A Review on CRISPR/Cas9 as a Novel Technique for Cancer Therapy

Introduction

Cancer is a broad term for a series of diseases characterized by irregular cell development with the ability to infiltrate and disseminate to other body parts [1]. It’s one of the most common causes of death worldwide and a significant public health issue. In 2020, 19.3 million new cases of cancer and over ten million deaths from cancer were registered, globally [2].

Cancer is featuring by the aggregation of many genetic and non-genetic alterations in the cancer cell genome, which lead to carcinogenesis and malignant growth [3]. These alterations may include inactivated tumor suppression, oncogene activation, epigenetic factor mutations, and chemoresistance mutations [4].

Despite the significant advancements in cancer treatment, such as irradiation, chemotherapy, and surgery, the high likelihood of rejection and primary or acquired chemo-radiation tolerance usually leads to inadequate treatment [5]. As a result, the ability to repair or destroy certain DNA regions of a cancer cells which can be achieved by genome editing, can provide an important method for cancer therapy [5].

Genome editing is a kind of genetic modification in which artificially modified nucleases or molecular scissors are used to insert, substitute, or delete DNA from a genome [6]. However, the gene editing technologies are divided into three methodological generations: zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regulatory interspaced short palindromic repeat (CRISPR) system [7].

CRISPR is an adaptive immune system in bacteria that comprises a bank of foreign genetic information and a process for identifying and killing the invading foreign agents like plasmids and viruses [8]. The CRISPR systems are found in 70% of bacteria and 90% of Archaea, and some contain several CRISPR areas on their chromosomes. However, following discovery of CRISPR system as a natural defensive mechanism in bacteria, the researchers tried to modify it to make it a useful tool for gene editing [9]. The CRISPR system comprises a single guide RNA (sgRNA) that targets the specific gene and the Cas9 protein, which is now the most widely used gene editing tool [9]. Moreover, the CRISPR technology has been used in oncology testing and cancer therapy trials since it allows for accurate and effective genome engineering [10, 11].

CRISPR Background

In 1987, CRISPR was first discovered in Escherichia coli when researchers were looking for the gene that controls alkaline phosphatase isozyme conversion [12]. Also, CRISPRs were discovered in Archaea, especially Haloferax mediterranei, in 1993, and have subsequently been found in multiple bacterial and archaeal genomes [13].

In the mid-2000s, the discovery of similarities between the spacer regions of CRISPRs and the succession of archaea, plasmids, and bacteriophages provided an insight that CRISPRs could play an essential role, e.g., in immune system [14]. Later, in 2002, Cas (CRISPR-Linked) genes were assigned to genes that were predicted to encode DNA repair proteins for hyperthermophilic Archaea and were found to be strongly associated with CRISPR [15]. Meanwhile, CRISPR is a term has been universally launched. Similarly, in the eukaryotic RNA interference (RNAi) system, comparative genomic studies have suggested that CRISPR and its proteins function together, forming a supposed immunity mechanism to protect prokaryotic cells from invading pathogens and plasmids [16]. Spacer repeats are transcribed into CRISPR RNAs (crRNAs) that lead the Cas enzyme to the invader's target DNA [17]. In 2012, Jennifer Doudna and Emmanuelle Charpentier proved that the CRISPR-Cas9 can be programmed with RNA in order to edit genomic DNA [18]. However, the use of CRISPR/Cas9 in the modification of human genomes was then declared, thus paving the way for CRISPR use in medicine [19]. Moreover, in 2016, CRISPR/Cas9 modified immune cells were utilized in order to treat people with lung cancer in the first human clinical study using CRISPR [20]. In 2020, for their development of CRISPR/Cas9 technology, the Nobel Prize in Chemistry was given to Emmanuelle Charpentier and Jennifer Doudna. Figure 1 shows, in brief, the time course of CRISPR technology evolution.

Figure 1. Timeline of the CRISPR technology evolution

CRISPR/Cas system classification

There are two classes of CRISPR systems that are divided into six different types and several subtypes. Class 1 includes I, III, and IV types. While class 2 includes II, V, and VI types, being classified according to structural and functional properties [Table 1]. Also, the CRISPR system contains many associated proteins with distinct type of CRISPR [Table 2]. The CRISPR/Cas class 1 system employs a mixture of many Cas proteins, while the class 2 system only employs one Cas protein with several domains. Therefore, the class 2 CRISPR/Cas system is preferable for gene engineering due to its easiness and simplicity. The type II CRISPR/Cas9 system is the most commonly used and studied among the different types of CRISPR class 2 systems [8, 21].

Table 1. Classes of CRISPR system [8, 21]

Abbreviation: PAM: Protospacer adjacent motif, crRNA: CRISPR RNAs, tracrRNA: Trans-activating crisper RNA.

Table 2. Proteins in CRISPR system [8, 21]

CRISPR/Cas9 as an editorial tool

Cas9 is a CRISPR protein type II, class 2, targets DNA molecular. It is a crRNA-guided endonuclease with HNH and RuvC nuclease regions that cleaves the genomic dsDNA [18]. The HNH nuclease region splits the strand of DNA complementary to the gRNA array, whereas the RuvC nuclease region splits the strand of DNA [18]. The most frequently used type of CRISPR/Cas9, Streptococcus pyogenes Cas9 (SpCas9), targets DNA by recognizing the protospacer adjacent motif (PAM) [22]. The Cas9 protein size is variable for different bacterial species, with 1053 amino acid residues (a.a) in Streptococcus aureus and 1368 a.a in Streptococcus pyogenes [23]. The CRISPR/Cas9 system is composed of crRNA, tracrRNA, and Cas9. Artificially, tracrRNA and crRNA can be turned into sgRNA, which guides Cas9 to the target region [24].

Mechanism of CRISPR/Cas9 system action

CRISPR is a natural defense mechanism that helps bacteria and Archaea to resist viral or exogenous plasmid invasion [25]. When a virus infects bacteria, remnants of the viral DNA are embedded into the bacterial CRISPR gene, thus serving as a memory. I.e., when the same virus infects the bacterium again, it can recognize the virus by using this marker. Moreover, bacteria use the Cas9 endonuclease to trigger a double-strand break (DSB) in the viral DNA, which can result in viral inactivation [26]. At the molecular level, the mechanism of CRISPR-Cas9 action can be presented into three major phases: Adaptation, Biogenesis, and Interference, as illustrated in Table 3.

Table 3. The three phases of CRISPR/Cas9 mechanism [27, 28, 29]

At the technical level, the CRISPR type II system is made up of the Cas9 protein and single guide RNA (sgRNA). Cas9 acts as a nuclease that triggers DSBs in the DNA molecule, while sgRNA can identify the target site, particularly through homologous recombination of the 20-bp DNA sequence [30, 31]. Thus, when the CRISPR/Cas9 system is introduced into a cell, the gRNAs direct the Cas9 nuclease to a particular DNA site with a protospacer adjacent motif (PAM) that corresponds to the gRNA. Then, the Cas9 nuclease breaks the DNA double strands and produces a DSB [32]. As shown in Figure 2, an endogenous repair mechanism, e.g., non-homologous end joining (NHEJ) and homology directed repair (HDR) can mostly repair the DSBs caused by Cas9 nuclease [33]. NHEJ is effective but not precise and could cause genetic mutations such as deletions or insertions [34]. Meanwhile the HDR path is ineffective and proceeds through mitosis only. However, HDR allows for precise DNA repair based on homologous sequences [35]. Notably, the CRISPR/Cas9 system is used to edit genes in a variety of cells, and successful transfer of the CRISPR-Cas9 system into cells is still a major challenge.

Figure 2. Endogenous repair mechanisms; non-homologous end joining (NHEJ) and homology directed repair (HDR)

Delivery systems of CRISPR/Cas9

There are several CRISPR/Cas9 genome editing strategies: sgRNA and Cas9-mRNA, sgRNA and Cas9 protein, and a plasmid-based CRISPR-Cas9 system [36]. The benefits and disadvantages of these strategies are illustrated in Table 4. However, the efficient distribution of the CRISPR/Cas9 system to cancer cells is essential for the CRISPR/Cas9 system to be successful in treating cancer. Therefore, the CRISPR/Cas9 system for cancer gene treatment has been studied using three delivery approaches: physical approaches, non- viral vectors, and viral vectors.

Table 4. Features of various CRISPR/Cas9 genome editing strategies

Physical methods of gene transduction

Physical methods do not depend on the utilization of vectors, but rather on making pores in the cell membrane [38]. However, the physical method provides a delivery process that is unaffected by the type of cell or package size [39]. Electroporation is a common physical method used to deliver CRISPR/Cas9 with great efficiency. It employs electrical current pulses to promote transient holes in plasma membranes, allowing the cargo to be delivered into cells [40]. On the other hand, in vitro electroporation has successfully delivered a CRISPR/Cas9 plasmid into cancer cells [41, 42]. Despite the benefits of electroporation, cell damage induced by electroporation may be a major concern for in vitro experiments [40]. Moreover, the delivery of CRISPR/Cas9 may be performed by other common physical methods, e.g., microinjection, membrane deformation, and hydrodynamic injection [43, 44]. Herein, Table 5 shows some studies that used physical methods to deliver CRISPR/Cas9.

Table 5. CRISPR/Cas9 delivery by means of physical methods

Viral vectors

Viral vectors are broadly utilized as gene delivery tools because of their great effectiveness and potentially, long-term effects due to their integration with the host DNA [50]. However, there are various viral types for CRISPR/Cas9 delivery, i.e., adenovirus (AdV), retroviruses (RV), adeno-associated virus (AAV), lentivirus (LV), Epstein-Barr virus, Sendai virus, and baculovirus. The loading capacity of viruses is variable (4.7-38 kb), thus defining the package of genes encoding the CRISPR/Cas systems enzyme [39]. However, AAVs have mostly been employed for CRISPR genome editing in vivo due to their unique features, e.g., being less immunogenic, having low toxicity, and having many AAV serotypes [51]. On the other hand, lentivirus (LV) is often used to deliver CRISPR/Cas9 in vitro because of its capacity to permeate the nuclear membrane without causing cell division [39]. Table 6 depicts some trials that used viral vectors to deliver CRISPR/Cas9.

Table 6. Different viral vectors used for the in vitro CRISPR/Cas9 delivery

Non-viral vectors

CRISPR/Cas9 may also be introduced to the cells using non-viral vectors. These approaches provide lower immune response, are not restricted by packaging limits, are simpler to synthesize, and can deliver many sgRNAs at once [50]. Furthermore, compared to viral vectors, non-viral vectors have fewer off-target effects [58]. Non-viral vectors, on the other hand, have limited in vivo applications due to their low transduction efficiency, despite their safety and ease of use [51]. Table 7 shows some trials that used non-viral delivery systems to introduce CRISPR/Cas9.

Table 7. CRISPR/Cas9 delivery via non-viral vectors

CRISPR/Cas9 Applications

CRISPR/Cas9 technology has paved the way for novel opportunities in human gene editing. Recently, it has been used in a variety of areas, including the treatment of genetic diseases, detection of disease- related gene and diagnosis, tumor therapy, genetic engineering of plants and animals, and the suppression and management of harmful bacteria [65].

CRISPR/Cas9 Application in cancer therapy

Despite been some advances in recent years, the rate of deaths due to cancer continues to rise, demonstrating the essential need for new and more effective treatment approaches. CRISPR/Cas9 technology seems to be a potential tool for cancer treatment. Due to its multiple applications in targeting cancer cells, such as cancer immunotherapy, oncolytic virotherapy, stromal-targeting therapies, etc. The CRISPR/Cas9 technology could be a promising tool of cancer treatment [66]. By using a variety of CRISPR/Cas9 strategies such as base editing and gene knockout/in, CRISPR/Cas9 can be utilized to replace, remove, or correct undesirable genes that cause genetic diseases [32]. Moreover, CRISPR/Cas9 is used in the treatment of different types of cancer such as lung, breast, liver, and others malignancies.

1. Lung cancer
Lung cancer is the major cause of fatality-related cancer in both men and women [2]. Various genes like EGFR, CD38, FAK, RSF1, and others are thought to be proto-oncogenes linked to lung cancer. Likewise, GOT1, MFN2, miR-1304, and others are recognized as suppressor genes in this malignancy [67]. The overexpression of oncogenes and suppressor gene mutations may promote the tumor development. In this respect, the CRISPR/Cas9 technology has the potential to effectively eradicate lung cancer [68]. By targeting the oncogenes CD38 and KRAS, CRISPR/Cas9 knockout/down decreased cell proliferation and tumor growth in vivo [69, 70]. Moreover, the knockout of the MFN2 suppressor gene enhances cell activity and colony formation by activating the mTORC2/Akt pathway [71]. Another study found that knockout of the suppressor gene Plakophilin 1 (PKP1) in the A549 cell line increased cell dissemination while decreasing their reproduction [72].

2. Breast cancer
Breast cancer is the most common cause of mortality in women worldwide. Over 2 million new cases of breast cancer are reported globally [73]. The genetic profile of breast cancer shows high clinical heterogeneity and presence of various molecular subtypes [74]. The complexity of breast cancer is represented by the fact that it comprises a variety of cells, including stem and progenitor cells, instead of a single cell population [75]. Relying on estrogen receptor (ER) expression, the breast epithelial cancer is divided into four subtypes: luminal A, B, triple-negative breast cancer (TNBC), and Her2-positive [76]. Simultaneously, the luminal subtypes are the more fatal and common forms of breast cancer, accounting for around 70% of cases, with 30% of patients resistant to endocrine treatments [77]. Therefore, cytoreductive therapy is critical in the malignancy treatment. In this regard, CRISPR/Cas9 has emerged as a novel and efficient therapeutic tool in the therapy of breast cancer [33]. The knockout of APOBEC3G and CDK4 oncogenes by CRISPR/Cas9 in MCF10A and MDA-MB-231 cell lines, respectively, leads to the inhibition of growth and proliferation of breast cancer cells [78, 79]. On the contrary, knocking down the RLIP and PSMD12 oncogenes in BC and MDA-MB-231 cell lines resulted in decreased breast cell reproduction and development, both in vitro and in vivo [80, 81].

3. Colorectal cancer
Colorectal cancer (CRC) is a cancer that arises in the rectum and colon, being is the world's ninth most common cancer [73]. Over 90% of all colorectal carcinomas are adenocarcinomas (ADC). Nevertheless, squamous cell, spindle-cell, adenosquamous, and neuroendocrine carcinomas account for the remaining 10% of carcinomas [82]. Mutations in many oncogenes and suppressor genes, including ATF3, NAT1, RBX2, DRD2, and AMPKa1, contribute to colorectal cancer. Thus, the knockout of these genes in the HCT116 cell line by CRISPR/Cas9 could be a promising therapeutic target, and inhibiting them could be useful in the patients with advanced colorectal cancer [83, 84].

4. Liver cancer
Liver cancer is the world's fifth most prevalent cancer and the second leading cause of cancer death, and it is more common in males [73]. Hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) are the two major types of liver cancer. HCC and ICC represent 75% and 12-15% of all cases, respectively [85]. However, liver cancer patients have a poor diagnosis and few therapy choices [86]. Therefore, CRISPR/Cas9 technology may be a useful way to find novel therapeutic tools for this malignancy. Various oncogenes, like NCOA5 and Sphk1, were targeted in human HCC cell lines by CRISPR/Cas9 knockout, resulting in decreased cell proliferation, growth, and dissemination, thus reducing tumor development [87, 88]. In contrast, targeting the phosphatase and tension homolog (PTEN) gene in vitro by knocking it out promoted the invasion capacity of HCC cells [89]. Hence, with more experiments, CRISPR/Cas9 could have a promising future in fighting hepatocellular cancer.

5. Prostate and bladder cancer
Prostate cancer is the fourth most common cancer-related cause of mortality among males [73]. The prevalence and death rates in PC patients are significantly linked to age, with the peak incidence reported in the elderly (> 65 years) [90]. Prostate cancer is detected by relying on levels of prostate-specific antigen (PSA more than 4 ng/mL), a glycoprotein usually secreted by prostate cells. Albeit, patients without cancer are also found to have high PSA levels. Therefore, tissue biopsy is still the standard method for confirming this type of cancer [90]. Usage of CRISPR/Cas9 to fix the mutations caused by genomic changes might be a promising direction for PC treatment. In particular, it has been found that the knockout of the PTEN gene in PC by CRISPR/Cas9 mobilizes many critical genes for the survival of tumor cells. Moreover, the PTEN cell line showed increased cell proliferation and colony formation [91]. Deficiency of PTEN, a tumor suppressor gene, is associated with the progression, development, and metastasis of prostate cancer. Hence, PTEN knockout by CRISPR/Cas9 in vivo could explain the role of many genes with altered expression in PTEN-deficient cells in the development of prostate cancer [91].

On the contrary, bladder cancer accounts for 4.4% of all cancer incidence worldwide, and it is more common in males than in females [73]. Urothelial cell carcinoma causes 90% of all cases, while squamous cells cause the remaining 10% of bladder cancer cases [92]. However, lncRNA UCA1 has an important role in promoting bladder cancer as an oncogene [93]. In fact, the roles of the UCA1 gene in bladder cancer include increased cell cycle, apoptosis repression, and increased MMP [94]. Therefore, UCA1 knockdown by CRISPR/Cas9 in T24 and 5637 cell lines was shown to reduced cell reproduction, migration, and invasion in vivo and in vitro. As a result, the cell cycle was arrested at G1 phase, along with significant increase in apoptosis, and decreased MMP activity [93].

6. Cervical and ovarian cancer
Cervical cancer is another common cancer in women, being the third most prevalent cancer among women with a mortality rate of 7.7% [73]. Human papillomavirus (HPV) is among the most common causes of cervical cancer. The HPV produces cervical malignant cells by oncoprotein E7, which inhibits the activity of retinoblastoma family proteins (pRB), and oncoprotein E6, which destructs the tumor suppressor protein p53 [95]. However, the CRISPR/Cas9 technology can destroy HPV E6 and E7, by employing CRISPR-sgRNA to target E7 and E6 in vitro. This resulted in reduction in E7 and E6 mRNA and protein expression and accumulation of p21 and p53 proteins. Furthermore, cell growth has slowed and apoptosis has increased, particularly in vitro [96].

On the contrary, ovarian cancer is the ninth most frequent malignancy in women and the eighth most fatal among women [73]. About 95% of ovarian cancers are epithelial ovarian malignancies cancers, whereas non-epithelial cancers account for up to 5% of ovarian cancers [97]. In ovarian cancer, the epithelial to mesenchymal transition (EMT) pathway is linked to tumor metastasis, treatment resistance, and a low patient survival rate [98]. Moreover, high expression of the baculoviral IAP repeat containing 5 (BIRC5) gene leads to changes in EMT and tumor growth. Therefore, CRISPR/Cas9-mediated knockout of the BIRC5 gene in SKOV3 and OVCAR3 ovarian cells inhibited EMT, dramatically decreased cell proliferation, and their invasion, prompting cell apoptosis. Hence, in tumors, targeting the overexpressed BIRC5 gene could be an effective anti-cancer therapy [99].

A number of in vitro and in vivo experimental trials that used the CRISPR/Cas9-based gene knockout technologies in the therapy of various cancers, including lung, breast, colorectal, prostate, liver, and other malignancies are listed in Table 8.

Table 8. Some relevant works on CRISPR applications in potential cancer treatment

Abbreviations: AV: Adenovirus, CD8: Cluster of differentiation 38, CDK4: Cyclin-dependent kinase 4, LV: Lentivirus, TS: Tumor suppressor, OG: Oncogene, NV: Not available.

Benefits and disadvantages

In terms of simplicity, flexibility, and low price, the CRISPR/Cas9 system has many benefits over other gene editing technologies like ZFN and TALENs. However, the most significant distinction is that the CRISPR method depends on DNA-RNA recognition instead of DNA-protein interaction [18]. Thus, constructing a customized CRISPR/Cas9 system by simply modifying the guide-RNA (gRNA) sequence rather than designing a novel protein is more feasible and simpler than designing a novel protein [19, 113]. Nevertheless, the huge size of the Cas9 protein is one of the disadvantages of CRISPR-Cas9. Because of Cas9's large size (4-7 kb), it's difficult to pack the protein into low immunogenic AVV vectors used for gene delivery in vivo and in vitro [114]. Thus, to resolve this issue, the delivery method must be redesigned with a larger cargo capacity, or smaller Cas9 types can be used [115]. Furthermore, clinical trials have shown that Cas9 from S. aureus and S. pyogenes may cause an immune response within the body [116]. One probable way to override this problem is to upgrade Cas9 or use another bacterial protein that can evade the host's immune system. Another issue with the CRISPR system are the off-target effects that makes it hard to focus on a specific genomic locus [117]. Thus, one of the strategies that may include selection of an appropriate delivery tool that will help to reduce off-target effects while still increasing target performance, such as RNP delivery [118].

Conclusion and future directions

The emergence of the CRISPR/Cas9 system as a bacterial defense response against pathogens, as well as its use as a potent tool for generating selective genomic modifications, has opened new avenues for molecular biology. As an effective editing tool, CRISPR-Cas9 technology has considerable therapeutic potential for improving anticancer approaches, although with certain challenges. Moreover, CRISPR-Cas9 has a wide range of possible applications, including combating oncogenic diseases, modulating gene expression, and immunotherapy. As such, because of CRISPR's medicinal potential, it is regarded as a critical tool in combatting severe cancer disorders. CRISPR is only capable of correcting a single human mutation. However, by driving the technique to its extremes, many genes may be fixed, deleted, substituted, or implanted in vivo concurrently with one single strike. Moreover, the development of cas9 forms with no or minimal off-target effects must be considered for future CRISPR uses. Finally, the improvement of non-viral and viral delivery systems will be required to enhance CRISPR/Cas9 in vivo application, providing a basis for CRISPR therapeutic use.

Conflict of interest

None declared.

Abbreviations

References

1. National Cancer Institute. What is cancer. Cancer.Gov. Retrieved August 6, 2021, from https://www.cancer.gov/about-cancer/understanding/what-is-cancer
2. Ferlay J, Colombet M, Soerjomataram I, Parkin DM, Piñeros M, Znaor A, et al. Cancer statistics for the year 2020: An overview. Intl J Cancer. 2021; 149(4): 778-789. doi: 10.1002/ijc.33588
3. Del Sol A, Balling R, Hood L, Galas, D. Diseases as network perturbations. Curr Opin Biotechnol. 2010; 21(4): 566-571. doi: 10.1016/j.copbio.2010.07.010
4. Khan MNM, Islam KK, Ashraf A, Barman NC. A review on genome editing by CRISPR-CAS9 technique for cancer treatment. World Cancer Res J. 2020; 7, e1510. doi: 10.32113/wcrj_20203_1510
5. Yi L, Li J. CRISPR-Cas9 therapeutics in cancer: promising strategies and present challenges. Biochim Biophs Acta. 2016; 1866(2): 197-207. doi: 10.1016/j.bbcan.2016.09.002
6. Doudna, JA, Charpentier E. The new frontier of genome engineering with CRISPR-Cas9. Science. 2014; 346(6213): 1258096.
   doi: 10.1126/science.1258096
7. Mirzaei HR, Pourghadamyari H, Rahmati M, Mohammadi A, Nahand JS, Rezaei A, et al. Gene-knocked out chimeric antigen receptor (CAR) T cells: tuning up for the next generation cancer immunotherapy. Cancer Lett. 2018: 423: 95-104. doi: 10.1016/j.canlet.2018.03.010
8. Clark DP, Pazdernik NJ, McGehee MR. Molecular Biology (Genome Defense), 3^th Edition. Academic Press (Imprint of Elsevier), London 2019; pp. 622-653. doi: 10.1016/B978-0-12-813288-3.00020-3
9. 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. doi: 10.1038/nprot.2013.143
10. Heckl D, Kowalczyk MS, Yudovich D, Belizaire R, Puram RV, McConkey ME, et al. Generation of mouse models of myeloid malignancy with combinatorial genetic lesions using CRISPR-Cas9 genome editing. Nat Biotechnol. 2014; 32(9): 941-946. doi: 10.1038/nbt.2951
11. McKenna A, Findlay GM, Gagnon JA, Horwitz MS, Schier AF, Shendure J. Whole-organism lineage tracing by combinatorial and cumulative genome editing. Science 2016; 353(6298): aaf7907. doi: 10.1126/science.aaf7907
12. Ishino Y, Shinagawa H, Makino K, Amemura M, Nakata A. Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. J Bacteriol. 1987; 169(12), 5429-5433.
    doi: 10.1128/jb.169.12.5429-5433.1987
13. Mojica FJM., Juez G, Rodriguez VF. Transcription at different salinities of Haloferax mediterranei sequences adjacent to partially modified PstI sites. Mol Microbiol. 1993; 9(3): 613-621. doi: 10.1111/j.1365-2958.1993.tb01721.x
14. Daisy PS, Shreyas KS, & Anitha TS. Will CRISPR-Cas9 have cards to play against cancer? An update on its applications. Mol Bio. 2021; 63(2), 93-108. doi: 10.1007/s12033-020-00289-1
15. Jansen R, van Embden JDA, Gaastra W, Schouls LM. Identification of genes that are associated with DNA repeats in prokaryotes. Mol Microbiol. 2002; 43(6): 1565-1575. doi: 10.1046/j.1365-2958.2002.02839.x
16. Makarova KS, Grishin NV, Shabalina SA, Wolf YI, Koonin EV. A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. Biol Direct. 2006; 1(1), 1-26. doi: 10.1186/1745-6150-1-7
17. Brouns SJ, Jore MM, Lundgren M, Westra ER, Slijkhuis RJ, Snijders AP, et al. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 2008; 321(5891): 960-964. doi: 10.1126/science.1159689
18. 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. doi: 10.1126/science.1225829
19. Shalem O, Sanjana NE, Zhang F. High-throughput functional genomics using CRISPR-Cas9. Nat Rev Genet. 2015; 16(5): 299-311.
    doi: 10.1038/nrg3899
20. Cyranoski, D. Chinese scientists to pioneer first human CRISPR trial. Nat News. 2016; 535(7613): 476. doi: 10.1038/nature.2016.20302
21. Van Der Oost J, Westra ER, Jackson RN, Wiedenheft B. Unravelling the structural and mechanistic basis of CRISPR-Cas systems. Nat Rev Microbiol. 2014, 12(7): 479-492. doi: 10.1038/nrmicro3279
22. Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata SI, Dohmae N, et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell. 2014; 156(5): 935-949. doi: 10.1016/j.cell.2014.02.001
23. Friedland AE, Baral R, Singhal P, Loveluck K, Shen S, Sanchez M, et al. Characterization of Staphylococcus aureus Cas9: a smaller Cas9 for all-in-one adeno-associated virus delivery and paired nickase applications. Genome Biol. 2015; 16(1): 1-10.
    doi: 10.1186/s13059-015-0817-8
24. Knott GJ, Doudna JA. CRISPR-Cas guides the future of genetic engineering. Science. 2018; 361(6405): 866-869.
    doi: 10.1126/science.aat5011
25. Mohammadzadeh I, Qujeq D, Yousefi T, Ferns GA, Maniati M, Vaghari‐Tabari M. CRISPR/Cas9 gene editing: A new therapeutic approach in the treatment of infection and autoimmunity. IUBMB Life. 2020; 72(8): 1603-1621. doi: 10.1002/iub.2296
26. Hille F, Richter H, Wong SP, Bratovič M, Ressel S, Charpentier E. The biology of CRISPR-Cas: backward and forward. Cell. 2018; 172(6): 1239-1259. doi: 10.1016/j.cell.2017.11.032
27. Hille F, Charpentier E. CRISPR-Cas: biology, mechanisms and relevance. Philos Trans R Soc Lond B Biol Sci. 2016, 371(1707): 20150496. doi: 10.1098/rstb.2015.0496
28. Jiang F, Doudna JA. CRISPR–Cas9 structures and mechanisms. Annu Rev Biophys. 2017; 46: 505-529. doi: 10.1146/annurev-biophys-062215-010822
29. Akram F, Ul Haq I, Ahmed Z, Khan H, Ali MS. CRISPR-Cas9, A promising therapeutic tool for cancer therapy: A Review. Protein Pept Lett. 2020; 27(10): 931-944. doi: 10.2174/0929866527666200407112432
30. Gasiunas G, Barrangou R, Horvath P, Siksnys V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci. 2012; 109(39): E2579-E2586. doi: 10.1073/pnas.1208507109
31. Doench JG, Fusi N, Sullender M, Hegde M, Vaimberg EW, Donovan KF, et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol. 2016; 34(2): 184-191. doi: 10.1038/nbt.3437
32. Zhang B. CRISPR/Cas gene therapy. J Cell Physiol. 2021; 236(4): 2459-2481. doi: 10.1002/jcp.30064
33. 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. doi: 10.1016/j.drudis.2019.02.011
34. Rodgers K, McVey M. Error-prone repair of DNA double-strand breaks. J Cell Physiol. 2016; 231(1): 15-24. doi: 10.1002/jcp.25053
35. Tang XD, Gao F, Liu MJ, Fan QL, Chen DK, Ma WT. Methods for enhancing clustered regularly interspaced short palindromic repeats/Cas9-mediated homology-directed repair efficiency. Front Genet. 2019; 10: 551. doi: 10.3389/fgene.2019.00551
36. Liu C, Zhang L, Liu H, & Cheng K. Delivery strategies of the CRISPR-Cas9 gene-editing system for therapeutic applications. J Control Release. 2017; 266: 17-26. doi: 10.1016/j.jconrel.2017.09.012
37. Karimian A, Azizian K, Parsian H, Rafieian S, Shafiei-Irannejad V, Kheyrollah M, et al. CRISPR/Cas9 technology as a potent molecular tool for gene therapy. J Cell Physiol. 2019; 234(8): 12267-12277. doi: 10.1002/jcp.27972
38. Meacham JM, Durvasula K, Degertekin FL, Fedorov AG. Physical methods for intracellular delivery: practical aspects from laboratory use to industrial-scale processing. J Lab Autom. 2014; 19(1): 1-18. doi: 10.1177/2211068213494388
39. Fajrial AK, He QQ, Wirusanti NI, Slansky JE, Ding X. A review of emerging physical transfection methods for CRISPR/Cas9-mediated gene editing. Theranostics. 2020; 10(12): 5532. doi: 10.7150/thno.43465
40. Song X, Liu C, Wang N, Huang H, He S, Gong C, Wei Y. Delivery of CRISPR/Cas systems for cancer gene therapy and immunotherapy. Adv Drug Deliv Rev. 2021; 168: 158-180. doi: 10.1016/j.addr.2020.04.010
41. Feng Y, Sassi S, Shen JK, Yang X, Gao Y, Osaka E., et al. Targeting Cdk11 in osteosarcoma cells using the CRISPR/Cas9 system. J Orthop Res. 2015; 33(2): 199-207. doi: 10.1002/jor.22745
42. Valletta S, Dolatshad H, Bartenstein M, Yip BH, Bello E, Gordon S, et al. ASXL1 mutation correction by CRISPR/Cas9 restores gene function in leukemia cells and increases survival in mouse xenografts. Oncotarget. 2015; 6(42): 44061. doi: 10.18632/oncotarget.6392
43. Han X, Liu Z, Chan Jo M, Zhang K, Li Y, Zeng Z, et al. CRISPR-Cas9 delivery to hard-to-transfect cells via membrane deformation. Sci Adv. 2015; 1(7): e1500454. doi: 10.1126/sciadv.1500454
44. Zhu W, Xie K, Xu Y, Wang L, Chen K, Zhang L, Fang J. CRISPR/Cas9 produces anti-hepatitis B virus effect in hepatoma cells and transgenic mouse. Virus Res. 2016, 217: 125-132. doi: 10.1016/j.virusres.2016.04.003
45. Hansen M, de Ávila BEF, Beltrán M, Zhao J, Ramírez DE, Angsantikul P, et al. Active Intracellular delivery of a Cas9/sgRNA complex using ultrasound-propelled nanomotors. Angew Chem Int Ed. 2018; 57(10): 2657-2661. doi: 10.1002/anie.201713082
46. Sessions JW, Skousen CS, Price KD, Hanks BW, Hope S, Alder JK, Jensen BD. CRISPR-Cas9 directed knock-out of a constitutively expressed gene using lance array nanoinjection. Springerplus. 2016; 5(1): 1-11. doi: 10.1186/s40064-016-3037-0
47. Su S, Hu B, Shao J, Shen B, Du J, Du Y, et al. CRISPR-Cas9 mediated efficient PD-1 disruption on human primary T cells from cancer patients. Sci Rep. 2016; 6(1): 1-14. doi: 10.1038/srep20070
48. Hung KL, Meitlis I, Hale M, Chen CY, Singh S, Jackson SW, et al. Engineering protein-secreting plasma cells by homology-directed repair in primary human B cells. Mol Ther 2018; 26(2): 456-467. doi: 10.1016/j.ymthe.2017.11.012
49. Kararoudi MN, Dolatshad H, Trikha P, Hussain SRA, Elmas E, Foltz JA, et al. Generation of knock-out primary and expanded human NK cells using Cas9 ribonucleoproteins. J Vis Exp. 2018; (136): e58237. doi: 10.3791/58237
50. Wang HX, Li M, Lee CM, Chakraborty S, Kim HW, Bao G, Leong KW. CRISPR/Cas9-based genome editing for disease modeling and therapy: challenges and opportunities for nonviral delivery. Chem Rev. 2017; 117(15): 9874-9906. doi: 10.1021/acs.chemrev.6b00799
51. Xu CL, Ruan MZ, Mahajan VB, Tsang SH. Viral delivery systems for CRISPR. Viruses. 2019; 11(1): 28. doi: 10.3390/v11010028
52. Yoshiba T, Saga Y, Urabe M, Uchibor R, Matsubara S, Fujiwara H, & Mizukami H. CRISPR/Cas9‑mediated cervical cancer treatment targeting human papillomavirus E6. Oncol Lett. 2019; 17(2): 2197-2206. doi: 10.3892/ol.2018.9815
53. Kennedy EM, Kornepati AV, Goldstein M, Bogerd HP, Poling BC, Whisnant AW, et al. Inactivation of the human papillomavirus E6 or E7 gene in cervical carcinoma cells by using a bacterial CRISPR/Cas RNA-guided endonuclease. J Virol. 2014; 88(20): 11965.
    doi: 10.1128/JVI.01879-14
54. Wu W, Duan Y, Ma G, Zhou G, Park-Windhol C, D'Amore PA, Lei H. AAV-CRISPR/Cas9–mediated depletion of VEGFR2 blocks angiogenesis in vitro. Invest Ophthalmol Vis Sci. 2017; 58(14): 6082-6090. doi: 10.1167/iovs.17-21902
55. Chu HW, Rios C, Huang C, Wesolowska-Andersen A, Burchard EG, O’Connor BP, et al. CRISPR–Cas9-mediated gene knockout in primary human airway epithelial cells reveals a proinflammatory role for MUC18. Gene Ther. 2015; 22(10): 822-829. doi: 10.1038/gt.2015.53
56. Liu Q, Fan D, Adah D, Wu Z, Liu R, Yan QT, et al. CRISPR/Cas9-mediated hypoxia inducible factor-1α knockout enhances the antitumor effect of transarterial embolization in hepatocellular carcinoma. Oncol Rep. 2018; 40(5): 2547-2557. doi: 10.3892/or.2018.6667
57. Koo T, Yoon AR, Cho HY, Bae S, Yun CO, Kim JS. Selective disruption of an oncogenic mutant allele by CRISPR/Cas9 induces efficient tumor regression. Nucleic Acids Res. 2017; 45(13): 7897-7908. doi: 10.1093/nar/gkx490
58. Mout R, Ray M, Lee YW, Scaletti F, Rotello VM. In vivo delivery of CRISPR/Cas9 for therapeutic gene editing: progress and challenges. Bioconjug Chem. 2017; 28(4): 880-884. doi: 10.1021/acs.bioconjchem.7b00057
59. Hu Z, Yu L, Zhu D, Ding W, Wang X, Zhang C, et al. Disruption of HPV16-E7 by CRISPR/Cas System Induces Apoptosis and Growth Inhibition in HPV16 Positive Human Cervical Cancer Cells. Biomed Res Int. 2014; 1-9. doi: 10.1155/2014/612823
60. Kretzmann JA, Ho D, Evans CW, Plani-Lam JH, Garcia-Bloj B, Mohamed AE, et al. Synthetically controlling dendrimer flexibility improves delivery of large plasmid DNA. Chem Sci. 2017; 8(4): 2923-2930. doi: 10.1039/C7SC00097A
61. Ho YK, Zhou LH, Tam KC, Too HP. Enhanced non-viral gene delivery by coordinated endosomal release and inhibition of β-tubulin deactylase. Nucleic Acids Res. 2017; 45(6): e38-e38. doi: 10.1093/nar/gkw1143
62. Zhang L, Wang P, Feng Q, Wang N, Chen Z, Huang Y, et al. Lipid nanoparticle-mediated efficient delivery of CRISPR/Cas9 for tumor therapy. NPG Asia Mater. 2017; 9(10): e441-e441. doi: 10.1038/am.2017.185
63. Kim SM, Yang Y, Oh SJ, Hong Y, Seo M, Jang M. Cancer-derived exosomes as a delivery platform of CRISPR/Cas9 confer cancer cell tropism-dependent targeting. J Control Release. 2017; 266: 8-16. doi: 10.1016/j.jconrel.2017.09.013
64. He ZY, Zhang YG, Yang YH, Ma CC, Wang P, Du W, et al. In vivo ovarian cancer gene therapy using CRISPR-Cas9. Hum Gene Ther 2018; 29(2): 223-233. doi: 10.1089/hum.2017.209
65. Barrangou R, & Doudna JA. Applications of CRISPR technologies in research and beyond. Nat Biotechnol. 2016; 34(9): 933-941.
    doi: 10.1038/nbt.3659
66. Chen M, Mao A, Xu M, Weng Q, Mao J, Ji J. CRISPR-Cas9 for cancer therapy: Opportunities and challenges. Cancer Lett. 2019; 447: 48-55. doi: 10.1016/j.canlet.2019.01.017
67. Jiang C, Lin X, Zhao Z. Applications of CRISPR/Cas9 technology in the treatment of lung cancer. Trends Mol Med. 2019; 25(11): 1039-1049. doi: 10.1016/j.molmed.2019.07.007
68. Nair J, Nair A, Veerappan S, Sen D. Translatable gene therapy for lung cancer using Crispr CAS9 – an exploratory review. Cancer Gene Ther. 2020; 27(3): 116-124. doi: 10.1038/s41417-019-0116-8
69. Bu X, Kato J, Hong JA, Merino MJ, Schrump DS, Lund FE, et al. CD38 knockout suppresses tumorigenesis in mice and clonogenic growth of human lung cancer cells. Carcinogenesis. 2018; 39(2): 242-251. doi: 10.1093/carcin/bgx137
70. Gao Q, Ouyang W, Kang B, Han X, Xiong Y, Ding R, et al. Selective targeting of the oncogenic KRAS G12S mutant allele by CRISPR/Cas9 induces efficient tumor regression. Theranostics. 2020; 10(11): 5137. doi: 10.7150/thno.42325
71. Xu K, Chen G, Li X, Wu X, Chang Z, Xu J, et al. MFN2 suppresses cancer progression through inhibition of mTORC2/Akt signaling. Sci Rep. 2017; 7(1): 1-13. doi: 10.1038/srep41718
72. Martin-Padron J, Boyero L, Rodriguez MI, Andrades A, Díaz-Cano I, Peinado P, et al. Plakophilin 1 enhances MYC translation, promoting squamous cell lung cancer. Oncogene. 2020; 39(32): 5479-5493. doi: 10.1038/s41388-019-1129-3
73. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021; 71(3): 209-249. doi: 10.3322/caac.21660
74. Sørlie T, Perou CM, Tibshirani R, Aas T, Geisler S, Johnsen H, et al. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci. 2001, 98(19): 10869-10874. doi: 10.1073/pnas.191367098
75. Visvader JE, Stingl J. Mammary stem cells and the differentiation hierarchy: current status and perspectives. Genes Dev. 2014; 28(11): 1143-1158. doi: 10.1101/gad.242511.114
76. Goldhirsch A, Winer EP, Coates AS, Gelber RD, Piccart-Gebhart M, Thürlimann B, et al. Personalizing the treatment of women with early breast cancer: highlights of the St Gallen International Expert Consensus on the Primary Therapy of Early Breast Cancer 2013. Ann Oncol 2013; 24(9): 2206-2223. doi: 10.1093/annonc/mdt303
77. Padua MB, Bhat-Nakshatri P, Anjanappa M, Prasad MS, Hao Y, Rao X, et al. Dependence receptor UNC5A restricts luminal to basal breast cancer plasticity and metastasis. Breast Cancer Res. 2018; 20(1): 1-18. doi: 10.1186/s13058-018-0963-5
78. Mendes dAR, Bandarra S, Clara RA, Mascarenhas P, Bekman E, Barahona I. Inactivation of APOBEC3G gene in breast cancer cells using the CRISPR/Cas9 system. Ann Med. 2019; 51(sup1): 40-40. doi: 10.1080/07853890.2018.1561848
79. Ahmed A, Ashraf D, Bahaa A, El-Tayebi H, & Adwan H. Impact of CDK4 knock out using CRISPR/Cas9 gene editing technology on breast cancer progression. Eur J Cancer. 2020; 138: S70-S71. doi: 10.1016/S0959-8049(20)30716-4
80. Singhal J, Chikara S, Horne D, Awasthi S, Salgia R, Singhal SS. Targeting RLIP with CRISPR/Cas9 controls tumor growth. Carcinogenesis. 2021; 42(1): 48-57. doi: 10.1093/carcin/bgaa048
81. Du X, Shen X, Dai L, Bi F, Zhang H, Lu C. PSMD12 promotes breast cancer growth via inhibiting the expression of pro-apoptotic genes. Biochem Biophys Res Commun. 2020; 526(2): 368-374. doi: 10.1016/j.bbrc.2020.03.095
82. Fleming M, Ravula S, Tatishchev SF, Wang HL. Colorectal carcinoma: Pathologic aspects. J Gastrointest Oncol. 2012; 3(3): 153.
    doi: 10.3978/j.issn.2078-6891.2012.030
83. Kline CLB, Ralff MD, Lulla AR, Wagner JM, Abbosh PH, Dicker DT, et al. Role of dopamine receptors in the anticancer activity of ONC201. Neoplasia. 2018; 20(1): 80-91. doi: 10.1016/j.neo.2017.10.002
84. Wu XY, Fang J, Wang ZJ, Chen C, Liu JY, Wu GN, et al. Identification of RING-box 2 as a potential target for combating colorectal cancer growth and metastasis. Am J Clin Cancer Res. 2017, 7(6): 1238-1251. PMID: 28670488
85. Petrick JL, McGlynn KA. The changing epidemiology of primary liver cancer. Curr Epidemiol Rep. 2019; 6(2): 104-111.
    doi: 10.1007/s40471-019-00188-3
86. Goossens N, Sun X, Hoshida Y. Molecular classification of hepatocellular carcinoma: potential therapeutic implications. Hepat Oncol. 2015; 2(4): 371-379. doi: 10.2217/hep.15.26
87. He J, Zhang W, Li A, Chen F, Luo R. Knockout of NCOA5 impairs proliferation and migration of hepatocellular carcinoma cells by suppressing epithelial-to-mesenchymal transition. Biochem Biophys Res Commun. 2018, 500(2): 177-183. doi: 10.1016/j.bbrc.2018.04.017
88. Cai H, Xie X, Ji L, Ruan X, Zheng Z. Sphingosine kinase 1: A novel independent prognosis biomarker in hepatocellular carcinoma. Oncol Lett. 2017; 13(4): 2316-2322. doi: 10.3892/ol.2017.5732
89. Zhou X, Li R, Jing R, Zuo B, Zheng Q. Genome-wide CRISPR knockout screens identify ADAMTSL3 and PTEN genes as suppressors of HCC proliferation and metastasis, respectively. J Cancer Res Clin Oncol. 2020; 146(6): 1509-1521. doi: 10.1007/s00432-020-03207-9
90. Rawla P. Epidemiology of prostate cancer. World J Oncol 2019; 10(2): 63. doi: 10.14740/wjon1191
91. Takao A, Yoshikawa K, Karnan S, Ota A, Uemura H, De VMA, et al. Generation of PTEN‑knockout (‑/‑) murine prostate cancer cells using the CRISPR/Cas9 system and comprehensive gene expression profiling. Oncol Rep. 2018; 40(5): 2455-2466.
    doi: 10.3892/or.2018.6683
92. Saginala K, Barsouk A, Aluru JS, Rawla P, Padala SA, Barsouk A. Epidemiology of bladder cancer. Med Sci. 2020; 8(1): 15.
    doi: 10.3390/medsci8010015
93. Zhen S, Hua L, Liu YH, Sun XM, Jiang MM, Chen W, et al. Inhibition of long non-coding RNA UCA1 by CRISPR/Cas9 attenuated malignant phenotypes of bladder cancer. Oncotarget. 2017; 8(6): 9634. doi: 10.18632/oncotarget.14176
94. Fu X, Liu Y, Zhuang C, Liu L, Cai Z, Huang W. Synthetic artificial microRNAs targeting UCA1-MALAT1 or c-Myc inhibit malignant phenotypes of bladder cancer cells T24 and 5637. Mol Biosyst. 2015; 11(5): 1285-1289. doi: 10.1039/C5MB00127G
95. Thatte J, Banks L. Human papillomavirus 16 (HPV-16), HPV-18, and HPV-31 E6 override the normal phosphoregulation of E6AP enzymatic activity. J Virol. 2017; 91(22): e01390-17. doi: 10.1128/JVI.01390-17
96. Ling K, Yang L, Yang N, Chen M, Wang Y, Liang S, et al. Gene Targeting of HPV18 E6 and E7 synchronously by nonviral transfection of CRISPR/Cas9 system in cervical cancer. Hum Gene Ther. 2020; 31(5-6): 297-308. doi: 10.1089/hum.2019.246
97. Torre LA, Trabert B, DeSantis CE, Miller KD, Samimi G, Runowicz CD, et al. Ovarian cancer statistics, 2018. CA Cancer J Clin. 2018; 68(4): 284-296. doi: 10.3322/caac.21456
98. Vergara D, Merlot B, Lucot JP, Collinet P, Vinatier D, Fournier I, Salzet M. Epithelial–mesenchymal transition in ovarian cancer. Cancer Lett. 2010; 291(1): 59-66. doi: 10.1016/j.canlet.2009.09.017
99. Zhao G, Wang Q, Gu Q, Qiang W, Wei JJ, Dong P, et al. Lentiviral CRISPR/Cas9 nickase vector mediated BIRC5 editing inhibits epithelial to mesenchymal transition in ovarian cancer cells. Oncotarget. 2017; 8(55): 94666. doi: 10.18632/oncotarget.21863
100. Annunziato S, Kas SM, Nethe M, Yücel H, Del BJ, Pritchard C, et al. Modeling invasive lobular breast carcinoma by CRISPR/Cas9-mediated somatic genome editing of the mammary gland. Genes Dev. 2016; 30(12): 1470-1480. doi: 10.1101/gad.279190.116
101. Yan F, Le YXL, Qiao B, Meng Q, Yu L, Yuan X, et al. Overexpression of the transcription factor ATF3 with a regulatory molecular signature associates with the pathogenic development of colorectal cancer. Oncotarget. 2017; 8(29): 47020. doi: 10.18632/oncotarget.16638
102. Dai C, Zhang X, Xie D, Tang P, Li C, Zuo Y. Targeting PP2A activates AMPK signaling to inhibit colorectal cancer cells. Oncotarget. 2017; 8(56): 95810. doi: 10.18632/oncotarget.21336
103. Wang L, Minchin RF, & Butcher NJ. Arylamine N-acetyltransferase 1 protects against reactive oxygen species during glucose starvation: role in the regulation of p53 stability. PloS One 2018; 13(3): e0193560. doi: 10.1371/journal.pone.0193560
104. Mizuno Y, Shimada S, Akiyama Y, Watanabe S, Aida T, Ogawa K, et al. DEPDC5 deficiency contributes to resistance to leucine starvation via p62 accumulation in hepatocellular carcinoma. Sci Rep. 2018; 8(1): 1-11. doi: 10.1038/s41598-017-18323-9
105. Su B, Zhang L, Zhuang W, Zhang W, Chen X. Knockout of Akt1/2 suppresses the metastasis of human prostate cancer cells CWR22rv1 in vitro and in vivo. J Cell Mol Med. 2021; 25(3): 1546-1553. doi: 10.1111/jcmm.16246
106. Batır MB, Şahin E, Çam FS. Evaluation of the CRISPR/Cas9 directed mutant TP53 gene repairing effect in human prostate cancer cell line PC-3. Mol Biol Rep. 2019, 46(6): 6471-6484. doi: 10.1007/s11033-019-05093-y
107. Chen D, Li H, Zhang H, Li Q, Huang Y, & Liu H. MTHFD2 Regulates the AKT/MYC Signaling Pathway in Bladder Cancer and Promotes Proliferation, Viability and Migration in Vitro. Res Sq. 2020. doi: 10.21203/rs.3.rs-96109/v1
108. Huo W, Zhao G, Yin J, Ouyang X, Wang Y, Yang C, et al. Lentiviral CRISPR/Cas9 vector mediated miR-21 gene editing inhibits the epithelial to mesenchymal transition in ovarian cancer cells. J Cancer. 2017; 8(1): 57. doi: 10.7150/jca.16723
109. You MH, Jeon MJ, Kim SR, Lee WK, Cheng SY, Jang G., et al. Mitofusin-2 modulates the epithelial to mesenchymal transition in thyroid cancer progression. Sci Rep. 2021; 11(1): 1-12. doi: 10.1038/s41598-021-81469-0
110. Fan Y, Li J, Wei W, Fang H, Duan Y, Li N, et al. Ku80 gene knockdown by the CRISPR/Cas9 technique affects the biological functions of human thyroid carcinoma cells. Oncol Rep. 2019; 42(6): 2486-2498. doi: 10.3892/or.2019.7348
111. Ercolano G, De CP, Rubino V, Terrazzano G, Ruggiero G, Carriero R, et al. Knockdown of PTGS2 by CRISPR/CAS9 system designates a new potential gene target for melanoma treatment. Front Pharmacol. 2019; 10: 1456. doi: 10.3389/fphar.2019.01456
112. Pan D, Kobayashi A, Jiang P, de ALF, Tay RE, Luoma AM, et al. A major chromatin regulator determines resistance of tumor cells to T cell-mediated killing. Science. 2018; 359(6377): 770-775. doi: 10.1126/science.aao1710
113. Liu B, Xu H, Miao J, Zhang A, Kou X, Li W, et al. CRISPR/Cas: a faster and more efficient gene editing system. J Nanosci Nanotechnol. 2015; 15(3): 1946-1959. doi: 10.1166/jnn.2015.9832
114. Naso MF, Tomkowicz B, Perry WL, Strohl WR. Adeno-associated virus (AAV) as a vector for gene therapy. Biodrugs. 2017; 31(4): 317-334. doi: 10.1007/s40259-017-0234-5
115. Lino CA, Harper JC, Carney JP, Timlin JA. Delivering CRISPR: a review of the challenges and approaches. Drug Deliv. 2018; 25(1): 1234-1257. doi: 10.1080/10717544.2018.1474964
116. Crudele JM, Chamberlain JS. Cas9 immunity creates challenges for CRISPR gene editing therapies. Nat Commun. 2018; 9(1): 1-3.
     doi: 10.1038/s41467-018-05843-9
117. Tsai SQ, Joung JK. Defining and improving the genome-wide specificities of CRISPR-Cas9 nucleases. Nat Rev Genet. 2016; 17(5): 300-312. doi: 10.1038/nrg.2016.28
118. Doudna JA. The promise and challenge of therapeutic genome editing. Nature. 2020; 578(7794): 229-236.
     doi: 10.1038/s41586-020-1978-5