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19Cellular Therapy and Transplantation (CTT). Vol. 11, No. 3-4, 2022 doi: 10.18620/ctt-1866-8836-2022-11-3-4-10-24 Submitted: 31 October 2022, accepted: 25 November 2022
A Review on CRISPR/Cas9 as a Novel Technique for Cancer Therapy
Ali A. Saleem 1, Khalida K. Al-Kelaby 2
1 Medical Laboratory Techniques, Pathological Analysis, Al-Hakim Hospital, Najaf, Iraq, 54001
2 Department of Clinical and Laboratory Sciences, Faculty of pharmacy, Kufa University, Najaf, Iraq, 54001
Ali A. Saleem, Medical Laboratory Techniques, Pathological Phone: (+964) 7711667130 Analysis, Al-Hakim Hospital, Najaf, Iraq, 54001 E-mail: [email protected]
Citation: Saleem AA, Al-Kelaby KK. A Review on CRISPR/Cas9 as a Novel Technique for Cancer Therapy. Cell Ther Transplant 2022; 11(3-4): 10-24.
Summary
Cancer is a disorder that, basically, occurs as a result of genetic and epigenetic abnormalities. It's one of the leading causes of death in the globe, and it's still a major social and economic problem. According to statistics, over 10 million people die with malignancies, and cancer rates are expected to increase by 50% in the next ten years, culminating in approximately 15 million deaths. Single or multiple gene mutations, chromosomal abnormalities may cause cancer. Although numerous treatment options are used to treat cancer, they are still insufficient against malignant diseases. Therefore, a variety of novel strategies for early cancer therapy are examined. One of the most recent and potentially effective technologies that has been used in last years for genetic modification and cancer therapy is Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-asso-
ciated protein-9 (Cas9), a unique RNA domain-containing endonuclease-based genome engineering technology. In simple words, CRISPR/Cas9 has been derived from a bacterial defensive mechanism against viral infection. Recently, this approach has proved its usefulness in cancer therapy and gene editing. In general, this report presents a review of this key technology and its components. Specifically, in this work, we address the probable prospective uses and recent breakthroughs of CRISPR/Cas9 technology in cancer treatment, as well as the problems that can be encountered during clinical investigations. In this regard, we intend to contribute to optimizing work on CRISPR/Cas9 as well as to focus on the probable future paths of this technology.
Keywords
CRISP, Cas9, modern technology, 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-radi-ation 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 mediterra-nei, 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, CRIS-PR 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]
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 nu-clease 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 crR-NA, tracrRNA, and Cas9. Artificially, tracrRNA and crRNA can be turned into sgRNA, which guides Cas9 to the target region [24].
CRISPR class Type Subtypes Associated Protein Target General Characteristics
1 I I- A, B, C, D, E, F and U Casi, 2, 3, 4, 5, 6, 7, 8, 10 DNA Requires a PAM sequence.
III III-A, B, C, D Casi, 2, 5, 6, 7, 10, Csm, Cmr RNA and DNA - Make single-stranded nicks for both RNA and DNA targets. - A PAM repeat is not needed.
IV None characterized Casi, 5, 7 DNA Many preserved Cas genes are missing, as well as a CRISPR array.
2 II II- A, B, C Casi, 2, 4, 9, RNase 111 DNA - Both tracrRNA and crRNA are needed. - Makes double-stranded nicks in the target DNA. - A PAM repeat is not needed.
V V- A, B, C Cas2, 4, 12 DNA - Makes double-stranded fractures in the target DNA. - A PAM repeat is needed. - Both crRNA and tracrRNA are needed by the Type V-B effector (C2c1).
VI VI- A, B, C Cas1, 2, 13 RNA - Makes single-stranded nicks in target RNAs. - A PFS is needed.
CRISPR Protein Function
Casi DNA nuclease
Cas2 RNA nuclease
Cas3 DNA nuclease and helicase
Cas4 DNA nuclease
Cas5 Ribonuclease responsible for converting pre-crRNA to mature crRNA.
Cas6 Ribonuclease responsible for converting pre-crRNA to mature crRNA.
Cas7 It has an RNA identification motif and joins crRNA, which is usually found in multiple copies.
Cas8 Big subunit of effector component in type I
Cas9 DNA nuclease
CasiO Big subunit of effector component in type III
Cas12 (Cpfi) DNA nuclease, crRNA processing
Cas13 (C2c2) RNA nuclease, crRNA processing
Csm, Cmr RNA nuclease and single-stranded DNA
RNase III Processes tracrRNA and promotes with crRNA maturation
Abbreviation: PAM: Protospacer adjacent motif, crRNA: CRISPR RNAs, tracrRNA: Trans-activating crisper RNA.
Table 2. Proteins in CRISPR system [8, 21]
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.
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.
Table 3. The three phases of CRISPR/Cas9 mechanism [27, 28, 29]
Adaptation Biogenesis Interference
When a virus infects a microbe, like a bacteria or archaea, the immune system captures and incorporates a piece of the invading virus's DNA into its own genome in the shape of a "spacer" at the CRISPR locus. Subsequently, it acts as a virus's memory, allowing microbes to program their defenses against related phages in the future. As a result, these spacers are used to destroy viral particles as part of their defensive response [27]. In this phase, the CRISPR sequence is transcribed, resulting in a long precursor CRISPR-RNA (pre-crRNA). This pre-crRNA is a long transcript with a spacer and a repeat chain, which is processed further with another type of RNA called tracrR-NA (trans-activating crRNA) to form the crRNA. This crRNA contains the invader's genome code, which is later used to signal the Cas protein to cut off the phage or plasmid DNA [26, 28] Interference is the final stage of the CRISPR mechanism. It includes cutting phage genetic material with the aid of the crRNA/tracrRNA duplex and the Cas9 molecule [29]
Nonhomologous end joining (NHEJ) Homology directed repair (HDR)
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.
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.
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
Table 4. Features of various CRISPR/Cas9 genome editing strategies
Strategies Advantages Disadvantages Article
sgRNA and Cas9-mRNA Low off-target effects, rapid onset, and transitory expression Poor stability
sgRNA and Cas9 protein Rapid onset, short duration, low offtarget effects, and high efficiency Endotoxin contamination and high prices [36, 37]
plasmid-based CRISPR-Cas9 system Good stability and low price Inefficiency, integration issue, a late onset
Study type Type Carried substance Cell Line Action Article
In vitro Electroporation CRISPR/Cas9 plasmid Human osteosarcoma U2OS cells repressed CDK11 expression [41]
Human myeloid leukemia KBM5 cells repaired ASXL1 gene expression [42]
Membrane deformation • Cas9 and sgRNA EGFP • Cas9 and sgRNA-AAVSl • Cas9 and sgRNA-NUAK2 • MDA-MB231 cells • MCF7 cells • Human cervical HeLa cells • EGFP knockout • Split at the AAVS1 locus with indels • NUAK2 indels [43]
Ultrasound- propelled nanomotors Cas9/sgRNA complex Murine melanoma B16F10 cells GFP knockout [45]
lance array nano injection (LAN) CRISPR/Cas9 plasmid GFP+/FRT HeLa cell Disruption of EGFP gene [46]
Ex vivo Electroporation CRISPR/Cas9 plasmid Human primary T cell PD-1 gene knockout [47]
Cas9/sgRNA complex Human primary B cell secretion of therapeutic proteins [48]
Cas9/sgRNA complex Human primary NK cell TGFBR2 and HPRT1 genes knockout [49]
In vivo Hydrodynamic injection CRISPR/Cas9 plasmid M-TgHBV mouse model Remove two open reading frames (ORFs) of HBV reproduction template [44]
Table 5. CRISPR/Cas9 delivery by means of physical methods
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.
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
Table 6. Different viral vectors used for the in vitro CRISPR/Cas9 delivery
Study type Virus Target gene Cell line Action Article
In vitro Adeno-associat-ed virus (AAV) HPV-E6 protein HCC cell lines (HeLa, HCS-2, SKG) Increase p53 expression, apoptosis, and inhibited development [52]
Lentivirus E6- or E7-specific sgRNA HCC Hela cell HPV E6 or E7 inhibition in HeLa cell [53]
Ex vivo AAV VEGFR2 HRE-Cells Inhibition of VEGFR2 [54]
Lentivirus MUC18 Human primary nasal airway epithelial cells MUC18 knockout [55]
In vivo Lentivirus HIF-1a SMMC-7721 xenograft HCC model HIF-1a knockout [56]
Adenovirus EGFR gene H1975 cells EGFR gene disruption [57]
Table 7. CRISPR/Cas9 delivery via non-viral vectors
Study type Type Target Cell line Action Article
In vitro X-tremeGENE HP DNA Transfection Reagent E7 gene Human cervical cancer Siha cells Increase apoptosis and inhibit the viability of cells [59]
flexible den-drimer MASPIN gene Human breast cancer MCF-7 cells Activation of MASPIN suppressor [60]
Ex vivo LPEI Mesenchymal stem cells (MSC) Mesenchymal stem cells Highly efficient cytokine overexpression [61]
In vivo PLNP Plkl gene Subcutaneous A375-derived melanoma Reduce Plkl protein expression; decrease tumor progress. [62]
SKOV3 cell-derived exosomes PARP-1 gene SKOV3 cell PARP-1 suppression; increase the chemo-sensitivity to cisplatin [63]
Folate-modified liposomes DNMT1 gene SKOV3 cell Reduce DNMT1 expression; decrease tumor growth [64]
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.
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 on-cogenes 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, cytoreduc-tive 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 hepatocellu-lar 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 glycopro-tein 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 on-cogene [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
Cancer Type Target Gene Cell Line CRISPR effect Study type Vector Action Gene function Article
Lung cancer CD 38 A549 Knockout In vitro & In vivo NV Decrease cell formation, invasion and metastasis; inhibit tumor growth OG [69]
KRAS A549 Knock-down In vitro & In vivo AV Proliferation of cancer cells is reduced. OG [70]
PKP1 SqCLC Knockout In vitro & In vivo LV Sufficiently reduce cell reproduction and cell dissemination TS [72]
MFN2 A549 cells Knockout In vitro & In vivo NV Enhance colony formation, cell activity, and metastasis by up-regulation of mTORC2/Akt pathway TS [71]
Breast cancer APOBEC3G MCF10A Knockout In vitro Plasmid Repress cell proliferation OG [78]
RLIP BC cells Knock-down In vitro & In vivo LV Reduce the BC cell proliferation OG [80]
PSMD12 MDA-MB-231 Knock-down In vivo LV Decreased development and emigration of breast cells OG [81]
CDK4 MDA-MB 231 cells Knockout In vitro Plasmid Cellular viability, the ability of cells to replicate and growth, and mobility are generally reduced OG [79]
Cdh1 ILC- initiating cells Knock-in In vitro & In vivo LV In particular, stimulate PTEN gene disruption TS [100]
Colorectal DRD2 HCT116 Knockout In vitro LV Decreased activity of anticancer 0NC201 TS [83]
ATF3 HCT116 Knockout In vitro LV Decreased invasion and metastasis of tumors OG [101]
AMPKa1 HCT116 Knockout In vitro & In vivo LV Activating AMPK signaling by targeting PP2A reduces colorectal cancer cells TS [102]
NAT1 HT-29 Knockout In vitro NV Under glucose deprivation, increased apoptosis and reduced cell production OG [103]
RBX2 HCT116, HT29 Knockout In vitro & In vivo NV Decreased cell colony forming and migration OG [84]
Abbreviations: AV: Adenovirus, CD8: Cluster of differentiation 38, CDK4: Cyclin-dependent kinase 4, LV: Lentivirus, TS: Tumor suppressor, OG: Oncogene, NV: Not available.
Cancer Type Target Gene Cell Line CRISPR effect Study type Vector Action Gene function Article
Liver cancer NCOA5 Human HCC cells Knockout In vitro LV Decreased cell proliferation and migration, reduced tumor growth and EMT OG [87]
DEPDC5 Human HCC cells Knockout In vitro & In vivo LV Increased cellular ROS resistance, decreased PFS and OS in patients TS [104]
PTEN Hep3B Knockout In vitro LV Increasing the invasion capacity of Hep3B cells TS [89]
Sphkl L02 cells Knockout In vitro NV Reduced proliferation and tumor growth of liver cancer OG [88]
Prostate cancer Aktl/2 CWR22rv1 cells Knockout In vitro & In vivo LV Decreased metastasis of prostate cancer OG [105]
TP53 PC-3 cells Knockout In vitro Plasmid Reduce cell proliferation and promote apoptosis. TS [106]
PTEN APTEN Knockout In vivo NV Increased cell growth and colony-forming ability TS [91]
Bladder cancer UCA1 5637 and T24 cells Knock-down In vitro & In vivo NV Suppressed cell proliferation, migration and invasion OG [93]
MTHFD2 EJ cells Knock-down In vitro NV Suppressed cell growth, reproduction, and emigration OG [107]
Cervical cancer E6 & E7 SiHa & HeLa cells Knockout In vitro & In vivo NV Decreased cell growth and increased cell apoptosis OG [96]
PTEN EC cells Knockout In vitro & In vivo LV Produced PARP/PI3K inhibition in cells, resulting in DNA damage increase and repair defects TS [74]
Ovarian cancer BIRC 5 SKOV3and OVCAR3 cells Knockout In vitro LV Dramatically decreased cell proliferation, and invasion, and prompted cell apoptosis. OG [99]
miR-21 SKOV3 and OVCAR3 cells Knock-down In vitro LV The EMT in ovarian cancer cells is suppressed OG [108]
Thyroid cancer MFN2 Cal62 and HTH83 cells Knockout In vitro & In vivo Electro-poration MFN2 overexpression inhibited EMT, with reduced thyroid cancer cell proliferation and emigration TS [109]
Ku80 K1 and B-CPAP cells Knock-down In vitro LV Excessive apoptosis and decreased growth, invasion, and colony formation OG [110]
Melanoma PTGS2 B16F10 cell Knock-down In vitro & In vivo NV Decreased tumor growth and metastasis OG [111]
Pbrml B16F10 cell Knockout In vivo LV Increased response to T-cell immu-notherapy and decreased resistance OG [112]
Benefits and disadvantages
In terms of simplicity, flexibility, and low price, the CRIS-PR/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
Abbreviation Meaning
AAV Adeno-associated virus
ADC Adenocarcinomas
AV Adenovirus
BC Breast cancer
BIRC5 Baculoviral IAP Repeat Containing 5
C2c1 type V-B CRISPR-Cas system
Cas9 (CRISPR)-associated protein-9
CD38 Cluster of differentiation 38
CDK4 Cyclin-dependent kinase 4
CRISPR Clustered Regularly Interspaced Short Palindromic Repeat
crRNAs CRISPR RNAs
DSB Double-strand break
EMT Mesenchymal transition
HCC Hepatocellular carcinoma
HCC cell lines Hepatocellular carcinoma cell lines
HDR Homology directed repair
HPV Human papillomavirus
LV Lentivirus
MMPs Matrix metalloproteinase
NHEJ Non-homologous end joining
OG Oncogene
ORFs Open reading frames
PAM Protospacer adjacent motif
pRB Retinoblastoma family proteins
pre-crRNA Precursor CRISPR-RNA
PSA Prostate-Specific Antigen
PTEN Phosphatase and Tension Homolog
RNAi RNA interference
RV Retrovirus
sgRNA A single guide RNA
SpCas9 Streptococcus pyogenes Cas9
tracrRNA Trans-activating crisper RNA
TS Tumor suppressor
ZFNs Zinc-finger nucleases
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I CRISPR/Cas9 как новая технология терапии рака
Али А. Салем ', Халида К. Аль-Келаби 2
1 Лаборатория медицинских технологий и патологического анализа, Госпиталь Аль-Хаким, Наджаф, Ирак
2 Департамент клинических и лабораторных исследований, Факультет фармации, Университет Куфа, Наджаф, Ирак
Резюме
Рак является заболеванием, обусловленным в основном, генетическими и эпигенетическими нарушениями. Эти заболевания - одна из ведущих причин смерти в мире и представляет собой крупную социальную и экономическую проблему. Согласно статистическим данным, более 10 миллионов человек погибают от злокачественных опухолей, и ожидается 50%-ное повышение частоты их возникновения в следующие 10 лет, приводя к 15 миллионам смертельных исходов. Единичные или множественные генные мутации, хромосомные аберрации могут вызывать раковые заболевания. Хотя для лечения рака используют многочисленные варианты лечения, они все же недостаточны против этих заболеваний. Поэтому изучается ряд новых стратегий ранней терапии злокачественных опухолей. Одной из наиболее современных и потенциально эффективных технологий, применяемых в последние годы для генных модификаций и онкотерапии является система Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-ассоциированного протеина-9 (Cas9) - уникальная
технология геномной инженерии, основанная на применении уникальной РНК-содержащей эндону-клеазы. Исходно, CRISPR/Cas9 возникла из противовирусного механизма защиты бактерий от вирусных инфекций. В настоящее время этот подход оказался полезным в лечении рака и генном редактировании. В целом, это сообщение является обзором этой ключевой технологии и ее компонентов. В частности, в этой работе мы касаемся возможных перспективных приложений и нынешних прорывов в технологии CRISPR/Cas9 для лечения рака, а также тех проблем, которые могут возникнуть при клинических исследованиях. В этом отношении мы намерены сделать вклад в оптимизацию работ по CRISPR/Cas9, а также сделать акцент на возможные будущие пути развития этой технологии.
Ключевые слова
CRISP, Cas9, современная технология, рак, терапия.
