v
Search
Advanced Search

Publications > Journals > Journal of Exploratory Research in Pharmacology > Article Full Text

  • Review Article
  • OPEN ACCESS

Frontiers of CRISPR-Cas9 for Cancer Research and Therapy

  • Anamika Banerjee1,
  • Sunil Kumar Malonia2,*  and
  • Shubham Dutta1,* 
 Author information  Cite
Journal of Exploratory Research in Pharmacology   2021;6(3):96-104

doi: 10.14218/JERP.2020.00033

Abstract

In recent years, gene editing technologies have made significant progress in understanding gene function and regulation. The Clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9) system has emerged as a versatile tool for gene editing and genome engineering. In the last few years, CRISPR-Cas9 technology has been widely applied to cancer research, mainly to understand the mechanisms of oncogenesis, drug-target identification, and the development of various cell-based therapies. When combined with genome sequence information, this technology has also shown promise to cure heritable genetic disorders. This review summarizes some of the recent developments and preclinical applications of CRISPR-Cas9 technology in cancer research and therapy. We will discuss how CRISPR based approaches have been used as a tool to identify cancer-specific vulnerabilities and potential applications in cancer therapy.

Keywords

CRISPR, Cas9, Cancer, Gene editing, Non-coding RNA

Introduction

Cancer is one of the leading causes of death worldwide.1 It is a complex disease originating from various genetic alterations/epigenetic aberrations. Due to its mechanistic complexity, current cancer-specific therapies have certain limitations, emphasizing the need for alternative therapeutic approaches. The Cancer Genome Atlas2–7 has described over 15,000 tumors that have generated multi-dimensional data that illuminate the complexities of oncogenesis. Researchers have made a significant effort to comprehend the underlying biology of tumorigenesis, which has helped in the development of various chemotherapeutic agents, small molecule drugs, and antibodies. However, different cancer patients have vastly different genetic alterations/aberrations that can drastically alter tumor progression and susceptibility to therapy. As a result, there have been a significant thrusts and emphasis on identifying various genetic/epigenetic mutations, comprehending the genomic landscape, and developing more productive and safe therapeutic approaches for cancer.8,9 RNA interference (RNAi) and random mutagenesis were initially used to discover genetic alterations.10,11 Subsequently, the discovery of programmable nucleases such as zinc-finger nucleases (ZFNs) and Transcription activator-like effector nucleases (TALENs) provided an immense opportunity to study the function of genes in cancer. These genome editing technologies have enabled researchers to target the genome at specific sites to generate gene-knockouts.12,13 Both TALENs and ZFNs are engineered nucleases and are created by fusing DNA binding domains with a DNA cleavage domain. They can target and cleave specific DNA sequences, which are then repaired by the endogenous DNA repair machinery, thereby precisely altering DNA sequences in the complex genome.12,13 However, TALENs and ZFNs are considered to have low editing efficiency and are expensive tools. The recent identification of RNA-guided programmable nucleases of the bacterial adaptive immune system has shown tremendous success and proven to be a highly efficient tool for gene editing/genome engineering.13–17

The Clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9) system of gene editing has gained significant attention in recent years for its precision and effectiveness in genome editing in several model systems and human cells. Not only that, CRISPR-Cas9 is also used to introduce transcriptional and epigenetic modifications in different mammalian systems. Direct somatic editing using CRISPR-Cas9 has transformed the druggable space by helping researchers to correct any altered genomic aberrations or modify regulatory elements and splicing patterns.18,19

The technological demonstration of CRISPR-Cas9 and the molecular basis of editing has been covered elsewhere.20–22 In short, a single guide RNA (sgRNA) directs Cas9 endonuclease to introduce a double-stranded break at a specific site in the genome.23 The endogenous DNA repair mechanisms later repair the genome using two different mechanisms: namely, the predominant error-prone non-homologous end joining24–26 and less frequent homology-directed repair (HDR),27–31 which requires a donor template. Non-homologous end joining has been used to disrupt the genome sequences to create deletion and insertions, while HDR has been used to alter specific regions of the genome using exogenous repair templates. In addition to this, a modified version of Cas9 protein, catalytically dead Cas9 (nuclease-dead Cas9), has been utilized to either activate or repress the target genes; these mechanisms are known as CRISPR activation and CRISPR interference respectively (Fig. 1).32–34

Schematic representation of CRISPR-Cas9-mediated genome editing.
Fig. 1  Schematic representation of CRISPR-Cas9-mediated genome editing.

(a) The Cas9 nuclease is directed to the target DNA by complementary base pairing with its bound guide RNA in which the target site is followed by a PAM sequence (NAG, NAG). Cleavage of the dsDNA promotes either error-prone NHEJ or HDR. (b) Two guide RNAs (targeting two different sites, namely target A and target B) can be used simultaneously to remove a longer stretch of DNA and a new DNA can be inserted using a donor DNA using HDR. (c) Catalytically dead Cas9 (dCas9) can be tagged with transcription activators (or repressors) and fluorescent proteins (such as GFP) to regulate gene expression or visualization. CRISPR, clustered regularly interspaced short palindromic repeats; Cas9, CRISPR associated protein 9; dCas9, dead CRISPR associated protein 9; gRNA, guide RNA; mRNA, messenger RNA; dsDNA, double stranded deoxyribonucleic acid; PAM, protospacer adjacent motif; NHEJ, non-homologous end joining; HDR, homology directed repair; GFP, green fluorescent protein.

In this review, we will discuss how CRISPR-Cas9 is revolutionizing cancer research and therapy. The role of CRISPR-Cas9 system in understanding cancer genomics, exploring non-coding regions, in vivo gene editing, and the generation of novel organoid models will also be discussed. The recent advances in the delivery of CRISPR-Cas9 for in vivo gene editing will be reviewed Lastly, we will summarize the preclinical and clinical applications of CRISPR-Cas9 technology in cancer.

CRISPR-Cas9 as a tool for target discovery in cancer

CRISPR-Cas9 is a powerful tool to identify novel targets in cancer. Due to precise gene editing capabilities, CRISPR has become the tool of choice to study the function and regulation of specific genes. It has been used in high-throughput screening approaches, where CRISPR-Cas9 is used to generate a large number of genetic knockouts using which one can monitor whether there is a phenotypic effect due to knockout of a particular gene in a cell population. In cancer, this strategy has been used to perform high-throughput screens and look at gene-drug interactions when used in conjunction with small molecules.35–38 The recent advancement in next-generation DNA sequencing and genome-wide association studies has provided a more comprehensive understanding of variations in the human genome. This has unraveled thousands of single nucleotide polymorphisms and mutations attributed to various diseases and their predisposition. Several genomic repositories (such as The Cancer Genome Atlas, the Cancer Cell Line Encyclopedia or the Encyclopedia of DNA Elements39,40) have been developed, containing a catalog of disease-specific variation at the genome level. This has also encouraged personalized medicine by integrating available patient data and genetic information. However, the hypothesis generated by these high-throughput genomics data requires thorough testing and validation using accurate genetic models to develop new treatment standards. It is often seen that mutations in genes with known functions do not always directly correlate with the disease phenotype. To understand such relationships, researchers often need a large number of matched patient and regular tissue samples, which can be difficult to obtain due to ethical concerns. The advent of CRISPR technology has helped researchers to generate isogenic human knockout/genetically modified cells to address this problem.41 Likewise, CRISPR technology has been used in many cell types, particularly cancer cell lines, primary cells, organoids, and induced pluripotent stem cells.41–44 One example where CRISPR is used in cancer biology is if a tumor-derived cell line with a genetic lesion is thought to be sensitized to a particular therapeutic drug, one can use CRISPR to test the hypothesis of synthetic lethality in the appropriate cell line.45–47

Although this technique shows a lot of promise, the rates of CRISPR-mediated cell modification vary from cell type to cell type, especially HDR. Primary cells such as neurons and other non-mitotic cells are challenging to modify especially using HDR. The development of microhomology-mediated integration has shown promise to bypass HDR-mediated cell line modification.48–50 Furthermore, the discovery of newly engineered Cas enzymes has also enabled researchers to modify primary cells, alter the genome, and change specific bases. For example, the fusion of cytidine deaminases to Cas9 has enabled researchers to modify RNA-guided base editing,51,52 which has shown promise in editing a wide variety of cell types.

CRISPR-Cas9 as a tool to investigate non-coding regulatory regions in cancer

The majority of the human genome is comprised of non-protein coding regions that consist of various regulatory elements such as enhancers, silencers, and insulators. Due to the dearth of robust molecular biology tools, these regions have not been fully characterized. The importance of these non-coding regions is highlighted by the fact that dysregulation of these elements could contribute to oncogenesis.53 Therefore, a comprehensive understanding of these regulatory elements may help to understand the genomic landscape of cancer cells. Several studies have utilized CRISPR-Cas9 technology to interrogate the non-coding regions of the genome, in particular various cis-regulatory elements such as enhancers, super-enhancers, and trans-acting factors.

In one of the landmark studies, researchers utilized pooled CRISPR-Cas9 libraries to identify functional non-coding regions around CUL3, NF1, and NF2 genes, which have been previously shown to be responsible for promoting Vemurafenib resistance to melanoma cells harboring the BRAF V600E mutation.54,55 Notably, the authors found significantly more regulatory regions around the CUL3 gene compared to the NF1 and NF2. Furthermore, the authors used chromosome conformation capture studies to show a direct interaction between these non-coding regions to the 5′ end of the CUL3 gene.54

In another interesting study, researchers have performed CRISPR-Cas9 screens to identify several functional elements that regulate two transcription factors TP53 and ESR1, which play essential roles in tumor initiation and progression.56,57 These pooled CRISPR-Cas9 screens identified six different enhancer elements, one of which promoted the activation of the CDKN1A gene during a cell cycle arrest program called oncogene-induced senescence, and the other one mediated the expression of the CCND1 gene in response to ERS1 signaling. The identification of these regulatory elements and their associated target genes could lead to tumor diagnostics and therapeutics.56

The advent of CRISPR-Cas9 high-throughput screens has also provided researchers a versatile tool to interrogate the non-coding genome at high resolution, particularly the characterization of several non-coding RNAs in cancer and other diseases. Studies have utilized genome-wide deletion screens of long-noncoding RNA (lncRNAs) using paired sgRNAs to show that knockout of specific lncRNAs significantly altered cell proliferation eluding to their role in oncogenesis.58 Other studies have used CRISPR interference screens on various cell lines to study the effects of lncRNAs on cell viability.59 One of these studies identified 499 lncRNAs essential for cell proliferation. CRISPR-Cas9 has also been used to visualize lncRNAs in scenarios where the conventional fluorescence in-situ hybridization approaches have failed. One study used catalytically dead Cas9-GFP-sgRNA fusion probes to visualize X-inactive specific transcript lncRNA which has a complex secondary structure and was highly condensed for the access of fluorescence in-situ hybridization probes. The authors used this modified Cas9 to efficiently visualize the X-inactive specific transcript lncRNA along with repressive H3K27me3 histone marks in female fibroblasts.60

Although the role of CRISPR-Cas9-mediated identification of lncRNAs shows promise, there is one caveat; since various lncRNAs have bidirectional promoters and are located near/within protein-coding genes, targeting them with CRISPR-Cas9 affects the expression of these genes.61 Therefore, more complex CRISPR-Cas9 approaches need to be developed to interrogate the molecular function of lncRNAs.

CRISPR-Cas9 as a tool to generate organoid models of cancer

In recent years, organoids have become popular in vitro models over two-dimensional cell cultures to study human diseases. Adult human stem cells are isolated and stimulated with specific growth factors to help them proliferate and subsequently differentiate into three-dimensional organoids.62 Traditionally, small molecule therapeutic assays were performed on two-dimensional cell cultures, but this did not mirror normal physiological conditions where cells are in a three-dimensional microenvironment. These organoids mimicked cell-matrix and cell-cell environments compared to cells in two-dimensional cultures which helped researchers to develop physiologically relevant in vitro systems for their studies.

The advent of CRISPR-Cas9 has aided researchers to generate tumorigenic organoid models to interrogate putative mechanisms of oncogenesis. The first proof of concept study for genome editing using CRISPR-Cas9 was carried out in intestinal stem cell organoids where researchers repaired the CFTR locus from a Cystic Fibrosis patient.63 A few years later, healthy human intestinal stem cell-derived organoids were transformed into their tumorigenic counterparts by introducing several tumor-inducing mutations using CRISPR-Cas9. The activation of oncogenes (such as PI3K and KRAS, etc.) and the disruption of tumor-suppressors (such as TP53, APC, SMAD4, etc.) using CRISPR-Cas9-mediated gene editing helped establish organoid models of cancer in vitro.42,44 Another group used CRISPR-Cas9 to delete an essential DNA repair gene called MLH1 from healthy human stem cell organoids to accurately model oncogenesis induced by replication errors and mismatch repair.64 Thus the use of CRISPR-Cas9 in generating various stem-cell derived organoid models can be used to mimic patient-derived samples for functional assays of small-molecule and other high-throughput screens.65

CRISPR-Cas9 as a tool to overcome drug resistance in cancer

With the development of CRISPR-Cas9, researchers can modify the genome to understand the role of specific genes and molecular mechanisms underlying drug resistance in different types of cancer such as breast cancer, lung cancer, liver cancers, and gliomas.

Using a genome-wide CRISPR-Cas9 approach, researchers showed that deletion of transcriptional factors such as MED1, CREBBP, and EP300 increased the efficacy and synergy between two drugs, namely erlotinib (an epidermal growth factor receptor (EGFR) inhibitor) and TZH1 (a CDK7/12 inhibitor).66 These drugs are used in the treatment of EGFR-dependent lung adenocarcinoma. Another study used a combination of CRISPR, short-hairpin RNA (shRNA), and expression screen in non-small cell lung cancer cells to identify a protein called PBRM1 (a subunit of SWI/SNF complex) which attenuated the gefitinib mediated EGFR inhibition for prolonged cell survival.67

A genome-wide association study in women treated with selective estrogen modulators for breast cancer prevention identified a single nucleotide polymorphism (rs9940645) in a gene called ZNF423. Using CRISPR-Cas9 editing, the authors modified ZR75-1 cells to harbor the same variant. Studies on these cell lines showed that this variant containing the cell line was more sensitive to raloxifene, olaparib, and cisplatin.68 Another genome-wide CRISPR screen revealed the role of BAK in breast cancer. In this study, when breast cancer cells with high expression of MCL-1 were treated with S63845 (an MCL-1 inhibitor), a synergistic relationship with other drugs such as docetaxel, trastuzumab, and lapatinib which are used in the treatment of triple-negative breast cancer and HER2-amplified breast cancer was revealed. The CRISPR screen revealed that the deletion of BAK or double deletion of BAK and BOX showed resistance to S63845.69

Other genome-wide CRISPR-Cas9 screens have identified factors involved in drug resistance in liver cancers and glioblastoma. One screen identified a protein called SCOL1 (a protein involved in mitosis), the loss of which led to sorafenib resistance in hepatocellular carcinoma. Furthermore, it was shown that loss of SGOL1 from hepatocarcinoma cell lines decreased the cytotoxicity of sorafenib.70 CRISPR-Cas9-mediated knockout of a glioblastoma-associated oncogene called GLI1 showed increased apoptosis when combined with an antipsychotic drug Penfluridol.71 CRISPR-Cas9-mediated depletion of a centriole satellite protein PCM1 revealed that its removal inhibited glioblastoma cell proliferation and had increased sensitivity to temozolomide in patient-derived glioblastoma.72 Saber et al.73 and Liu et al.74 have reviewed these studies.

Delivery of CRISPR-Cas9 for in vivo editing

One of the significant challenges in CRISPR-mediated gene therapy is how editing components can be delivered into specific tissues in humans without leading to off-targets and activating the immune system. Broadly, there are two ways to deliver CRISPR-Cas9 for in vivo gene editing, namely the non-viral method and viral based method using adeno-associated virus (AAV).

Non-viral delivery of CRISPR-Cas9

Several non-viral methods of CRISPR-Cas9 delivery have been developed. One of the simplest delivery methods utilizes cell-penetrating peptides conjugated to sgRNA and Cas9 protein. It has been shown to edit HEK293T cells with an efficiency of 6.2%75 and can be a potential delivery approach for in vivo applications in future studies. Another delivery method is Lipid nanoparticles (LNPs) based. LNPs are produced using a combination of ionizable lipids, cholesterol, and PEGylated lipids. These LNPs are then used to deliver sgRNA and Cas9 to the mouse liver to target the transthyretin gene. This approach showed a more than 97% reduction in serum transthyretin levels.76 Polymer-based synthetic nanomaterials are also being developed for CRISPR-Cas9 delivery. Cationic polymers such as polyethyleneimine are conjugated with CRISPR-Cas9 vectors and injected directly into tumors in EGFP expressing mice and a significant drop in EGFP has been shown, exhibiting high knockout efficiency.77 Additionally, colloidal gold nanoparticles are also being used to successfully deliver gRNA-Cas9 ribonucleoproteins along with HDR template in mice to treat Duchenne muscular dystrophy with minimal off-target effects.78 Although all these approaches have shown promise, further research is required to improve the scalability and efficacy of these methods in preclinical and clinical studies.

AAV based delivery of CRISPR-Cas9

In the last decade, a lot of research has been undertaken to study and characterize AAVs. These studies have led to the discovery of multiple serotypes of AAVs with distinct tropisms to selectively target different tissues in humans for gene therapy.79 Therefore, AAVs have become a potent delivery tool for CRISPR-Cas9 to specific tissues for gene modification.80 For example, CRISPR-Cas9 harboring AAVs have been subretinally injected to knockdown the NRL gene in post-mitotic retinal photoreceptors to prevent retinal degeneration in mice81 and to the striatum to edit the huntingtin gene to alleviate Huntington’s disease.82 For systemic delivery, two crucial things need to be considered: (a) the serotype of the AAV and (b) tissue-specific promoters under which the Cas9 is cloned. Once these conditions are determined, the engineered virus can be used to deliver Cas9 and sgRNA for expression in specific tissues. This dual AAV system has been used to edit the ornithine transcarbamylase gene in mice liver83 and the dystrophin gene in the mouse model for Duchene muscular dystrophy and muscle stem cells in mice.84,85 Although the editing efficiency was between 10–70%, these experiments had significant phenotypic improvements in mice. Recent studies have also shown that although AAVs and Cas9 induce an immune response in mice, they do not inflict extensive cellular damage,86 making them potentially safer delivery methods over other delivery systems.

High titer AAV libraries have been used to target mouse tissues in vivo to generate tumors with several mutations in glioblastoma and liver tissue.87,88 This approach can help researchers robustly study the oncogenic role of individual mutations, helping develop patient-specific treatments with genetically matched tumors. This technique may also help in the development of tumor heterogeneity models, which has been recognized as an important contributor to drug resistance and cancer relapse.89,90 Therefore, this technique could be used to model, predict and investigate tumor clonal dynamics and eventually serve as a platform for novel cancer therapies.

One limitation of using AAV as a delivery tool is that AAVs have a limited cargo size, as two separate vectors are used for delivery, both directly to the target organ and systemically.91

Researchers are also using LNPs, cell penetrating peptides, and polymers-based synthetic nanomaterials to deliver CRISPR-Cas9 as well (discussed above). Recent studies have used lipid nanoparticles to efficiently deliver CRISPR-Cas9 into mouse liver to knockout specific genes76,92,93 and can deliver donor templates for HDR-mediated gene editing.78 There are two significant advantages of using lipid nanoparticles as a method of delivery: (a) they were previously used to deliver siRNA and mRNA in clinical trials94,95 and (b) they can be industrially manufactured.94 Additional research is required to improve the efficiency of ex vivo and in vivo CRISPR-Cas9 editing, and new screens are being performed to gain new insights on cancer-associated genes.

Clinical trials using CRISPR-Cas9

The idea of somatic gene therapy has been proposed long before the advent of gene editing technology was discovered. Somatic gene therapy is described as introducing genetic material into somatic cells, which will then express that novel gene product. These cells can then be put back into patients for therapeutic purposes. Before the introduction of modern gene therapy techniques, these early trials have been mostly unsuccessful due to problems associated with the host immune response, gene silencing, and random mutagenesis.96 Modern techniques have allowed researchers to overcome these problems along with reliable and permanent modification of patient somatic cells.

The first clinical trials were performed using ZFNs to target the human immunodeficiency virus (HIV) receptor called CCR5.97 ZFNs were used to modify HIV-infected patient’s T-cells ex vivo, which were then introduced back into the patients to promote resistance to HIV infection. This resulted in a significant reduction of viral particles compared to the control and was well tolerated by the patients. Although this treatment did not have a lasting effect,98 it ushered in an era for several other gene therapy clinical trials.

The first CRISPR-Cas9 trial was performed on non-small cell lung cancer patient T-cells, ex vivo modified by CRISPR-Cas9 to knockout programmed cell-death protein (PD-1) (NCT02793856).99 PD-1 ligand (PD-L1) is highly expressed on various cancer cell types, and it binds PD-1 receptors on the activated T cells, which leads to the inhibition of the cytotoxic T cells. The PD-1/PD-L1 pathway thus represents an immune checkpoint mechanism exerted by tumor cells in response to endogenous immune anti-tumor activity.100 Neutralizing antibodies of PD-1 (or the PD-L1) have been recently used to treat different types of cancer.101 In this gene therapy approach, patient peripheral blood was collected and CRISPR-Cas9 was used to knock-out PD-1 ex vivo. After rigorous testing, the PD-1 knockout cells were introduced back into the patients (Fig. 2). There are speculations as to whether the PD-1 modified T-cells are better compared to therapeutic antibodies against PD-L1 or PD-1 since the production of engineered T-cells is a costly and laborious process.99

CRISPR-Cas9 is used to knockout PD-1 in T cells from patients.
Fig. 2  CRISPR-Cas9 is used to knockout PD-1 in T cells from patients.

T-cells are isolated from the blood of a cancer patient. CRISPR-Cas9 ribonuclear proteins are electroporated into the T-cells and the TRAC, TRBC1, TRBC2, and PDCD1 loci are edited. The cells are then transduced with a lentiviral vector to express TCR-specific cancer testis antigens NY-ESO-1 and LAGE-1. These cells are then infused back into the patients and are safely monitored to check the safety and efficacy of treatment. T cell, thymus lymphocyte cell; PD-1, programmed cell death protein 1; TCR, T cells receptor; CRISPR, clustered regularly interspaced short palindromic repeats; Cas9, CRISPR associated protein 9; PAM, protospacer adjacent motif; NY-ESO-1, New York esophageal squamous cell carcinoma-1; TRAC, T cell receptor alpha constant; TRBC, T cell receptor beta constant; PDCD1, programmed cell death protein 1.

Another clinical trial in its Phase I/II has the PD-1 gene knocked out in an Epstein-Barr virus specific autologous T-cell for cancers positive for Epstein-Barr virus (NCT03044743). Researchers are now generating chimeric antigen receptor (CAR)-T cells with CRISPR-Cas9.102,103 Another preclinical study was conducted where CAR was delivered to the T-cell receptor α-chain loci using CRISPR-Cas9 that enhanced its tumor rejection capability compared to conventionally produced CAR-T cells in a mouse model.104 Multiple other clinical trials have also been registered in patients with other types of cancer such as renal, esophageal, and bladder cancer, some of which are phase II clinical trials (clinicaltrials.gov) (Table 1).

Table 1

A table summarizing the different clinical trials using CRISPR-Cas9 in progress

Clinical trial identifierCancer typePhaseTreatment strategy
NCT02867345Hormone refractory prostate cancerIPD-1 knockout T cells
NCT02867332Renal cell carcinomaIPD-1 knockout T cells
NCT03081715Esophageal cancerIIPD-1 knockout T cells
NCT02863913Bladder cancerIPD-1 knockout T cells
NCT02793856Non-small cell lung cancerIPD-1 knockout T cells
NCT03044743EBV positive advanced stage malignanciesI/IIPD-1 knockout EBV-CTL
NCT03166878B cell lymphoma / leukemiaI/IICRISPR-Cas9 edited CAR-T Cells Targeting CD19 and CD20 or CD22
NCT03057912HPV related cervical intraepithelial neoplasiaICRISPR-Cas9-sg HPV E6/E7 gel to disrupt HPV DNA
NCT03398967B cell lymphoma / leukemiaI/IICRISPR-Cas9 edited CAR-T cells targeting CD19
NCT03057912HPV related cervical intraepithelial neoplasiaICRISPR-Cas9-sg HPV E6/E7 gel to disrupt HPV DNA

NCT, national clinical trial; EBV, Epstein–Barr virus; HPV, human papillomavirus; PD-1, programmed cell death protein 1; EBV-CTL, Epstein-Barr virus (EBV) specific cytotoxic T cells; CAR-T, chimeric antigen receptor T cells; CD, cluster of differentiation; CRISPR, clustered regularly interspaced short palindromic repeats; Cas9, CRISPR associated protein 9.

Although a fair number of studies are ongoing with clinical trials using ex vivo genome editing, only two are being implemented for in vivo clinical trials. One trial (NCT03057912), which is still in the process of recruiting patients, involves the delivery of constructs targeting HPV16 and HPV18 using either TALENs or CRISPR-Cas9 using a gel that will be locally applied to the patient’s infected cervix. Further research is needed to improve the specificity and delivery of CRISPR-Cas9 to target specific tissues, and this will promote the development of CRISPR based clinical trials in the future.

Future perspectives

CRISPR/Cas9 technology has evolved as a versatile tool to manipulate and edit the genome and epigenome across a broad range of cell types and organisms. The applications of CRISPR/Cas9 in both basic and translational cancer research are beginning to unfold. In the future, applications of various CRISPR-based genome-wide screens may help to unravel novel genes, new biomarkers, and regulatory pathways(s) that can be therapeutically targeted in cancer. Integrating the information from these screens with available data on the genetic and epigenetic landscape of various cancer types may help identify synthetic lethal interactions and discover novel drug targets. However, this will require considerable research to thoroughly dissect the biological mechanisms underlying a multitude of genetic/epigenetic interactions revealed by CRISPR screens.

Furthermore, the ability of CRISPR/Cas9 to manipulate non-coding regions of the genome will augment the functional exploration of these less characterized features of the cancer genome. The precise engineering of pathogenic and driver mutations will enable researchers to gain deeper insights into the biological changes elicited by these mutations in cancer. Although there has been an increasing interest of the scientific community to examine and broaden the translational potential of CRISPR/Cas9 tools, translating these into the clinic successfully remains a challenge. Some of the major concerns are safety, including uncontrolled off-target effect, the immunogenicity of Cas nucleases, and the carcinogenic effect of CRISPR components, adverse immunological effects, and so forth. Many challenges associated with CRISPR technology still exist, particularly in clinical use. The future use of CRISPR/Cas9 technology will rely on its precision and efficacy and efforts to develop novel Cas9 variants with minimal off-target effects and enable precise editing of the genome. Furthermore, new advancements in the methods to deliver CRISPR components, improvements in viral and non-viral delivery methods will accelerate the in vivo application of the CRISPR-Cas9 system.

Conclusions

The CRISPR-Cas9 technology has evolved rapidly and has revolutionized the scientific field from basic research to clinical applications. Undoubtedly, this technology has provided many promising avenues for a greater understanding of cancer and treatments. Innovations in the CRISPR technology are likely to increase its precision, effectiveness, and safety for its future preclinical and clinical applications in cancer. In the next decade or so these technologies will play a pivotal role in identifying new drug targets, and developing dependable therapeutic strategies for several diseases, including cancer.

Abbreviations

ZFN: 

zinc finger nucleases

TALENs: 

Transcription activator-like effector nucleases

CRISPR-Cas9: 

clustered regularly interspaced short palindromic repeats CRISPR associated protein 9

sgRNA: 

single guide RNA

HDR: 

homology directed repair

lncRNA: 

long non-coding RNA

AAV: 

adeno associated virus

LNP: 

lipid nanoparticles

HIV: 

human immunodeficiency virus

CAR: 

chimeric antigen receptor

Declarations

Acknowledgement

None.

Funding

This research received no external funding.

Conflict of interest

The authors declare no conflict of interest.

Authors’ contributions

Conceptualization (AB, SD and SKM), original draft preparation (AB and SD), review and editing (AB, SD and SKM), visualization (AB and SD), supervision (SD and SKM). All authors have read and agreed to the published version of the manuscript.

References

  1. Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global cancer statistics, 2012. CA Cancer J Clin 2015;65(2):87-108 View Article
  2. Barretina J, Caponigro G, Stransky N, Venkatesan K, Margolin AA, Kim S, et al. The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature 2012;483(7391):603-607 View Article
  3. Bell D, Berchuck A, Birrer M, Chien J, Cramer DW, Dao F, et al. Integrated genomic analyses of ovarian carcinoma. Nature 2011;474(7353):609-615 View Article
  4. Hammerman PS, Lawrence MS, Voet D, Jing R, Cibulskis K, Sivachenko A, et al. Comprehensive genomic characterization of squamous cell lung cancers. Nature 2012;489(7417):519-525 View Article
  5. Koboldt DC, Fulton RS, McLellan MD, Schmidt H, Kalicki-Veizer J, McMichael JF, et al. Comprehensive molecular portraits of human breast tumours. Nature 2012;490(7418):61-70 View Article
  6. McLendon R, Friedman A, Bigner D, Van Meir EG, Brat DJ, Mastrogianakis GM, et al. Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature 2008;455(7216):1061-1068 View Article
  7. Muzny DM, Bainbridge MN, Chang K, Dinh HH, Drummond JA, Fowler G, et al. Comprehensive molecular characterization of human colon and rectal cancer. Nature 2012;487(7407):330-337 View Article
  8. Hudson TJ, Anderson W, Aretz A, Barker AD, Bell C, Bernabé RR, et al. International network of cancer genome projects. Nature 2010;464(7291):993-998 View Article
  9. Weinstein JN, Collisson EA, Mills GB, Shaw KRM, Ozenberger BA, Ellrott K, et al. The cancer genome atlas pan-cancer analysis project. Nat Genet 2013;45(10):1113-1120 View Article
  10. Agrawal N, Dasaradhi PV, Mohmmed A, Malhotra P, Bhatnagar RK, Mukherjee SK. RNA interference: biology, mechanism, and applications. Microbiol Mol Biol Rev 2003;67(4):657-685 View Article
  11. Nguyen N, Judd LM, Kalantzis A, Whittle B, Giraud AS, van Driel IR. Random mutagenesis of the mouse genome: a strategy for discovering gene function and the molecular basis of disease. Am J Physiol Gastrointest Liver Physiol 2011;300(1):G1-11 View Article
  12. Joung JK, Sander JD. TALENs: A widely applicable technology for targeted genome editing. Nat Rev Mol Cell Biol 2013;14(1):49-55 View Article
  13. Urnov FD, Rebar EJ, Holmes MC, Zhang HS, Gregory PD. Genome editing with engineered zinc finger nucleases. Nat Rev Genet 2010;11(9):636-646 View Article
  14. Cho SW, Kim S, Kim JM, Kim JS. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol 2013;31(3):230-232 View Article
  15. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome engineering using CRISPR/Cas systems. Science 2013;339(6121):819-823 View Article
  16. Jinek M, East A, Cheng A, Lin S, Ma E, Doudna J. RNA-programmed genome editing in human cells. ELife 2013;2:e00471 View Article
  17. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, et al. RNA-guided human genome engineering via Cas9. Science 2013;339(6121):823-826 View Article
  18. Cox DBT, Platt RJ, Zhang F. Therapeutic genome editing: Prospects and challenges. Nat Med 2015;21(2):121-131 View Article
  19. Hopkins AL, Groom CR. The druggable genome. Nat Rev Drug Discov 2002;1(9):727-730 View Article
  20. Doudna JA, Charpentier E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 2014;346(6213):1258096 View Article
  21. Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell 2014;157(6):1262-1278 View Article
  22. Wright AV, Nuñez JK, Doudna JA. Biology and Applications of CRISPR Systems: Harnessing Nature’s Toolbox for Genome Engineering. Cell 2016;164(1-2):29-44 View Article
  23. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 2012;337(6096):816-821 View Article
  24. Guirouilh-Barbat J, Huck S, Bertrand P, Pirzio L, Desmaze C, Sabatier L, et al. Impact of the KU80 pathway on NHEJ-induced genome rearrangements in mammalian cells. Mol Cell 2004;14(5):611-623 View Article
  25. Moore JK, Haber JE. Cell cycle and genetic requirements of two pathways of nonhomologous end-joining repair of double-strand breaks in Saccharomyces cerevisiae. Mol Cell Biol 1996;16(5):2164-2173 View Article
  26. Roth DB, Wilson JH. Nonhomologous recombination in mammalian cells: role for short sequence homologies in the joining reaction. Mol Cell Biol 1986;6(12):4295-4304 View Article
  27. Jasin M, de Villiers J, Weber F, Schaffner W. High frequency of homologous recombination in mammalian cells between endogenous and introduced SV40 genomes. Cell 1985;43(3 Pt 2):695-703 View Article
  28. Lin FL, Sperle K, Sternberg N. Model for homologous recombination during transfer of DNA into mouse L cells: role for DNA ends in the recombination process. Mol Cell Biol 1984;4(6):1020-1034 View Article
  29. Orr-Weaver TL, Szostak JW, Rothstein RJ. Yeast transformation: A model system for the study of recombination. PNAS 1981;78(10):6354-6358 View Article
  30. Resnick MA. The repair of double-strand breaks in DNA: A model involving recombination. J Theor Biol 1976;59(1):97-106 View Article
  31. Szostak JW, Orr-Weaver TL, Rothstein RJ, Stahl FW. The double-strand-break repair model for recombination. Cell 1983;33(1):25-35 View Article
  32. Gilbert LA, Horlbeck MA, Adamson B, Villalta JE, Chen Y, Whitehead EH, et al. Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation. Cell 2014;159(3):647-661 View Article
  33. Gilbert LA, Larson MH, Morsut L, Liu Z, Brar GA, Torres SE, et al. XCRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 2013;154(2):442-451 View Article
  34. Konermann S, Brigham MD, Trevino AE, Joung J, Abudayyeh OO, Barcena C, et al. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 2015;517(7536):583-588 View Article
  35. Zeng H, Castillo-Cabrera J, Manser M, Lu B, Yang Z, Strande V, et al. Genome-wide CRISPR screening reveals genetic modifiers of mutant EGFR dependence in human NSCLC. Elife 2019;8:e50223 View Article
  36. Xu S, Zhan M, Jiang C, He M, Yang L, Shen H, et al. Genome-wide CRISPR screen identifies ELP5 as a determinant of gemcitabine sensitivity in gallbladder cancer. Nat Commun 2019;10(1):5492 View Article
  37. Lord CJ, Quinn N, Ryan CJ. Integrative analysis of large-scale loss-of-function screens identifies robust cancer-associated genetic interactions. Elife 2020;9:e58925 View Article
  38. Makhov P, Sohn JA, Serebriiskii IG, Fazliyeva R, Khazak V, Boumber Y, et al. CRISPR/Cas9 genome-wide loss-of-function screening identifies druggable cellular factors involved in sunitinib resistance in renal cell carcinoma. Br J Cancer 2020;123(12):1749-1756 View Article
  39. ENCODE Project Consortium. The ENCODE (ENCyclopedia Of DNA Elements) Project. Science 2004;306(5696):636-640 View Article
  40. ENCODE Project Consortium. An integrated encyclopedia of DNA elements in the human genome. Nature 2012;489(7414):57-74 View Article
  41. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc 2013;8(11):2281-2308 View Article
  42. Drost J, van Jaarsveld RH, Ponsioen B, Zimberlin C, van Boxtel R, Buijs A, et al. Sequential cancer mutations in cultured human intestinal stem cells. Nature 2015;521(7550):43-47 View Article
  43. Grobarczyk B, Franco B, Hanon K, Malgrange B. Generation of Isogenic Human iPS Cell Line Precisely Corrected by Genome Editing Using the CRISPR/Cas9 System. Stem Cell Rev Rep 2015;11(5):774-787 View Article
  44. Matano M, Date S, Shimokawa M, Takano A, Fujii M, Ohta Y, et al. Modeling colorectal cancer using CRISPR-Cas9-mediated engineering of human intestinal organoids. Nat Med 2015;21(3):256-262 View Article
  45. Kasap C, Elemento O, Kapoor TM. DrugTargetSeqR: a genomics- and CRISPR-Cas9-based method to analyze drug targets. Nat Chem Biol 2014;10(8):626-628 View Article
  46. Shi J, Wang E, Milazzo JP, Wang Z, Kinney JB, Vakoc CR. Discovery of cancer drug targets by CRISPR-Cas9 screening of protein domains. Nat Biotechnol 2015;33(6):661-667 View Article
  47. Smurnyy Y, Cai M, Wu H, McWhinnie E, Tallarico JA, Yang Y, et al. DNA sequencing and CRISPR-Cas9 gene editing for target validation in mammalian cells. Nat Chem Biol 2014;10(8):623-625 View Article
  48. Nakade S, Tsubota T, Sakane Y, Kume S, Sakamoto N, Obara M, et al. Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9. Nat Commun 2014;5:5560 View Article
  49. Paix A, Wang Y, Smith HE, Lee CY, Calidas D, Lu T, et al. Scalable and versatile genome editing using linear DNAs with microhomology to Cas9 Sites in Caenorhabditis elegans. Genetics 2014;198(4):1347-1356 View Article
  50. Sakuma T, Nakade S, Sakane Y, Suzuki KT, Yamamoto T. MMEJ-assisted gene knock-in using TALENs and CRISPR-Cas9 with the PITCh systems. Nat Protoc 2016;11(1):118-133 View Article
  51. Komor AC, Kim YB, Packer MS, Zuris JA, Liu DR. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 2016;533(7603):420-424 View Article
  52. Nishida K, Arazoe T, Yachie N, Banno S, Kakimoto M, Tabata M, et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science 2016;353(6305):aaf8729 View Article
  53. Huarte M. The emerging role of lncRNAs in cancer. Nat Med 2015;21(11):1253-1261 View Article
  54. Sanjana NE, Wright J, Zheng K, Shalem O, Fontanillas P, Joung J, et al. High-resolution interrogation of functional elements in the noncoding genome. Science 2016;353(6307):1545-1549 View Article
  55. Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelson T, et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 2014;343(6166):84-87 View Article
  56. Korkmaz G, Lopes R, Ugalde AP, Nevedomskaya E, Han R, Myacheva K, et al. Functional genetic screens for enhancer elements in the human genome using CRISPR-Cas9. Nat Biotechnol 2016;34(2):192-198 View Article
  57. Stolte B, Iniguez AB, Dharia NV, Robichaud AL, Conway AS, Morgan AM, et al. Genome-scale CRISPR-Cas9 screen identifies druggable dependencies in TP53 wild-type Ewing sarcoma. J Exp Med 2018;215(8):2137-2155 View Article
  58. Zhu S, Li W, Liu J, Chen CH, Liao Q, Xu P, et al. Genome-scale deletion screening of human long non-coding RNAs using a paired-guide RNA CRISPR-Cas9 library. Nat Biotechnol 2016;34(12):1279-1286 View Article
  59. Liu SJ, Horlbeck MA, Cho SW, Birk HS, Malatesta M, He D, et al. CRISPRi-based genome-scale identification of functional long noncoding RNA loci in human cells. Science 2017;355(6320):aah7111 View Article
  60. Wasko U, Zheng Z, Bhatnagar S. Visualization of Xist Long Noncoding RNA with a Fluorescent CRISPR/Cas9 System. Methods Mol Biol 2019;1870:41-50 View Article
  61. Goyal A, Myacheva K, Groß M, Klingenberg M, Duran Arqué B, Diederichs S. Challenges of CRISPR/Cas9 applications for long non-coding RNA genes. Nucleic Acids Res 2017;45(3):e12 View Article
  62. Clevers H. Modeling Development and Disease with Organoids. Cell 2016;165(7):1586-1597 View Article
  63. Schwank G, Koo BK, Sasselli V, Dekkers JF, Heo I, Demircan T, et al. Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell Stem Cell 2013;13(6):653-658 View Article
  64. Drost J, van Boxtel R, Blokzijl F, Mizutani T, Sasaki N, Sasselli V, et al. Use of CRISPR-modified human stem cell organoids to study the origin of mutational signatures in cancer. Science 2017;358(6360):234-238 View Article
  65. Driehuis E, Clevers H. CRISPR/Cas 9 genome editing and its applications in organoids. Am J Physiol Gastrointest Liver Physiol 2017;312(3):G257-G265 View Article
  66. Terai H, Kitajima S, Potter DS, Matsui Y, Quiceno LG, Chen T, et al. ER Stress Signaling Promotes the Survival of Cancer “Persister Cells” Tolerant to EGFR Tyrosine Kinase Inhibitors. Cancer Res 2018;78(4):1044-1057 View Article
  67. Liao S, Davoli T, Leng Y, Li MZ, Xu Q, Elledge SJ. A genetic interaction analysis identifies cancer drivers that modify EGFR dependency. Genes Dev 2017;31(2):184-196 View Article
  68. Qin S, Ingle JN, Liu M, Yu J, Wickerham DL, Kubo M, et al. Calmodulin-like protein 3 is an estrogen receptor alpha coregulator for gene expression and drug response in a SNP, estrogen, and SERM-dependent fashion. Breast Cancer Res 2017;19(1):95 View Article
  69. Merino D, Whittle JR, Vaillant F, Serrano A, Gong JN, Giner G, et al. Synergistic action of the MCL-1 inhibitor S63845 with current therapies in preclinical models of triple-negative and HER2-amplified breast cancer. Sci Transl Med 2017;9(401):eaam7049 View Article
  70. Sun W, He B, Yang B, Hu W, Cheng S, Xiao H, et al. Genome-wide CRISPR screen reveals SGOL1 as a druggable target of sorafenib-treated hepatocellular carcinoma. Lab Invest 2018;98(6):734-744 View Article
  71. Ranjan A, Srivastava SK. Penfluridol suppresses glioblastoma tumor growth by Akt-mediated inhibition of GLI1. Oncotarget 2017;8(20):32960-32976 View Article
  72. Hoang-Minh LB, Deleyrolle LP, Nakamura NS, Parker AK, Martuscello RT, Reynolds BA, et al. PCM1 Depletion Inhibits Glioblastoma Cell Ciliogenesis and Increases Cell Death and Sensitivity to Temozolomide. Transl Oncol 2016;9(5):392-402 View Article
  73. Liu B, Saber A, Haisma HJ. CRISPR/Cas9: a powerful tool for identification of new targets for cancer treatment. Drug Discov Today 2019;24(4):955-970 View Article
  74. Saber A, Liu B, Ebrahimi P, Haisma HJ. CRISPR/Cas9 for overcoming drug resistance in solid tumors. Daru 2020;28(1):295-304 View Article
  75. Ramakrishna S, Kwaku Dad AB, Beloor J, Gopalappa R, Lee SK, Kim H. Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA. Genome Res 2014;24(6):1020-1027 View Article
  76. Finn JD, Smith AR, Patel MC, Shaw L, Youniss MR, van Heteren J, et al. A Single Administration of CRISPR/Cas9 Lipid Nanoparticles Achieves Robust and Persistent In Vivo Genome Editing. Cell Rep 2018;22(9):2227-2235 View Article
  77. Sun W, Ji W, Hall JM, Hu Q, Wang C, Beisel CL, et al. Self-assembled DNA nanoclews for the efficient delivery of CRISPR-Cas9 for genome editing. Angew Chem Int Ed Engl 2015;54(41):12029-12033 View Article
  78. Lee K, Conboy M, Park HM, Jiang F, Kim HJ, Dewitt MA, et al. Nanoparticle delivery of Cas9 ribonucleoprotein and donor DNA in vivo induces homology-directed DNA repair. Nat Biomed Eng 2017;1:889-901 View Article
  79. Zincarelli C, Soltys S, Rengo G, Rabinowitz JE. Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection. Mol Ther 2008;16(6):1073-1080 View Article
  80. Luo J, Luo Y, Sun J, Zhou Y, Zhang Y, Yang X. Adeno-associated virus-mediated cancer gene therapy: current status. Cancer Lett 2015;356(2 Pt B):347-356 View Article
  81. Yu W, Mookherjee S, Chaitankar V, Hiriyanna S, Kim JW, Brooks M, et al. Nrl knockdown by AAV-delivered CRISPR/Cas9 prevents retinal degeneration in mice. Nat Commun 2017;8:14716 View Article
  82. Yang S, Chang R, Yang H, Zhao T, Hong Y, Kong HE, et al. CRISPR/Cas9-mediated gene editing ameliorates neurotoxicity in mouse model of Huntington’s disease. J Clin Invest 2017;127(7):2719-2724 View Article
  83. Yang Y, Wang L, Bell P, McMenamin D, He Z, White J, et al. A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice. Nat Biotechnol 2016;34(3):334-338 View Article
  84. Bengtsson NE, Hall JK, Odom GL, Phelps MP, Andrus CR, Hawkins RD, et al. Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy. Nat Commun 2017;8:14454 View Article
  85. Tabebordbar M, Zhu K, Cheng JKW, Chew WL, Widrick JJ, Yan WX, et al. In vivo gene editing in dystrophic mouse muscle and muscle stem cells. Science 2016;351(6271):407-411 View Article
  86. Chew WL, Tabebordbar M, Cheng JK, Mali P, Wu EY, Ng AH, et al. A multifunctional AAV-CRISPR-Cas9 and its host response. Nat Methods 2016;13(10):868-874 View Article
  87. Wang G, Chow RD, Ye L, Guzman CD, Dai X, Dong MB, et al. Mapping a functional cancer genome atlas of tumor suppressors in mouse liver using AAV-CRISPR-mediated direct in vivo screening. Sci Adv 2018;4(2):eaao5508 View Article
  88. Chow RD, Guzman CD, Wang G, Schmidt F, Youngblood MW, Ye L, et al. AAV-mediated direct in vivo CRISPR screen identifies functional suppressors in glioblastoma. Nat Neurosci 2017;20(10):1329-1341 View Article
  89. McGranahan N, Swanton C. Clonal Heterogeneity and Tumor Evolution: Past, Present, and the Future. Cell 2017;168(4):613-628 View Article
  90. Schmitt MW, Loeb LA, Salk JJ. The influence of subclonal resistance mutations on targeted cancer therapy. Nat Rev Clin Oncol 2016;13(6):335-347 View Article
  91. Senis E, Fatouros C, Große S, Wiedtke E, Niopek D, Mueller AK, et al. CRISPR/Cas9-mediated genome engineering: an adeno-associated viral (AAV) vector toolbox. Biotechnol J 2014;9(11):1402-1412 View Article
  92. Jiang C, Mei M, Li B, Zhu X, Zu W, Tian Y, et al. A non-viral CRISPR/Cas9 delivery system for therapeutically targeting HBV DNA and pcsk9 in vivo. Cell Res 2017;27(3):440-443 View Article
  93. Miller JB, Zhang S, Kos P, Xiong H, Zhou K, Perelman SS, et al. Non-Viral CRISPR/Cas Gene Editing In Vitro and In Vivo Enabled by Synthetic Nanoparticle Co-Delivery of Cas9 mRNA and sgRNA. Angew Chem Int Ed Engl 2017;56(4):1059-1063 View Article
  94. Mehnert W, Mäder K. Solid lipid nanoparticles: production, characterization and applications. Adv Drug Deliv Rev 2001;47(2-3):165-196 View Article
  95. Pardi N, Hogan MJ, Pelc RS, Muramatsu H, Andersen H, DeMaso CR, et al. Zika virus protection by a single low-dose nucleoside-modified mRNA vaccination. Nature 2017;543(7644):248-251 View Article
  96. Wirth T, Parker N, Yla-Herttuala S. History of gene therapy. Gene 2013;525(2):162-169 View Article
  97. Tebas P, Stein D, Tang WW, Frank I, Wang SQ, Lee G, et al. Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV. N Engl J Med 2014;370(10):901-910 View Article
  98. Cornu TI, Mussolino C, Cathomen T. Refining strategies to translate genome editing to the clinic. Nat Med 2017;23(4):415-423 View Article
  99. Cyranoski D. CRISPR gene-editing tested in a person for the first time. Nature 2016;539(7630):479 View Article
  100. Iwai Y, Ishida M, Tanaka Y, Okazaki T, Honjo T, Minato N. Involvement of PD-L1 on tumor cells in the escape from host immune system and tumor immunotherapy by PD-L1 blockade. PNAS 2002;99(19):12293-12297 View Article
  101. Reck M, Rodriguez-Abreu D, Robinson AG, Hui R, Csőszi T, Fülöp A, et al. Pembrolizumab versus Chemotherapy for PD-L1-Positive Non-Small-Cell Lung Cancer. N Engl J Med 2016;375(19):1823-1833 View Article
  102. Forsberg EMV, Lindberg MF, Jespersen H, Alsén S, Bagge RO, Donia M, et al. HER2 CAR-T Cells Eradicate Uveal Melanoma and T-cell Therapy-Resistant Human Melanoma in IL2 Transgenic NOD/SCID IL2 Receptor Knockout Mice. Cancer Res 2019;79(5):899-904 View Article
  103. Upadhyay R, Boiarsky JA, Pantsulaia G, Svensson-Arvelund J, Lin MJ, Wroblewska A, et al. A Critical Role for Fas-Mediated Off-Target Tumor Killing in T-cell Immunotherapy. Cancer Discov 2021;11(3):599-613 View Article
  104. Eyquem J, Mansilla-Soto J, Giavridis T, van der Stegen SJ, Hamieh M, Cunanan KM, et al. Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection. Nature 2017;543(7643):113-117 View Article
Copyright © 2021 Authors. This is an Open Access article distributed under the terms of the Creative Commons Attribution-Noncommercial 4.0 License (CC BY-NC 4.0), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
  • Journal of Exploratory Research in Pharmacology
  • pISSN 2993-5121
  • eISSN 2572-5505

Article Options

Metrics

Cite this Article

  • © 2024 Xia & He Publishing Inc.
  • Total visits : 2637917
  • Visits today : 3910
Back to Top

Frontiers of CRISPR-Cas9 for Cancer Research and Therapy

Anamika Banerjee, Sunil Kumar Malonia, Shubham Dutta
  • Reset Zoom
  • Download TIFF