key: cord-0684897-rp4i7s64
authors: Hazafa, Abu; Mumtaz, Muhammad; Farooq, Muhammad Fras; Bilal, Shahid; Chaudhry, Sundas Nasir; Firdous, Musfira; Naeem, Huma; Ullah, Muhammad Obaid; Yameen, Muhammad; Mukhtiar, Muhammad Shahid; Zafar, Fatima
title: CRISPR/Cas9: A powerful genome editing technique for the treatment of cancer cells with present challenges and future directions
date: 2020-10-05
journal: Life Sci
DOI: 10.1016/j.lfs.2020.118525
sha: a1032abf832d11f87b417802348d3e5ea42a074a
doc_id: 684897
cord_uid: rp4i7s64

Cancer is one of the most leading causes of death and a major public health problem, universally. According to accumulated data, every year, approximately 8.5 million people died because of the lethality of cancer. Recently, a new RNA domain-containing endonuclease-based genome engineering technology, namely the clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein-9 (Cas9) have been proved as a powerful technique in the treatment of cancer due to its multifunctional properties including high specificity, accuracy, time reducing and cost-effective strategies with minimum off-target effects. The present review investigates the overview of recent studies on the newly developed genome-editing strategy, CRISPR/Cas9, as an excellent pre-clinical therapeutic option in the reduction and identification of new tumor target genes in the solid tumors. Based on accumulated data, we revealed that CRISPR/Cas9 significantly inhibited the robust tumor cell growth (breast, lung, liver, colorectal, and prostate) by targeting the oncogenes, tumor-suppressive genes, genes associated to therapies by inhibitors, and genes associated to chemotherapies drug resistance, and suggested that CRISPR/Cas9 could be a potential therapeutic target in inhibiting the tumor cell growth by suppressing the cell-proliferation, metastasis, invasion and inducing the apoptosis during the treatment of malignancies in the near future. The present review also discussed the current challenges, and barriers and proposed future recommendations for a better understanding.

Globally cancer is considered one of the major leading causes of death. In 2018, approximately 9.6 million people had died because of cancer. Cancer is characterized by the heterogeneity and accumulation of different genetic mutations and alternations, including modification in epigenetic factors (DNMT1), variation in genes like multidrug resistance genes (MDR1/Pgp and ABC), activation in tumor suppressor (TP53) and oncogenes (RAS) [1] . The tumor heterogeneity is categorized into intratumoral and intertumoral heterogeneity. Intratumoral heterogeneity mainly occurs due to the unequal circulation of genetically distinct tumor subpopulations across single or different disease sites. In contrast, intertumoral heterogeneity is associated with patients' specific factors such as patients-somatic profile and germline alternations. Intertumoral heterogeneity also circulates between patients harboring tumors of the same histological type [2] .

However, genetic instability generated several critical substrates for tumor heterogeneity that are maintained by specific therapeutic processes. Resultingly, this heterogeneity triggers the sitespecific response and ultimately provides the seeds for the development of cancer resistance [3] .

During the past few decades, different therapies, including immunotherapy, targeted antibodies, hormone therapy, chemotherapy, targeted drug therapy, stem cell transplant, and surgery have been introduced into the medical field for the betterment of prognosis in cancer patients, which significantly eradicate or condense the cancerous cells and upsurges the maximum lifespan of about only five years [4, 5] . Even after these therapies, cancer continues to evolve and ultimately give rise to molecular heterogeneity attributed to various molecular events like epigenetic, genetic, phenotypic, and transcriptomic alternations. However, due to their several limitations, including toxicity, high cost, and adverse effects (affect the healthy cells and caused the partial or complete loss of organ functions) these therapies did not improve as much survival of patients. transcription activator-like effector nucleases (TALENs) and zinc-finger nucleases (ZFNs) have been extensively used for DNA repair by targeting the gene of interest. The efficiency of these techniques entirely depends on specificity and affinity of nucleases [17] . Traditionally, TALENs and ZFNs editing technologies were used to target several oncogenes like human Papillomavirus oncogenes. A study showed that Compo-Zr ZFNs significantly targeted the E6 gene in HPV-16 cell lines with the editing activity of 50% but showed less activity in CaSki and SiHa cervical cell lines. Similarly, TALENs effectively edited the E7 gene in SiHa cells but did not show the desirable activity in other cell lines [16] . Recently, a study revealed that ZFNs significantly removed the HIV-1 pro-viral DNA and gave a new hope to target the other viral genomes in single or combine form (TALEN/ZFN) [18] . Although these genome editing techniques showed somehow better results, but these both techniques exert off-target effects and also very time taking and less efficient techniques. So, there was a need to develop a highly efficient and accurate technique to compensate the old ones [19] .

Based on the emerging achievement of CRISPR/Cas9 in the molecular field, this technique has superiority over the other conventional genome editing techniques such as transcriptional activator-like effector nuclease (TALENs), and zinc finger nuclease (ZFNs) due to its limited off-target effects and less time-consuming target ability. The CRISPR-Cas9 only required the short complementary sequence of sg-RNA to target the DNA, which is a relatively cost-effective and much easier method than conventional tools (TALENs, ZFNs, and RNAi) to edit desired part of DNA [20, 21] . According to the accumulated data, the CRISPR/Cas9 system was initially identified by the prokaryotic (Escherichia coli) adaptive immune system, which is considered good anti-phages and antiviruses system [22] . The CRISPR/Cas9 comprises three distinct prime function in the specificity, efficiency, and accuracy of the CRISPR/Cas9 tool. The first [10] [11] [12] nucleotides of sgRNA at 3ˊ end next to a proto-spacer adjacent motif (PAM; see glossary)

known as seed sequence directed the Cas9 protein in the genome editing performance by binding to the target sites [29] . The recent evidence also suggested that the truncated sgRNA with less than 20 nucleotides effectively minimized the off-target effects of about 5000 folds without losing efficiency [22] . In addition, another study also reported that the development of about 5 base pairs in sgRNA duplex excellently enhanced the knockout proficiency of CRISPR/Cas9 [23] . The Cas9 is a CRISPR class 2 isolated (Streptococcus pyogenes) protein guided by RNAguided nuclease and responsible for DNA double-strand destruction [30] . The Cas9 protein contains two lobes, including alpha and nuclease lobes. The nuclease lobe consists of two extra domains, including RuvC and HNH domain. The RuvC (retrovirus integrase protein) cuts the non-targeted DNA double strands, whereas the HNH cleaved the specifically targeted strand of DNA. Both of these domains usually formed a DSB (double-strand break), but due to mutation at D10A and H840A in RuvC and HNH domains respectively, results in deactivation of Cas9 (dCas9) [31, 32] .

In the past few years, due to the specificity, efficiency, and accuracy of CRISPR/Cas9, this genome editing tool is excessively used in the research laboratories, which helped the researchers to detect the role of different oncogenes in the cancer cells [33] . The tumor-suppressor genes (ETS1, CPEB2, BRCA1, PGC1a, TP53, MiR-1205, and SOX15) comprise their significant role in tumorigenesis, which can suppress cell mitigation, cell proliferation, enhance cell differentiation, J o u r n a l P r e -p r o o f Journal Pre-proof and downregulate the cancer progression [34, 35] . The CRISPR/Cas9 technology targeted these tumor-suppressor genes to inhibit or reduce the tumorigenesis by restoring the activities of tumor-suppressor genes [36] . The CRISPR/Cas9 barcoding technology is used to identify the cancer heterogeneity and its therapeutic targets against different cancer cells [37] . Herein, we discussed the novel molecular therapeutic effects of CRISPR/Cas9 technology in the treatment of most accruing solid cancers, including breast, lung, colorectal, prostate, and liver cancers.

Breast cancer is a common leading cause of death among women, comprising 30% of all new diagnosis tumors, globally. The molecular profile of breast cancer reveals the heterogeneity and existence of several cancer subtypes with clinical consequences [38] . One of the major challenges of the breast tumor is the complexity of cancer, as cancer is not the form of a single cell; instead, it is composed of diverse cells including the appearances of progenitor or stem cells [39] . The breast epithelium is composed of four different subtypes based on the estrogen receptor (ER) expression, including luminal A, luminal B, Her2-enriched, and triple-negative breast cancer (TNBC) [40] . The luminal subtypes (ER-positive) are the most lethal and common forms of breast cancer of about 70% with resistance (30%) to endocrine therapies in affected patients [41] . However, a reduction of breast cancer is crucial, particularly in re-occurrence. Recently, the CRISPR/Cas9 has proved a new, revolutionary, and an effective therapeutic target in the chemotherapeutic drugs, including docetaxel, gemcitabine, and doxorubicin in the 2D tumor culture model as compared to 3D tumor-chip model. Similarly, another study revealed that the non-viral delivery of sgRNA (a component of CRISPR) into HCC1806 and MCF10A cell lines significantly knockout both alleles of APOBEC3G genes in the treatment of breast cancer via blocking the conversion of the G1 to S phase in the cell cycle and inhibiting the cell proliferation [43] . Yang and his research group exposed that CRISPR/Cas9 technology successfully deleted the individual and co-knockout of CXCR7 and CXCR4 genes (both alleles) in the TNBC cell line (MDA-MB-231) during the treatment of breast cancer by suppressing the tumor cell proliferation, invasion, tumor growth, and invasion [44] .

Hannafon and co-workers reported that the lentiviral delivery of CRISPR/Cas9 (sgRNA) into the xenografted nude mice (see glossary) significantly knockout both alleles of miRNA (miR-27b and miR-23b genes on chromosome 9) in the MCF7 mammary tumor cells to reduce tumor growth by inducing the cell-proliferation and mitigation at the endpoint by using a ki-67 marker (cell cycle marker) [45] . Similarly, another study exposed that in vivo lentiviral delivery of CRISPR/Cas-mediated somatic genome in mice successfully reduced the breast tumor susceptibility in triple-negative breast cancer (TNBC; see glossary) cell lines of p53 and K14Cre [46] . Alvares-Fernández et al. [47] stated that during the in vivo study, the CRISPR-based interruption of MASTL kinase effectively condensed the cell-proliferation in humans mammary tumor cell lines and showed its highest expression in cancer cells as a contrast to normal cells.

However, the suppression of MASTL could be an excellent target option in the treatment of breast cancer. Hence, the preclinical studies of breast cancer with CRISPR/Cas9 lead to identifying many new proteins and genes that could bring significant treatment outcomes in breast tumor suppression. 

Liver cancer is aligned as the fourth leading cause of death among both women and men of all cancer-related mortalities, globally. Unlike BRAF mutations in melanoma and EFGR mutation in lung tumors, the significant mutations in a liver tumor are still undruggable. Different therapies, including inhibitors (sorafenib and THZ1) are recently used in the treatment of liver cancer, but they are limited to drug resistance and molecular targets [48] . Currently, the new emerging genome editing technique, namely CRISPR/Cas9, is showing admirable results in the identification of new genes (CDK7, CD44, and Nf1) as therapeutic targets in the treatment of liver tumor [49] . The effect of CRISPR/Cas9 in the different liver tumor's oncogenes, tumorsuppressor genes, and drug resistance genes are presented in Fig. 2 .

Wang et al. [50] reported that an in vitro lentiviral delivery of sgRNA (a component of the CRISPR system) into two cell lines of liver, including Huh7 and Hep38, identified a cyclindependent kinase namely CDK7 (see glossary) as hits during the screening by next-generation sequence. They also revealed that CRISPR/Cas9-mediated CDK7 significantly affected the cellproliferation activity of Huh7 and Hep38 cell lines in both long-term colony formation assays and short-term IncuCyte cell-proliferation assay. Hence, CDK7 is identified as an important therapeutic target in the treatment of hepatocellular carcinoma by the CRISPR/Cas9 genomeediting tool. Similarly, Han and co-workers [51] the liver tumor treatment. They exposed that inhibition of ERK2 (MAPK1) alerted several liver tumor cell lines to sorafenib and suggested that patients with high basal p-ERK (>30% of all liver tumors) are most likely to assistance from such synergistic treatment. Therefore, the inhibition of kinases, including ERK and MEK could be a potent therapeutic target in the treatment of liver carcinoma. In addition, Song and his research group potently revealed that Plxnb1, B9d1, Flrt 2, and Nf1 genes are the liver tumor-suppressor genes using CRISPR/Cas9 technology. They exposed that the lentiviral delivery of sgRNA and Cas9 into p53 and MYC-Cas9 liver cells successfully knockout the NF1 gene in the mouse liver model. They also reported that the inactivation or suppression of these genes (previously thought they are not related to a liver tumor) by CRISPR/Cas9 leads to the acceleration of liver tumors in mice and upregulated the liver progenitor cell markers namely SOX9 and HMGA2. However, for the first time, they founded that Plxnb1, B9d1, Flrt 2, and Nf1 are the liver tumor-suppressive genes by using CRISPR/Cas9 technology [53] . However, the simultaneous inhibition of SOX9 and HMGA2 might be a potent therapeutic target in the reduction of liver carcinoma. Similarly, Zhu et al. [54] deleted the IncBRM gene in the Huh7 and Hep3B cell lines of a liver tumor by using CRISPR/Cas9 technology and reported that knockout of IncBRM gene impaired the serial sphere formation without affecting the expression of neighboring genes, which suggested that IncBRM gene perform its role in trans. They suggested that the IncBRM gene could also serve as a biomarker (see glossary) for the analysis and drug discovery against liver cancer. associated proto-oncogenes. Similarly, APC, MCC, RB, CDKN2A, TP53, and NM23 are known as cancer-suppressor genes in the lung tumor (see Fig. 2 ). A recent study reported that both oncogene overexpression and gene mutations are involved in lung tumor growth. However, the utilization of recently developed gene-editing technology (CRISPR/Cas9) could significantly remove lung cancer by targeting the tumor-suppressive genes, genes associated with therapies by inhibitors, oncogenes, and genes associated with chemotherapy drug resistance [35, 56] .

Most recently, Lu and co-workers performed a clinical trial-based experiment (NCT02793856) to investigate the feasibility and safety of CRISPR/Cas9-mediated T-cell therapy in the treatment of lung cancer by targeting the PD-1 gene. They revealed that the median overall survival rate was 42.6 weeks (CI: 95%, 10.3-74.9 weeks), with 0.05% off-target effects in 18 patients accessed by next-generation sequence. They also claimed that the lifespan of gene-editing T-cell was short, which suggested limited off-target effects, and proved that CRISPR/Cas9 genome editing technology is clinically feasible for lung tumor treatment [57] . However, in the future, more clinical trials should be performed to treat lung cancer. Similarly, Perumal et al. [58] experimented on Slug/Snail non-small lung cancer cells (NSLC; see glossary) by CRISPR/Cas9 genome editing tool. They reported that non-viral delivery (plasmid) of Cas9 into NCI-H460 and A549 cell lines of lung carcinoma effectively knockout the PTEN gene by using the Epithelialmesenchymal transition (EMT) marker. They revealed that the knockout of the PTEN gene showed high invasion, metastasis, and cancer cell growth than a PTEN wild type by promoting the expression of phosphorylation of GSK-3β and AKT pathway while suppressing the βcatenin. Hence, the inactivation of the PTEN gene could be contributing to the EMT marker by regulating the translocation of β-catenin (see glossary) in the lung carcinoma treatment.

A study revealed that the knockout of AMPK (α1 and α2) signaling pathway by CRISPR/Cas9 technology in murine lung tumor (KRAS gene) significantly reduced the size of lung carcinoma cells [59] . Similarly, Cheung et al. [60] experimented using CRISPR/Cas9 genome editing technology to target the point mutation in lung tumors (EGFR). They reported that CRISPR/Cas9 involved in specific genome targets in L858R mutant cells and suggested that this novel strategy could be used to target the specific area of lung cancer (15 to 35%) with T>G, A>G, and C>G point mutations. Moreover, the lentiviral delivery of Cas9 into A549 cell lines effectively knock out the MYC gene, which resultingly revealed that the silencing of long non-coding RNA EPIC1

(lnc-EPICI) by CRISPR/Cas9 that significantly reduced the cell-proliferation, tumor size, and cancer cell survival [61] . Another study showed that the knockout of p107 and closely related p130 genes by CRISPR/Cas9 strategy effectively enhance the cancer progression (see glossary).

They also suggested that this novel strategy could be feasible and safe to tumor suppressor genes in small cell lung cancer (SCLC; type of lung cancer), which might be anticipated in the development of a novel potential drug against the progression of lung carcinomas [62] .

Because of high-prevalence, genetic physiognomies (MSI, BRAF, NRAS, and KRAS), and clinicpathogenesis of colorectal cancer, it has attained the much attention during the last couple of decades, which imitated the preliminary step toward precision medicine [63, 64] .

Conventionally, several anti-tumor agents, including anti-VEGF and anti-EGFR have been used against colorectal cancer, but they are limited to restrained efficiency and metastatic settings [65] . However, most recently, the CRISPR/Cas9 has proven as a revolutionary technique in the molecular biology and oncology research field due to its multifunctional strategies, including high accuracy in genome editing and high specificity [66] . The effect of CRISPR/Cas9 in the 

Prostate cancer is ranked as the second most leading cause of cancer-related deaths among men, a commonly diagnosed tumor in European men. The development of tumor size leads to the many genomic alternations, including a mutation in function and structure of the genome, and these alternations depend on the tumor size and cancer-cell types [71] . Conventionally different strategies, including chemotherapies, have been used against these alternations, but they remain poorly characterized [72] . However, the CRISPR/Cas9 proves as a potential target against prostate cancer due to its limited off-target effects and high accuracy [10] .

Most recently, Fenner and his research group [73] demonstrated that the knockout of ER-β gene by CRISPR/Cas9 in mice genome effectively involved in regulating the prostate tumor size and act as a tumor-suppressor gene, that suggested its (ER-β gene) role in the prostate cancer which was previously not reported. Hence, the study claimed the role of the ER-β gene as a repressor gene in the prostate tumor androgen receptor, which could be led to the discovery of novel and better drugs against the lethality of prostate tumors. Batir et al. [74] The applications of CRISPR/Cas9 in the treatment of different solid cancer cells, including breast, lung, liver, colorectal, and prostate are presented in Table 1 .

Besides to Cas9 protein, researchers are also constantly trying to develop the new members of the CRISPR system. Recently some CRISPR-associated (Cas) proteins such as Cas12a, Cas12b, Cas13a, and Cas13b proteins have been developed and studied to minimize the off-target effects and enhance genome editing efficiency of CRISPR system. Cas12a is a simpler and smaller RNA-guided endonuclease protein as compare to Cas9 [19] . The type V of CRISPR/Cas system (Cas12a and Cas12b) represent some unique characteristics that make them distinguish from J o u r n a l P r e -p r o o f Cas9. The type V proteins members possess the RuvC nuclease domain instead HNH domain to identify the site-specific T-rich PAM (5') sequence just upstream to target region. In addition, Cas12 nucleases built staggered DSBs distal to PAM sequence unlike to Cas9 that helped to generate the unique knock-in strategies for genome editing through HNEJ mechanism [78] .

A study revealed that Cas12a (also known as Cpf1) showed less off-target effects and could be The emerging evidence revealed that due to small of Cas12b, it could easily be delivered to the target site by viral vector and Cas12b produced a long sticky end after double-strand DNA editing which could be expected to enhance the efficiency of ligation after DNA break [81] . It is also noteworthy that Cas12b is very sensitive and effective to single-base mismatch of about 20 nucleotides that make it highly specific CRISPR/Cas12b system [82] .

Like to type V CRISPR/Cas system, the type VI also possesses the two important Cas nucleases such as Cas13a and Cas13b. These two proteins also carried some unique features, unlike Cas9 like these proteins cleaved the ssRNA instead of DNA because they have no catalytical DNA J o u r n a l P r e -p r o o f domain. Recently, studies stated that Cas13 proteins possess two identified highly conserved prokaryotic and eukaryotic nucleotide-binding domains containing specific RNA cleavage site [78, 83] . Zhao et al. [84] stated that bacterial Cas13a and CRISPR-RNA (crRNA) showed the successful knockout effects in mutant KRAS mRNA expression with the knockout efficiency of about 94%. They also observed that in vitro delivery of CRISPR/Cas13a effectively knock down the KRAS-G12D mRNA expression (70%) via inducing the apoptosis and inhibiting the tumour cell proliferation. Fan et al. [85] reported that liposomes delivery of CRISPR/Cas13a into BCa cells significantly inhibited the bladder tumour genes and observed that codon-optimized LwaCas13a plays an important role in the target site regulation. Similarly, Li and co-workers [86] designed the dm 6 ACRISPR by fusing the ALKBH 5 to C or N-terminal of inactive Cas13b with the help of six amino acids sequence such as GSGGGG to target the RNA demethylation.

They found that dm 6 ACRISPR successfully targeted the N 6 -methyladenosine demethylation in cytochrome b 5 form A (CYB 5 A) mRNA which was responsible for coding the oncoproteins including EGFR and suggested that dm 6 ACRISPR could be a potential target to inhibit the tumor cell proliferation. Although the some recently emerged Cas protein systems such as Cas12 and Cas13 showed the remarkable results in the tumor field, but still extensive research is required to properly understand the mechanism of action, efficiency and off-target effects of these nucleases.

After the discovery of the CRISPR/cas9 genome editing technique, the delivery of CRISPRcomponents remains a major challenge. According to emerging evidence, the viral vector has been used over the decades in reverse genetic engineering (see glossary) to deliver the oncogene to the tissue to induce tumors. Initially, for this purpose, different viral-vectors have been used, but among all, the recombinant retroviral vectors got considerable attention due to their J o u r n a l P r e -p r o o f minimum off-target effects, high specificity, and accuracy to target cell genome. Since the repurposing of the CRISPR system into biotechnology, the viral vectors were rapidly developed for the delivery of sgRNAs or Cas9 to the target cell genome [87] . Initially, the lentiviral libraries were pooled to deliver the CRISPR components due to the substantial size of the Cas9 gene (4.1 kb), which could efficiently fit into the lentivirus [88, 89] . Nevertheless, several major concerns, including mutagenesis and immunogenicity, are associated with the use of adenoviral and lentiviral vectors as the Cas9 ribonucleoproteins delivery method (see Fig. 3 ) [90] .

Besides to viral vectors, the plasmid DNA is known as another alternative method for the delivery of CRISPR components as sgRNA and Cas9 for the genome editing approach in vitro and in vivo studies. In recent years the standard molecular cloning techniques have been used to develop the plasmid, including cell-type-specific transcriptional targeting elements, diverse Cas9 structures, and different sgRNAs [91, 92] . But the emerging evidence associated with the several problems with the plasmid DNA delivery system, including plasmid DNA, enhances the perseverance time of CRISPR RNPs inside the cell, which results in increased off-target ability.

Besides, the plasmid DNA persuades the mutagenesis effects when active promoter elements implanted into the host genome [93, 94] .

Although the viral vectors have been a significant option in the delivery of CRISPR component, there is a need to develop vector with both clinical and pre-clinical studies delivery strategies with restricted payload size. One of the major concerns with adeno-associated viruses (AAV) is the packing of Cas9 into separate viral particles, which resultingly reduced the accuracy [95] .

However, to tackle this problem, the scientist has discovered another effective delivery method of CRISPR components known as non-viral delivery to target genome editing, as presented in 

Cancer is one of the leading causes of death and the most studied field during the past few decades due to its complexity, heterogeneity of the disease, and high epidemiology among humans. The multi-mutated and multigenic characteristics of cancer give them a unique heterogeneity ability that could exist differently in different cancer-affected patients with the same cancer type. However, this unique characteristic of heterogeneity triggered the major challenge for clinical rehabilitation [98] . The specific, accurate, and simple genome editing ability of CRISPR/Cas9 attained the special attention of the scientific community around the globe, especially during the last five years in the cancer biology field. The discovery of Cas9 nuclease has broadened the era of molecular biology, specifically to treat the multifunctional J o u r n a l P r e -p r o o f Journal Pre-proof genes of cancer. The modern researchers have been using the CRISPR/Cas9 technology, particularly in suppressing the oncogenes in mice models in terms of cost-effective, high specificity, accuracy, and time length without applying multifunctional colonies of mice [99] .

No doubt, the CRISPR/Cas9 proved as a revolutionary tool and showed remarkable results in the treatment of cancer cells, but there are also still a few challenges that should be improved on urgent bases. One of the major challenges during the synthesis of the CRISPR system is to reduce the off-target effects of Cas9 nuclease [100] . The Cas9 nuclease is a promising factor to control the off-target characteristics and undesirable adverse effects of the CRISPR system. To regulate the off-target challenge of Cas9, the researchers should investigate the different physical or chemical agents, but not limited to tetracycline and doxycycline, as a promising approach to develop the desired expression of Cas9. In addition, the various online tools are available for the assistance of researchers to design sgRNA with minimum off-target effects to promote specificity toward the cell genome editing [101, 102] . Recently few genome editing variants of the CRISPR system, including truncated sgRNA, Cas9 nickase, and interaction of Fok1 with dCas9, proved as a promising tool in the reduction of off-target effects [22, 103] .

In addition to minimizing the off-target effects, the in vivo delivery system of Cas9 should also extend its application to the cervix and stomach to locate and treat the more cancers. The enormous size of Cas9 is associated with packing problems, particularly in low immunogenic adeno-associated viral (AAV) vectors, which are excessively used in the in vitro and in vivo gene deliveries [104] . The most recent studies claim that the persistent binding of Cas9 nuclease with DSBs suppressing the entry of repair proteins to the target site, which resultingly plummeting the repair efficiency. This remains the rate-limited step in a Cas9-DSB complex in the genome editing in vivo [105] . However, the translocating RNA polymerase could be used to dislocate the J o u r n a l P r e -p r o o f Journal Pre-proof template bounded Cas9, only after RNA polymerase locates the DSBs from an exact direction [104, 106] . Moreover, there are still some oncogenic genomic variations, including chromothripsis, aneuploidy, and LINE-1-mediated genome mutation, that are difficult to treat with the current version of the CRISPR/Cas9 system. However, the mutagenic efficiency of CRISPR/Cas9 should be improved in the near future by designing the potent Cas9, develop efficient delivery methods with prevailing sgRNA [103, 107] .

In summary, the present review made a comprehensive discussion on the structure and function, 

This research received no specific grant from any funding agency in public, commercial or notfor-profit sectors. 

CRISPR-Cas9 therapeutics in cancer: promising strategies and present challenges

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Tumour heterogeneity and resistance to cancer therapies

Old dogs, new trick: classic cancer therapies activate cGAS

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TALEN based HPV-E7 editing triggers necrotic cell death in cervical cancer cells

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CRISPR/Cas9-An evolving biological tool kit for cancer biology and oncology

Crystal structure of Cas9 in complex with guide RNA and target DNA

Differential effects of estrogen receptor β isoforms on glioblastoma progression

SIRT4 acts as a tumor suppressor in gastric cancer by inhibiting cell proliferation, migration, and invasion

Applications of CRISPR/Cas9 Technology in the Treatment of Lung Cancer

MFN2 suppresses cancer progression through inhibition of mTORC2/Akt signaling

CRISPR-barcoding for intratumor genetic heterogeneity modeling and functional analysis of oncogenic driver mutations

Mammary stem cells and the differentiation hierarchy: current status and perspectives

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

Dependence receptor UNC5A restricts luminal to basal breast cancer plasticity and metastasis

CRISPR/Cas9-Mediated Mutagenesis to Validate the Synergy between PARP1

Inhibition and Chemotherapy in BRCA1-Mutated Breast Cancer Cells

Inactivation of APOBEC3G gene in breast cancer cells using the CRISPR/Cas9 system

Impact of CXCR4 and CXCR7 knockout by CRISPR/Cas9 on the function of triple-negative breast cancer cells

Ding, miR-23b and miR-27b are oncogenic microRNAs in breast cancer: evidence from a CRISPR/Cas9 deletion study

PO-333 Somatic engineering of mammary gland epithelial cells using CRISPR/Cas9 for rapid testing of breast cancer susceptibility genes in mouse models

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Liver cancer gene discovery using gene targeting, Sleeping Beauty, and CRISPR/Cas9, Seminars in liver disease

CRISPR/Cas9-mediated p53 and Pten dual mutation accelerates hepatocarcinogenesis in adult hepatitis B virus transgenic mice

A CRISPR screen identifies CDK7 as a therapeutic target in hepatocellular carcinoma

Knock out CD44 in reprogrammed liver cancer cell C3A increases CSCs stemness and promotes differentiation

Phospho-ERK is a biomarker of response to a synthetic lethal drug combination of sorafenib and MEK inhibition in liver cancer

Genome-wide CRISPR screen identifies regulators of mitogen-activated protein kinase as suppressors of liver tumors in mice

LncBRM initiates YAP1 signalling activation to drive self-renewal of liver cancer stem cells

The role of polyphenol (Flavonoids) compounds in the treatment of cancer cells

Translatable gene therapy for lung cancer using Crispr CAS9-an exploratory review

Safety and feasibility of CRISPR-edited T cells in patients with refractory non-small-cell lung cancer

PTEN inactivation induces epithelial-mesenchymal transition and metastasis by intranuclear translocation of β-catenin and snail/slug in non-small cell lung carcinoma cells

Genetic analysis reveals AMPK is required to J o u r n a l P r e -p r o o f support tumor growth in murine Kras-dependent lung cancer models

Specific targeting of point mutations in EGFR L858R-positive lung cancer by CRISPR/Cas9

Long non-coding RNA EPIC1 promotes human lung cancer cell growth

CRISPR-mediated modeling and functional validation of candidate tumor suppressor genes in small cell lung cancer

Comprehensive intra-individual genomic and transcriptional heterogeneity: Evidence-based Colorectal Cancer Precision Medicine

Synthesis of 5-Fluorouracil Cocrystals with Novel Organic Acids as Coformers and Anticancer Evaluation against HCT-116 Colorectal Cell Lines

Prognosis of patients with peritoneal metastatic colorectal cancer given systemic therapy: an analysis of individual patient data from prospective J o u r n a l P r e -p r o o f randomised trials from the Analysis and Research in Cancers of the Digestive System (ARCAD) database

Modeling colorectal cancer using CRISPR-Cas9-mediated engineering of human intestinal organoids

Transcriptional activation of fucosyltransferase (FUT) genes using the CRISPR-dCas9-VPR technology reveals potent N-glycome alterations in colorectal cancer cells

Targeting HPV16 DNA using CRISPR/Cas inhibits anal cancer growth in vivo

Par3L enhances colorectal cancer cell survival by inhibiting

Genome editing of mutant KRAS through supramolecular polymer-mediated delivery of Cas9 ribonucleoprotein for colorectal cancer therapy

Cancer incidence and mortality patterns in Europe: Estimates for 40 countries and 25 major cancers

PGC1α suppresses prostate cancer cell invasion through ERRα transcriptional control

CRISPR-Cas9 ERβ deletion reveals roles in prostate

Evaluation of the CRISPR/Cas9 directed mutant TP53 gene repairing effect in human prostate cancer cell line PC-3

Generation of PTEN-knockout (-/-) murine prostate cancer cells using the CRISPR/Cas9 system and comprehensive gene expression profiling

CRISPR/Cas9 targeting of the androgen receptor suppresses the growth of LNCaP human prostate cancer cells

CRISPR/Cas9 targeting of GPRC6A suppresses prostate cancer tumorigenesis in a human xenograft model

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High-performance CRISPR-Cas12a genome editing for combinatorial genetic screening

CRISPR-Cas12a with an oAd Induces Precise and Cancer-Specific Genomic Reprogramming of EGFR and Efficient Tumor Regression

PAM-dependent target DNA recognition and cleavage by C2c1 CRISPR-Cas endonuclease

C2c1-sgRNA complex structure reveals RNA-guided DNA cleavage mechanism

C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector

A CRISPR-Cas13a system for efficient and specific therapeutic targeting of mutant KRAS for pancreatic cancer treatment

A Multifunction Lipid-Based CRISPR-Cas13a Genetic Circuit Delivery System for Bladder Cancer Gene Therapy

Targeted mRNA demethylation using an engineered dCas13b-ALKBH5 fusion protein

Applications of the CRISPR/Cas9 system in murine cancer modeling

High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells

Genome-scale CRISPR-Cas9 knockout screening in human cells

Knockdown of hypoxia-inducible factor-1 alpha by tumor targeted delivery of CRISPR/Cas9 system suppressed the metastasis of pancreatic cancer

Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects

Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex

Non-viral vectors for gene-based therapy

Poly (beta-amino ester) nanoparticles enable non-viral delivery of CRISPR/Cas9 plasmids for gene knockout and gene deletion

Nrl knockdown by AAV-delivered CRISPR/Cas9 prevents retinal degeneration in mice

Near-infrared optogenetic engineering of photothermal nanoCRISPR for programmable genome editing

Material solutions for delivery of CRISPR/Cas-based genome editing tools: Current status and future outlook

Intratumor heterogeneity in localized lung adenocarcinomas delineated by multiregion sequencing

CRISPR/Cas9: transcending the reality of genome editing

CRISPR-cas9: a powerful tool towards precision medicine in cancer treatment

Inducible in vivo genome editing with CRISPR-Cas9

Enhancing homologydirected genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA

Modeling cancer processes with CRISPR-Cas9

CRISPR-Cas9 a boon or bane: the bumpy road ahead to cancer therapeutics

Enhanced bacterial immunity and mammalian genome editing via RNA-polymerase-mediated dislodging of Cas9 from double-strand DNA breaks

CRISPR-Cas9 mediated efficient PD-1 disruption on human primary T cells from cancer patients

Applications of CRISPR-Cas enzymes in cancer therapeutics and detection

Modeling Breast Cancer Using CRISPR-Cas9-Mediated Engineering of Human Breast Organoids

CRISPR-mediated direct mutation of cancer genes in the mouse liver

Efficient in vivo liver-directed gene editing using CRISPR/Cas9

Transcriptional activation of fucosyltransferase (FUT) the CRISPR-dCas9-VPR technology reveals potent N-glycome alterations in colorectal cancer cells

CRISPR/Cas9-mediated gene knockout of NANOG and NANOGP8 decreases the malignant potential of prostate cancer cells

Anti-CRISPR protein applications: natural brakes for CRISPR-Cas technologies

Hacking the cancer genome: profiling therapeutically actionable long non-coding RNAs using CRISPR-Cas9 screening

ZEB1 is neither sufficient nor required for epithelial-mesenchymal transition in LS174T colorectal cancer cells

Application of the CRISPR/Cas9 system to drug resistance in breast cancer

Construction of Traf3 knockout liver cancer cell line using CRISPR/Cas9 system

Landscape of Noncoding RNA in Prostate Cancer

The functions and regulation of the PTEN tumour suppressor: new modes and prospects

Precision cancer mouse models through genome editing with CRISPR-Cas9

Distinct structural classes of activating FOXA1 alterations in advanced prostate cancer

Essentials of Cancer Genomic

Tumor suppressor role of cytoplasmic polyadenylation element binding protein 2 (CPEB2) in human mammary epithelial cells

Effects of SOX15 on the colorectal cancer cells via downregulation of the Wnt/β-catenin signaling pathway

MicroRNA-1205, encoded on chromosome 8q24, targets EGLN3 to induce cell growth and contributes to risk of castration-resistant prostate cancer

Probing the tumor suppressor function of bap1 in crispr-engineered human liver organoids

Delivery strategies of the CRISPR-Cas9 gene-editing system for therapeutic applications

the Cas9 used the molecular scissors to cut the affected part of the target DNA and insert the healthy or desired part of DNA (step 4-5). The double-strand break (DSB) is repaired by enzymes in two ways, either by homologous recombination (HR) or nonhomologous end-joining (NHEJ). In the present figure, the DNA is repaired by the HR method (step 6). Finally, if all the process succeeds, cancer (but not limited to cancer) patient become healthy

The authors declared no conflict of interest.

For this type of study, informed consent is not required.