical applications.^24,36 Here, we discuss some prospects of CRISPR/Cas9-based technology to target the cancer epigenome as potential cancer therapy.

Epigenetic Regulation

Epigenetic mechanisms, including DNA methylation and various histone modifications, constitute an essential environment for gene expression regulation, thus playing important roles in normal cell differentiation, proliferation, and function.³⁷ DNA methylation usually takes place at the 5′ carbon of the cytosine ring within CpG dinucleotides in the mammalian genomes, resulting in gene silencing.² In mammalian cells, there are three main DNA methyltransferases (DNMTs): DNMT1, DNMT3A, and DNMT3B.³⁷ Of those, DNMT1 is responsible for the maintenance of preexisting methylation patterns after DNA replication during cell divisions,³⁸ whereas DNMT3A and DNMT3B target previously unmethylated CpGs, yielding novel methylation patterns.^39,40 Another member of the DNMT3 family, DNMT3-like (DNMT3L), has no catalytic activity and functions as a regulator of DNMT3A and DNMT3B.^41,42 DNA methylation is reversible both at a specific genomic locus and on a global scale. The mechanisms of DNA demethylation can be either passive or active.⁴³ The passive process takes place on newly synthesized DNA strands during DNA replication when DNMT1 function is absent. Active DNA demethylation occurs via removal of a methyl group that has been modified in a DNA replication-independent manner. For example, it was shown that the ten–eleven translocation (TET) protein family could sequentially modify DNA methylation, which initiates an important active mechanism of DNA demethylation.⁴⁴ The TET family comprises at least three members, TET1–3, all of which can oxidize methylated cytosine into 5-hydroxymethylcytosine, and subsequently into carboxyl- and formylmethylcytosine.^45,46 These modified methyl groups may be actively removed via DNA repair pathways, resulting in active demethylation. Also, the oxidized methyl groups are not recognized by DNMT1, leading to passive demethylation during replication.⁴⁵ Dynamic and reversible DNA methylations through DNMTs and TETs are critical for gene regulation and various cellular functions.

Histone modification is another major type of epigenetic mechanism that occurs on specific amino acid residues of the histone proteins. Posttranslational histone modifications play fundamental roles in gene regulation and cell differentiation by forming synergistic signaling platforms within specific genomic loci, which are recognized by specific readers with diverse functions ranging from transcriptional regulation of specific genes to large-scale chromatin remodeling.⁴⁷ At present, a large body of histone modifications has been discovered, including lysine acetylation and methylation, arginine methylation, serine and threonine phosphorylation and glycosylation, ADP ribosylation, lysine ubiquitylation, as well as SUMOylation (posttranslational modification of small ubiquitin-like modifier [SUMO] proteins). Among them, acetylation and methylation are the best known histone modifications. Depending on which amino acids in the histone are modified and how many methyl groups are added, histone methylations can either increase or decrease gene transcription.^48,49 Methylation events such as H3K4me2/3 (di- and trimethylation of histone H3 at Lys-4) and H3K79me3 (trimethylation of histone H3 at Lys-79) weaken chemical attractions between histone tails and the DNA helix, thus facilitating DNA to uncoil from nucleosomes and to be available for transcription factor proteins and RNA polymerase. On the other hand, methylations such as H3K9me2/3, H3K27me2/3, and H4K20me3 are associated with repression. Histone methylation is relatively stable because its half-life is approximately equal to the half-life of histones themselves.⁵⁰ However, like DNA methylation, histone methylation is also reversible. The methyl groups on given histone amino acid residues can be removed by specific demethylases. LSD1, also known as KDM1A, was the first demethylase to be discovered, which performs the demethylation of di- and monomethyl Lys-4 and Lys-9 in histone H3.⁵¹ After that, more histone demethylases were identified that use similar or different reaction mechanisms in demethylation.⁵² Histone acetylation is another major type of histone modification that usually occurs at lysine residues within the N-terminal histone tails, and such acetylations are catalyzed by histone acetylases (HATs) and removed by histone deacetylases (HDACs).⁵³ Some acetylation and methylation can take place on the same amino acid residues of histone protein, and counteract each other. A good example is the acetylation and methylation of the histone H3 tail, in which H3K9 trimethylation (H3K9me3) is a crucial epigenetic mark of heterochromatin and has been associated with transcriptional repression,⁵⁴ whereas H3K9 acetylation is associated with transcriptional activation and marks euchromatin.⁵⁵ These histone and DNA modifications are tightly regulated by numerous epigenetic regulators, which bear mutations in many human diseases associated with abnormal epigenetic patterns.

Epigenetic Dysregulation in Cancer

It has been increasingly recognized that epigenetic abnormalities play essential roles in both the genesis and progression of cancer cells. Among them alterations in the methylation pattern of genomic DNA are common. A global loss of DNA methylation coupled with hypermethylation at specific loci characterizes a significant portion of human cancer. Global DNA hypomethylation could activate genes that are commonly silenced in normal cells, such as viral and parasitic transposons, and affect genome stability. On the other hand, gene-specific DNA hypermethylation is often found in the promoter regions and therefore causes abnormal silencing of tumor suppressor genes such as PTEN, TP53, BRCA1, ATM, and so on.⁵⁶ Maintenance of these examples of abnormal hypermethylation requires the continuous activity of DNMTs. Therefore, cancer cells may be more sensitive to changes in DNMT activity than are normal cells, which represents the key rationale for DNMT inhibition in cancer therapies.⁵⁷ In support of this notion, evidence from a mouse model has shown that haploinsufficiency of Dnmt1 was sufficient to delay progression of leukemogenesis and to impair leukemia stem cell self-renewal, but did not disturb normal hematopoiesis.⁵⁸

Histone modifications are also frequently dysregulated in cancer cells. As mentioned previously, some histone modifications counteract each other. Imbalanced histone modifications lead to dysregulated gene expression and cell growth, and are often found in cancerous cells. In some cases, such as prostate and ovarian tumors, a decreased level of H3K9 (histone H3, Lys-9) acetylation was observed and correlated with severe histological grading and clinical outcomes.^59
–61 However, in other cases, such as hepatocellular carcinoma and gastric adenocarcinoma, an increase in H3K9 acetylation levels and a decrease in H3K9me3 were associated with a poor prognosis.⁶² Consistent with these observations, numerous histone modification enzymes are mutated in cancerous cells and contribute to malignant phenotypes. For example, HDACs, LSD1, and EZH2, a histone methyltransferase responsible for H3K27 methylation, are often overexpressed in many human cancers, including prostate, breast, ovarian, and colon cancer, and lead to epigenetic alterations that promote tumor growth, invasion, and metastasis.^7,9,11,63 The rapidly growing list of mutant epigenetic factors found in cancer includes, but is not limited to, the DNA methyltransferase DNMT3B; DNA demethylation enzyme TET2; histone methylation enzymes MLL3, EZH2, and NSD2; and the isocitrate dehydrogenases IDH1 and IDH2, whose mutant proteins generate 2-hydroxyglutarate (2-HG) that inhibits several epigenetic enzymes including TET2 and JMJD3.^64

–69 Interestingly, mutations in histone proteins themselves can impact their epigenetic modifications. For example, mutations in histone H3 have been found to be recurrently mutated in several cancers.¹² The H3 mutations are usually heterozygous and take place on its K27, K36, and G34 amino acid residues, which were demonstrated to induce abnormal loss or insufficiency of methylations.^14,15 The epigenetic consequences of such genetic changes were further supported by in vitro functional testing and in vivo animal models.^70
–72 This evidence strongly supports the notion that epigenetic mechanisms may act as driving forces during carcinogenesis and support cancer cell survival and proliferation. On the basis of this notion, epigenetic regulatory enzymes are considered important targets for cancer therapy.³ Several drugs targeting DNA methylation and histone acetylation have been approved for cancer treatment in the clinic.³ DNMT inhibitors, such as the cytidine analogs 5-azacytidine and 5-aza-2′-deoxycytidine, can be incorporated into the daughter strands of DNA during DNA replication, which leads to DNA demethylation because they cannot be methylated by the DNMTs.^73,74 HDAC inhibitors directly block the action of HDAC and result in hyperacetylation of histones. Both have been shown to inhibit the growth of a variety of cancers or to restore the sensitivity of cancer cells to chemical drugs and radiation-induced cell cycle arrest and apoptosis.^57,63 At present, a number of additional small molecules with epigenetic modulation activity are undergoing preclinical evaluation and clinical trials, such as G9a, EZH2, and IDH inhibitors.^75

–79 However, most of these molecules showed limited specificity to their target enzymes and target cells, thus causing unpredictable side effects. Therefore, the traditional strategies of epigenetic therapy need to be further improved to target epigenetic programs in a tumor-specific manner.

Targeting Cancer Epigenetic Factors by Cas9 Nuclease

Advances in the development of genome-editing tools have made it more accessible to induce targeted DNA breaks in a sequence-specific and programmable manner. ZFNs, TALENs, and the CRISPR/Cas9 system can all be programmed to generate targeted double-strand breaks (DSBs) in genomic DNA of interest.^20,22,24,80 In most cases, DSBs are repaired by a nonhomologous end-joining (NHEJ) mechanism that can result in random insertions and/or deletions. In the presence of a repair template, DSBs may also be repaired by a homology-directed repair (HDR) mechanism that generates designed modifications, albeit at much lower frequency.⁸¹ ZFNs and TALENs are chimera nucleases that combine programmable and sequence-specific DNA-binding modules with the non-sequence-specific DNA cleavage domain from the Fok1 endonuclease.^20,80 On binding to their targets, ZFNs or TALENs form heterodimers and cause DSBs in regions between their binding sites. In contrast, CRISPR/Cas9 targets and cleaves its target DNA through a programmable short guide RNA binding to its complementary DNA strand and a Cas9 protein, respectively. In addition, Cas9 protein recognizes the protospacer adjacent motif (PAM) of target DNA that is usually a 2- to 5-bp unique sequence adjacent to the RNA-binding site.^22,25 Through the binding of guide RNA and Cas9 protein to the complementary strand and PAM motif, Cas9 cleaves the DNA double strands several base pairs upstream of the PAM motif to generate a DSB. For the most widely used Streptococcus pyogenes Cas9 (SpCas9) system, the cleavage site is about 3 or 4 bp upstream of the NGG PAM.²⁵ Reprogramming the CRISPR/Cas9 system for new targets is unprecedentedly easy, requiring only the presence of PAM in the target DNA and changing the first ∼20 bases in the guide RNA. When a donor template is given, this system can achieve precise genome correction.³¹ And this effect may be further enhanced by simultaneously inhibiting the NEJM pathway. In two independent reports, when the NEJM pathway was inhibited by the ligase IV inhibitor SCR7, the efficiency of template-guided HDR could be enhanced up to 20-fold.^82,83 These advancements have made the CRISPR/Cas9 system promising in a wide range of biomedical research.

As discussed previously, targeting epigenetic regulators with small molecules is effective in cancer therapy but with limitation in specificity. Therefore, it is attractive to design CRISPR/Cas9-based strategies to target these cancerous epigenetic regulators in a more specific manner. For example, DNMT1 and several other factors carrying cancer-related mutations or causing epigenetic silencing are good candidates for testing such strategies. As mentioned previously, because cancerous cells are more sensitive to the level of DNMT1 activity than normal cells, targeting this enzyme should cause limited side effects. Cancer-specific mutations, such as H3K27M and IDH1 R132H, have been demonstrated to be driving forces for the proliferation of malignant cells, and therefore reprogramming Cas9 nuclease to target these specific mutant sequences may achieve mutant allele-specific disruption.

Manipulating the Epigenetic Patterns of Target Loci by Nuclease-Inactivated (Dead) Cas9 Effectors

Besides introducing genetic mutations of epigenetic regulators, direct manipulation of the epigenetic patterns of target genes is also possible through nuclease-inactivated Cas9 (dead Cas9, dCas9)-based methods.^32,34
–36 Like zinc finger effectors and transcription activator–like (TAL) effectors,^84,85 dCas9 can also carry effectors and dock at specific loci to activate or repress gene expression. When fused to transcriptional activation domains such as the VP64 domain and VP64-p65-Rta fusion domain, dCas9 effectors activate gene expression at various levels depending on the strength of activator used and the dCas9-binding position relative to the transcription start site.^86,87 Interestingly, dCas9 can specifically interfere with transcriptional elongation and result in 10- to 300-fold repression of mRNA production when it is directed to the nontemplate strand of gene bodies, which is thought to be due to a “roadblocking” effect in the path of RNA polymerase during transcription elongation.⁸⁸ Likewise, dCas9 can also be redirected to regions near the initial occupation of RNA polymerase and inhibit transcription initiation.⁸⁸ To achieve transcriptional inactivation, dCas9 had also been fused with transcriptional repressor domains, such as the Krüppel-associated box (KRAB) repressor domain, that induce long-range transcriptional repression through the spread of heterochromatin.^89,90 Triggered by the positive results from the fusion of transcriptional regulators with those reprogrammable DNA-binding proteins, a number of epigenetic regulators have also been tested to determine whether they can be redirected to specific genomic regions and induce desired local epigenetic modifications.^91

–94 Histone methyltransferase domains from G9A and Suv39H1 have been fused to zinc finger proteins, which led to local methylation of histone H3K9 and the consequent repression of target gene expression.⁵⁴ The histone demethylase domain from LSD1 fused with TALE efficiently removed H3K4me2 and H3K27ac from the enhancers of target loci and caused downregulation of proximal genes. dCas9 had been fused with the histone acetyltransferase domain from p300 and the demethylase domain from LSD1 to specifically alter local histone modification, leading to robust transcriptional activation or repression of target genes.^91,95 DNA modification enzymes have also been fused with programmable nucleases to alter the methylation state of target loci.^92,96 The methyltransferase domain from DNMT3a fused with zinc finger protein caused site-specific DNA methylation and repression of the target gene,⁹⁶ and the TET1 hydroxylase catalytic domain fused with TALE led to DNA demethylation and transcriptional activation of the target genes.⁹² Unlike reprogrammable transcriptional effectors, whose function requires their presence at the target site, reprogrammable epigenetic factors that were redirected to local genomic regions altered the levels of nearby histone or DNA modifications and might have long-term effect on gene regulation. Especially in the case of DNA methylation, induced site-specific DNA methylation causes stable and heritable repression of target genes.

That dCas9 effectors can achieve either repression or activation of cancer-related genes makes them useful tools in cancer gene therapy by activating tumor suppressor genes or inhibiting tumor-driving genes. In many cancers, some epigenetic factors, such as TET families that function as tumor suppressors, are usually inactivated by epigenetic mechanisms, whereas other epigenetic factors, such as LSD1, EZH2, and NSD2 that function as tumor drivers, are overexpressed by either epigenetic or genetic mechanisms. Therefore, it may be effective to design CRISPR/Cas9-based transcriptional regulators to restore or suppress the expression of these enzymes in such cancers. Further, dCas9 effectors do not result in genetic changes and therefore their effects can be reversible. In theory, such methods can be applied to target the driver genes of cancer and the genes essential for cancer maintenance or drug resistance. Moreover, these methods can also target the epigenetically silenced tumor suppressors, which otherwise are “undruggable.” Thus, dCas9-fused epigenetic regulators can be a powerful tool to reversibly manipulate epigenetic patterns and thus regulate expression without genetic changes of either oncogenes or tumor suppressors for cancer treatment.

Delivery of the Crispr/Cas9 System to Cancer Cells

Theoretically, Cas9 can be delivered as mRNAs, proteins, nonviral DNA, and viruses (Fig. 1). mRNAs are not sufficiently stable for long-term gene therapy purposes. However, for the action of Cas9 nuclease, even transient expression and function can leave the genetic alteration permanent, which is why Cas9 mRNA is widely used in both cell culture and model organisms such as Drosophila, zebrafish, Xenopus, and mouse.^97

–100 Notably, ZFN mRNAs have been approved in clinical trial for the treatment of HIV infection by disrupting the CCR5 gene in isolated human CD4⁺ T lymphocytes (https://clinicaltrials.gov; identifier NCT02388594). It is therefore interesting to test the feasibility of delivering Cas9 mRNA for therapeutic use. Compared with mRNAs, proteins are much more stable and easier to be produced at a large scale. Although the cell-penetrating peptides (CPPs) and Cas9 fusion protein are difficult to express and purify by genetic methods, in vitro CPP-conjugated recombinant Cas9 protein and CPP-complexed guide RNAs have been shown to cause efficient gene disruptions.³⁰ Given that CPPs containing protein drugs are in clinical trials, the CPP-conjugated Cas9 system is worthy of further evaluation as well.¹⁰¹

Figure 1.

Overview of delivery systems for CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein-9 nuclease). The schematic summarizes the delivery of CRISPR/Cas9 through proteins, mRNAs, nonviral plasmid DNA, and various viruses. For viral and plasmid vector delivery, Cas9 and guide RNA (gRNA) have either been combined into a single vector or separated into two vectors. For mRNA delivery, Cas9 mRNA and single guide RNA (sgRNA) are synthesized in vitro and encapsulated in nanoparticles. For protein delivery, Cas9 proteins and sgRNAs are either conjugated with cell-penetrating peptides (CPPs) or encapsulated in nanoparticles. PAM, protospacer adjacent motif; rAAV, recombinant adeno-associated virus.

Nonviral plasmid DNAs are among the most stable formulations for gene transfer and are widely used for ex vivo delivery of Cas9 and guide RNA that are either combined in a single plasmid or separated into two plasmids. For in vivo delivery, only a hydrodynamic gene transfer method was reported, in which a relatively large volume of plasmid DNA solution was quickly injected into the mouse circulation via the tail vein to create hydrodynamic pressure to enable the plasmid DNA to escape the vasculature between the endothelial cells lining the hepatic sinusoids and allow plasmids to enter the cells, mainly hepatocytes.¹⁰² However, the efficiency was rather low. Besides, it is not clear whether this delivery method has specificity for cancerous cells, including primary hepatocellular carcinoma (HCC) or tumor cells metastasized from other organs to liver. Therefore, the development of gene transfer methods targeting cancer, which allow for efficient and specific expression of the CRISPR/Cas9 system in tumor tissues, would provide a major advancement in cancer treatment.

Basically, cancer-specific expression of transgenes can be achieved either by targeting delivery of therapeutic vector to tumor tissue or by controlling transgene expression with a tumor-specific promoter. Cancer-specific promoters, such as the promoter for the telomerase gene, are active specifically in cancerous cells and minimize transgene expression in normal cells.^103,104 Ligand-directed targeting is a more popular choice to delivery transgene specifically to cancerous cells that express corresponding receptors on their surface. By using cancer-specific ligands/receptors, this method can achieve high delivery efficiency and specificity, and thus has great potential in cancer gene therapy.¹⁰⁵ There has been rapid development of biodegradable nanocarriers based on lipid and various polymers for cancer-specific gene delivery.^106

–109 Folate-coated liposome, for example, showed a significantly greater ability to deliver DNA and small molecule drugs to cells expressing folate receptor than uncoated liposome.¹⁰⁹ Folate receptor is highly expressed in a wide range of epithelial malignant cells, such as ovarian, cervical, and lung cancer cells, and absent or weakly expressed in normal cells.¹⁰⁷ This property makes folate-targeted approaches promising in cancer-specific gene transfer. We tested a folate-coated liposome vector carrying the Cas9 plasmid as an approach to treating ovarian cancer. In a mouse ovarian cancer model, intraperitoneal injection of folate-conjugated liposome carrying Cas9 plasmids targeting DNMT1 resulted in 80–90% reduction in tumor volume without any detectable acute toxicity (our unpublished data). As the size of the plasmid is pivotal for efficient gene delivery through endosomal uptake and cytoplasmic transportation toward the nucleus, there is a great need to reduce the length of the Cas9-coding region. Fortunately, a smaller Cas9 ortholog from Staphylococcus aureus (SaCas9) was isolated and shown to edit the genome with efficiencies similar to those of SpCas9.¹¹⁰ On the basis of the fact that truncated SpCas9 protein with Rec2 domain deletion still preserved about 50% cleavage activity, there may still be room to reduce the size of SaCas9 while keeping its activity at an acceptable level.¹¹¹ In addition, minicircle plasmid has shown great benefit in reducing plasmid size and minimizing immunoreactions by removing bacteria-derived DNA sequences,¹¹² which can generate an SaCas9 plasmid less than 4 kb long. Therefore, by combining these techniques, we shall see a significant improvement in the nonviral delivery of the Cas9 system.

Adenoviral, retroviral, lentiviral, and adeno-associated viral (AAV) vectors are the major viral vectors used in gene therapy. Compared with nonviral vectors, viral vectors generally offer higher delivery efficiency and longer term transgene expression. Adenovirus and lentivirus have been used to deliver Cas9 ex vivo and achieved highly efficient gene disruption. However, a concern when using virus, especially adenovirus, is its immunogenicity, which may hamper its use in repeated treatments. AAV is relative small and causes a mild immune response, making it an attractive delivery vector for gene therapy. As mentioned previously, the discovery of SaCas9 of a smaller size makes it possible to use AAVs as Cas9 carriers. In in vivo experiments, AAV-mediated systemic delivery of Cas9 caused greater than 40% gene disruption in mouse liver.¹¹⁰ Given that AAV infection is usually tissue specific, AAV-based Cas9 delivery may be useful in treating malignant tumors from certain tissues.

Perspectives

The rapid progression of genome-editing technology has brought great benefits for both basic science and clinical research. It is hoped that in the near future, with further development of the CRISPR/Cas9 system, this system can be designed to be smaller and to recognize any possible PAM, and thus to target any DNA sequences of interest, and to function in a more specific manner. Although applications of CRISPR/Cas9 in the clinic are just taking place, rapid advances in this field and other additional technologies, such as targeted delivery and nanotechnology, will undoubtedly further expand its therapeutic use in cancer treatment.

Footnotes

Acknowledgments

This work was supported by the Hi-Tech Research & Development (863) Program of China (2015AA020309) and the National Natural Science Foundation of China (NSFC; 81372143).

Author Disclosure

The authors have no conflicts of interest to disclose.

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