CRISPR/Cas9: A revolutionary genome editing tool for human cancers treatment.

Introduction

Cancer, a complex disease, is the second largest cause of death across the globe. Primarily, cancer is a genome ailment, feeding off mutations in DNA that consequently result in activating oncogenes and inactivating tumor suppressors, along with dysregulating the epigenome, which regulates the normal expression of genes. Additionally, cancer can also be defined as the disease of a cell resulting from the changes in the structure of the cell, metabolism, and motility to permit growth in bleak and unreceptive conditions. It eventually becomes a sickness for the organism, absorbing normal cell types, tissue functions and outmaneuvering the host’s defense mechanisms. 1 With the development of high throughput sequencing technologies, a myriad of genes coupled with the initiation and progression of cancer have been identified during the past 2 decades. 2 , 3 Despite the thrilling accomplishments in cancer therapeutics which include surgery, targeted biotherapy, chemotherapy, and radiotherapy, increased rates of postoperative relapse, resistance to chemotherapy/radiation along with detrimental off-target effects continue to be a hurdle in life span and standard life quality of a cancer patient. 4 For the development of effective and efficient treatment options aiming to enhance the denouement for millions of people diagnosed with cancer annually, it is critical to understand how genetic alterations, cellular adaptations, and modifications in the microenvironment of tumor influence the initiation, development, and treatment response of certain malignancies. 5

In recent years, noteworthy advances in biotechnology have been made, and the field of genetic engineering is now progressing at an exponential rate, offering countless benefits. Genome editing tools have transformed biological and genetic research and exploration through their groundbreaking ability to squarely amend, alter and reconfigure the genomes of living organisms. In current times, diverse genome manipulating techniques have been utilized to study simple, intricate, and raveled genomes. 6 , 7 Since the introduction of genome editing technologies in the 1990s, various strategies for targeted genome editing have been devised. One important use of gene-editing technology is genome modification, which comprises gene inactivation, insertion of a new sequence, and/or rectification of mutant regions with proper nucleotide sequences. 8 To modify genes, many approaches are used, including zinc-finger endonucleases (ZFNs), 9 transcription activator-like effector nucleases (TALENs), 10 and the clustered regularly interspaced short palindromic repeats (CRISPR) associated nuclease system.

CRISPR, a dominant and powerful gene-editing technique, emerged in 1986 and quickly rose to prominence as the century's most important genetic tool outweighing the previous methods owing to its outstanding advantages such as simplicity, cost-effectiveness, speed, and high efficiency. 11 , 12 The CRISPR/Cas system was initially found in Escherichia coli , but it is now known to be prevalent across a vast population of prokaryotic species. 13 , 14 In bacteria and archaea, it is a sequence-specific adaptive immune system that provides resistance to viruses, phages, and other genetic materials. 15 Apart from its role in antiviral immunity, CRISPR serves as a barricade against horizontal gene transfer mechanisms and therefore plays an imperative role in sustaining the integrity of the genome. 16

The CRISPR/Cas9 framework is a hereditary defense system in bacteria and archaea that utilizes RNA-directed nucleases to cleave targeted DNA. 17 , 18 CRISPR locus is an assembly of short direct repeats spaced by spacer sequences. 19 The repeats within a particular locus are identical in sequence and length. The spacers are likewise uniform in length, although their sequence composition varies greatly. Repeats in various species range from 21 to 47 bp, with an average of 32 bp, while spacers range from 20 to 72 bp. Although related species may share identical repeat sequences, the overall sequence diversity of both spacers and repeats in archaea and bacteria is quite high. 20 This system is consisting of DNA endonuclease (Cas9) and single guide RNA (sgRNA). The Cas9/sgRNA complex binds specifically to target dsDNA which has a complementary sequence match to 5′ end of sgRNA (match of first 17 to 20 nt of sgRNA) and also has a PAM area (protospacer adjacent motif). The sgRNA guides the Cas9 endonuclease to break the DNA strands at the desired target site. 21 Cas9, DNA nuclease with two active domains, cleave the target DNA strands at 3 nt upstream of PAM and produce a double-stranded break (DSB). DSBs are repaired through a host natural repair mechanism, i.e., NHEJ method (nonhomologous end joining) or HDR method (homologous direct repair pathway). CRISPR/Cas9 mediated cleavage is not always specific and sometimes may result in unwanted binding of Cas9 to target DNA producing nonspecific cleavage, and giving-off target effects. CRISPR/Cas9 target efficiency and subsequent cleavage specificity depend on gRNA and PAM location to the target loci. Off targets effects and strategies to avoid them have been discussed in CRISPR/Cas9 challenges section. 22 , 23 To minimize the off-target effects, tools to design desirable sgRNA have been provided in Table 1 .

CRISPR/Cas9 technology offers a unique and striking future in laboratory cancer research, with the possibility to generate superior disease models for cancer, therapeutic targets, and the identification and exploration of treatment resistance. It is an affordable technology offering a high level of flexibility, potency, and ease to use in laboratory research. 35 CRISPR/Cas9 has effectively been exploited to develop cancer disease models, by inactivating multiple tumor suppressor genes such as Nf1, P53, Ptch1, and Pten in mouse brain which has resulted in the creation of medulloblastoma and glioblastoma disease models. 36 General overview of cancer modeling with CRISPR/Cas9 technology is given in Figure 1 .

Current status of CRISPR/Cas9 in Cancer Treatment

CRISPR/Cas9 Mediated CAR-T Centered Cancer Treatment

CAR-T cells are T lymphocytes (human immune cells) that have been genetically modified to express a chimeric antigen receptor (CAR). Chimeric antigen receptor, commonly known as CAR, is a synthetic receptor that can recognize specific antigens. It consists of an antibody-driven target-binding extracellular domain, a hinge region, a trans-membrane domain, and an intracellular signaling moiety that can activate T-cells. 38 , 39 A CAR-targeted antigen can be recognized by the CAR-bearing altered T-cells, this consequently provokes the proliferation of T-cells, manufacturing of cytokine, and critical and targeted cytotoxicity versus tumor cells. 40 The performance of CARs is determined by several factors. A few of these include the position of the target cell surface antigen 41 as well as careful adjustment of intracellular and extracellular domains, including the nonfunctional linking transmembrane domain and hinges. 42

CAR-T-cell-based clinical trials for cancer treatment have shown promise in eliciting long-term full remissions in patients with a range of hematologic and solid malignancies, including relapsed/refractory acute lymphoblastic leukemia (ALL) and multiple myeloma, with response rates ranging from 80% to 100%. 43 – 45 CAR-T cell therapy for hematological malignancies, particularly B-cell malignancies, has a high success rate. 46 , 47 However, their application in treating solid tumors has not been successful yet. One of the barriers to its extensive use is CAR-T cell dysfunction, counting both the senescence and exhaustion of the cells limiting their effectiveness. Other barriers are the higher prevalence of toxicities, such as cytokine release syndrome, the possibility of graft versus host disease. 48 Currently, only five CAR-T products have been approved, for hematologic malignancies, by the United States Food and Drug Administration (FDA) which are (i) Abecma (idecabtagene vicleucel) for patients with relapsed or refractory (R/R) myeloma, (ii) Breyanzi (lisocabtagene maraleucel) for R/R large B-cell lymphoma patients, (iii) Tecartus (brexucabtagene autoleucel) for R/R mantle-cell lymphoma patients, (iv) Kymriah (tisagenlecleucel) for patients especially peds and young adults suffering with CD19 + R/R B-cell, (v) Yescarta (axicabtagene ciloleucel) specific for refractory large B-cell lymphoma patients. 49

A big restrictive factor in CAR-T cell therapy for cancer treatment is that the patients must wait for long periods before the treatment can be applied since every step of harvesting, engineering, expanding, and transfusing cells is tailored to each patient and takes time. Moreover, this procedure requires logistical and scientific support. CRISPR/Cas9-mediated genetic manipulation has the potential to increase the efficacy of immunotherapy by generating a universal “off-the-shelf” cellular product or modifying immune cells for overcoming resistance to hematological or solid malignancies. 50 The idea of universal allogeneic CAR-T products eliminates the requirement of harvesting, modifying, expanding, and transfusing T-cells from each patient, conserving resources and time. By altering allogeneic (healthy donor) T-cells to express the CAR by knocking-in and then knocking-out the genes involved in immune recognition of these nonself cells, the cells can be effectively transferred into patients without the danger of immunological rejection. It may also be used to precisely alter some cytokine genes for boosting the production of cytokine and immune cell reaction, preventing T-cell depletion and autoimmune responses correlated with other techniques of cytokine increase like viral transduction. 51 To ensure long-lasting engagement of T-cells, the CRISPR/Cas9 system can be employed for knocking out checkpoint inhibitor genes, for instance, the genes that code for CTLA-4 and PD-1. 52 The CRISPR-manipulated cells have the ability to be mass-produced, lyophilized, and kept at medical centers across the country, alleviating the need of transporting living cells for every patient from the manufacturing sites. The precise insertion of CAR into the exact position in the genome of the T-cells to ensure its sufficient expression can be facilitated by CRISPR.

CRISPR/Cas9 Mediated Adoptive T-Cell Centered Cancer Treatment

In this therapy, the T-cells of patients are modified to express specific TCRs that can recognize antigens released by tumor cells and trigger T-cells to kill the tumor cells. Tumor-infiltrating lymphocyte (TIL) treatment and chimeric antigen receptor T-cell (CAR-T-cell) therapy are the two forms of adoptive cell therapy. ACT for cancer is a prime focus for the gene-editing trials. 53 During adoptive T-cell therapy, a biopsy is performed, and tumor cell are subjected to whole-exome sequencing and RNA sequencing. Sequencing results along with bioinformatic prediction algorithms help in identifying immunogenic neoantigens. T-cells specific for these immunogenic neoantigens are isolated from the patient (either from peripheral blood mononuclear cells [PBMCs] or tumor-infiltrating lymphocytes (TILs) of a patient). Later, T-cell receptors (TCRs) are sequenced and electroporated into the same patient's PBMCs along with Cas9 and gRNA. This results in the production of neoantigen-specific T-cells which can be expanded and introduced back into the same patient for cancer therapy. 54

In 2016, researchers from the Chinese PLA General Hospital started the first CRISPR phase I clinical research in humans by infusing PD-1-KO essential T-cells into patients with stage IV metastatic nonsmall cell cellular breakdown in the lungs ( NCT02793856 ). The clinical productivity of the treatment was not resolved thus the viability of this study is questionable. 55 Three further stage I clinical investigations are being led to assess PD-1-KO essential T-cells in stage IV bladder disease ( NCT02863913 ), hormone-resistant prostate malignant growth ( NCT02867345 ), and metastatic renal cell carcinoma ( NCT02867332 ), starting around the same time. Be that as it may, later every one of these was removed due to lack of funding, which resulted in the studies being withdrawn. Although TILs have shown viability in metastatic diseases, especially melanoma, 56 and have shown similarly more particularity and effectiveness than essential altered T-cells, hardly any preliminaries are following hereditarily adjusted TILs.

Two clinical preliminaries are, as of now, in progress utilizing CRISPR/Cas9 to disturb CISH in TILs taken from gastrointestinal growth areas (NCT04089891 and NCT04426669 ). The silencer of cytokine signaling (SOCS) protein, cytokine-induced SH2 (CISH), is a part of the SOCS family, CISH protein is expanded in CD8 + T-cells in light of TCR initiation, subsequently hindering T-cells hostile to growth action. In-vivo examinations have shown how CISH cancellation causes TIL development, capacity, and cytokine discharge, as well as growth backslide. 57 Currently, mispairing of α and β chains of the therapeutic TCR complex is a main problem with ACT. This results in lessened effectuality for engaging TCR with the target antigen. Moreover, PD-1 expression on T-cells has a negative regulatory effect, which lowers the antigen reaction and, thus, the effectiveness of T-cell-mediated tumor killing. 58

Role of CRISPR/Cas9 in Cancer Diagnosis and Treatment

For Diagnosis

Cancer may be detected and treated earlier, which reduces the risk of mortality and improves patients’ quality of life following the treatment. Many cancer detection techniques are extensively employed, however, they ought to be improved in terms of sensitivity, specificity, and speed. As a result, for cancer prevention identifying vulnerable genes by genetic diagnosis is critical. 59 CRISPR has remarkable potential to be used in diagnostics where it can detect genes involved in various cancer developments, their proliferation, and metastasis or in drug resistance. Detection of cancer-specific sequence changes can be done with the help of CRISPR-mediated enzymatic digestion which works as a diagnostic tool. CRISPR can detect microsatellites (a diagnostic marker in various cancers), which are made of short tandem repeats (STRs) through detecting these STRs. There are four Cas systems, i.e., Cas9, Cas12, Cas13, and Cas14, which are currently being used in CRISPR-based molecular diagnostics. These four CAS systems use different techniques for diagnosis such as Cas9 uses CRISPR-Chip and CRISDA, Cas12 uses DETECTR, HOLMES, and SHERLOCKv2, Cas13 uses SHERLOCK, and SHERLOCKv2 while Cas14 uses Cas14 DETECTR. In the processes of DNA recognition, shearing, and degradation, Cas12a works as a corresponding protein of Cas13a. Based on Cas12a, RPA was introduced as a form of new nucleic acid detection technique named DETECTR which compared with SHERLOCK can eliminate transcription of amplified DNA products into RNA step. 1 – 60

Researchers have also used Cas13a system as a basis and introduced RPA (temperature amplification of DNA technology) to create SHERLOCK (specific high sensitivity enzymatic reporter UnLOCKing). It has a site-specific amplification principle such as in the case of DNA; it will be followed by amplification through RPA and in the case of RNA detection; it will be amplified by RT-RPA. Target RNA transcribed from amplified DNA products is detected by Cas13a-crRNA and reporter RNA. This technique has its advantage as it is highly specific, sensitive in nature, and has many uses in CRISPR-based diagnostics. Its high sensitivity allows it to do single-base resolution based on its crRNA design. It has its applications in the detection of specific nucleic acid molecules, pathogen identification, detection of drug-resistant genes, liquid biopsy, virus and its subtype detection and differentiation, and cancer mutation analysis. 60 Similar to this, Cas12-DETECTR was designed due to four characteristics of Cas14: (1) small size of Cas14 makes its large-scale production easy; (2) target DNA is ssDNA; (3) it has no PAM sequence and thus any site sequence in the desired DNA can be targeted; (4) can detect DNA through a single base resolution. It has its applications in fast and efficient cancer and another disease diagnosis. SNP genotype analysis method was also made possible to establish due to the properties of Cas14. 61

Other CRISPR-based diagnostic techniques for cancer detection include improved SHERLOCK, which is used in fast liquid biopsies of cancer patient. It can detect very small amounts of viral RNA ≈2 aM. 62 HOLMES is another CRISPR-based diagnostic tool based on the Cas12a system and PCR. It is fast, highly specific, simple, low-cost, sensitive, and needs no professional equipment for its working. It has its applications in the detection of cancer mutations, SNPs, and pathogens. 63 CRISPR-Chip is another diagnostic technique that has a big advantage over SHERLOCK and DETECTR and that is it need no amplification of tested DNA. 60 Cas12 and Cas13 detect nucleic acids through SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) and DETECTR (DNA endonuclease-targeted CRISPR trans reporter) for an easy, affordable diagnosis of SARS-CoV-2 infection. Currently, CRISPR-DS is being clinically evaluated for p53 mutation detection in ovarian tumors. Thus, it can be freely said that CRISPR has its advantages in diagnosis as a personalized, sensitive, and safe monitoring system for cancer patients. 1 – 60

MicroRNAs (miRNAs) have a promising role in cancer pathologies by repression of protein-coding oncogenes or by impeding the expression of tumor suppressors. It can aid in the detection of and therapy for cancer. miRNAs are composed of a set of short, noncoding proteins, and small RNA molecules (20–24 nt in length). These RNA molecules are made from an endogenous transcript which contains a hairpin local structure, with the help of RNase-III-type enzyme Dicer. CRISPR/Cas9 technique targets the terminal loop or 50 regions of pre-RNA and thus suppressed the expression of miRNA. CRISPR/Cas9 targets desired DNA/ hsa-miR-17 gene with the help of sgRNA which binds with the target DNA at upstream of PAM (5′-NGG-3′). Cas9 nuclease introduces DSB in targeted DNA at Drosha or Dicer processing sites which are 3 bp upstream of PAM. This DSB is repaired by host own repair mechanisms. CRISPR/Cas9 with the help of sgRNA can also alter miRNA biogenesis process through intentional targeting of sequences either within or adjacent to Dicer and Drosha repairing sites in the secondary stem-loop structure of primary miRNAs. Mechanism of CRISPR/Cas9 mediated miRNA has been given in Figure 2 . 64

Functional genome screening techniques based on CRISPR system might show variations in the expression of a gene after medication and pinpoint genes linked to drug resistance thus adding novel biomarkers for precision therapy and offering an additional understanding of cancer development. 65 One promising example includes the screening of a gene related to cancer metastasis. 66 Where after mice were infected with lung cancer metastasis, genome-scale CRISPR knockout (mGeCKO) sgRNA library was created, and transduced cells were implanted into immuno-compromised mice. Six weeks later, mice with lung cancer metastasis were chosen for sequencing of the enhanced sgRNA. Finally, several candidate genes interrelated to lung metastasis were identified and verified, these included Pten , 67 miR-152, 68 and miR-345, 69 along with many novel genes such as Nf2 , Trim72 , and Fga .

CRISPR/Cas9 for Cancer Treatment

There are many types of cancer treatment methods, all have their own pros and cons. The type of treatment method one received mostly depends on cancer type, and its cancer progress stage. More often than not, combinations of treatment methods are suggested. Some of the most common cancer treatment methods are (1) surgery (to remove cancerous body tissue/part); (2) chemotherapy (drugs are prescribed to kill cancer cells); (3) hormone therapy (effective on hormones released during a certain type of cancer such as breast and prostate cancer); (4) Hypothermia (heat up to 113°F is provided to kill or stop cancer cell with little to no damage to normal cells); (5) Radiation therapy where radiations are used to treat cancer, etc. 70 Among all these methods, CRISPR/Cas-mediated genome editing offers vast promise in cancer therapy, to make quick progress in fundamental oncology research. Tumorigenesis is a multistep process in which cancer cells communicate with the host immune system. 71 Cancer immunotherapy is a well-known treatment option after surgery, chemotherapy, and radiation therapy. Adoptive T-cell immunotherapy, specifically chimeric antigen receptor (CAR) T-cell therapy, has marked a turning point in cancer treatment. 72

In the United States in 2019, the first clinical trial for the treatment of cancer using CRISPR was studied at The University of Pennsylvania, funded by the National Cancer Institute. In this type of immunotherapy, the immune cells of patients are genetically engineered to detect and destroy tumors in a better way. The treatment involves the introduction of four genetic changes into the T-cells. These are immune cells capable of battling and killing cancerous cell. First, a claw-like protein is provided to T-cells by the addition of a synthetic gene. This claw-like protein commonly known as a receptor recognizes NY-ESO-1, a chemical found on certain cancer cells. Subsequently, three genes are deleted with the help of the CRISPR system. Among these three, two have the ability to interact with the NY-ESO-1 receptor and one gene is capable to hinder the ability of cells to fight malignancy. It results in the abundant production of NYCE T-cells which are then injected into patients. In the long run, it is one of the precise and safe strategies to kill cancer cells. It also helps in improving the efficacy of T-cells. 73 A diagrammatic representation of the study performed at The University of Pennsylvania for the treatment of cancer using CRISPR Cas9 in the human body is illustrated in Figure 3 .

According to studies conducted by Edward Stadtmauer, an improved response was shown by NY-ESO-1–directed T-cells, and that too with minimal toxicity. Edward and his colleagues also tried to observe whether the function of T-cells is improved with the deletion of three genes using CRISPR. 75 The trials proved that the treatment so done employing CRISPR is harmless. The treatment was provided to three patients out of which two had severe multiple myeloma and one had metastatic sarcoma. All three patients had malignancies that contained the T-cell therapy's target (NY-ESO-1). This therapy was proven to be safe on the basis of the preliminary data. According to the researchers, some side effects occurred, but they were most likely caused by chemotherapy in patients before injecting them with NYCE cells. Furthermore, there was no evidence of an immune response to the CRISPR-edited cells. 74 List of ongoing CRISPR/Cas9 mediated cancer treatment clinical trials have been provided in Table 2 .

Role of CRISPR/Cas9 in Human Cancers

Cancer is the second most common cause of death across the world, representing around 8.8 million passing in 2015. Throughout the following 20 years, the quantity of new cases is anticipated to ascend by almost 70% internationally. 75 Current cancer-fighting methods are insufficient, and scientists are continually on the lookout for new technology that can help. CRISPR/Cas9 system offers new promise in this area. CRISPR technology allows humans to rewrite their genetic code. CRISPR induction is easier and faster than previous technologies, and it will likely speed up gene-editing processes all around the world. It’s been utilized in a variety of in-vivo and in-vitro malignant tumor models up to this point. Oncologists are especially excited about CRISPR's viability, precision, and potential in malignant tumor research. 77 CRISPR/Cas9 innovation has been utilized by researchers from one side of the planet to the other to address malignant tumor treatment from many examination perspectives.

Colorectal Cancer

Colorectal malignant growth (CRC) is a type of cancer that occurs in colon or rectum. To investigate the function of potential colorectal cancer-associated genes, in-vivo studies using genetically modified mouse models (GEMMs) are performed. 78 In GEMMs, the ApC and Trp53 tumor suppressor genes in colon epithelial cells were base edited by CRISPR/Cas9 and through orthotopic transplantation of ApC -edited colon organoids to produce tumor in mice. The study was successful in activation of an oncogene within the model mice. The study helped in cancer-associated genes characterization and to understand tumor progression and metastasis. 79 This gene editing technique can be utilized in numerous model-based cancer studies to investigate genes responsible for a certain cancer type and the associated changes due to its manifestation in the human body.

Breast Cancer

Bosom disease (BC) is another tumor that exterminates women all around the globe. TNBC has the most exceedingly terrible forecast of all the BC subtypes on the grounds that it needs articulation of the estrogen receptor, progesterone receptor85, and HER2/neu tyrosine kinase receptor. The CRISPR/Cas9 technology was utilized to restrict cancer advancement and lung metastasis by reducing Cripto -1, an early-stage undeveloped cell marker of cancer stem-like cells. In this review, Cripto -1 was found to be a potential therapeutic target for TNBC. The Brahma ( BRM ) and Brahma-related gene 1 ( BRG1 ) are both overexpressed in essential BCs. BRM and BRG1 work as ATPases inside mammalian SWI/SNF complexes and are important for their functioning. 80 The CRISPR/Cas9 gene editing machinery was used to take out the BRG1 or BRM genes, revealing that these two genes play an important role in BC cell multiplication. Therefore, both BRG1 and BRM could be utilized to treat BC.

One more type of human BC is intrusive lobular carcinoma (ILC). The deficiency of cell grip protein and methylation of the CDH1 gene advertiser are normal elements of this disease. 81 This recently made innovation can be utilized to examine potential cancer silencer genes embroiled in ILC in-vivo rapidly. Comparable frameworks could be utilized to make new in-vivo models for the ID and treatment of different BC subtypes. 82 Table 3 shows targeted genes and their functions in various cancer types.

Ovarian Cancer

Epithelial-to-mesenchymal translation (EMT) is an event during cancer metastasis. During this cycle, 102 epithelial cells lose their affiliations, and gene expression is changed. This shift is accomplished by different master regulators, including record factors like Snail1, TWIST, and zinc-finger E-box confining (ZEB). The Snail1 factor was taken out using CRISPR/Cas9 advancement, revealing that the actin cytoskeleton is changed when Snail1 is deleted. This approach was also used to analyze the components of Snail1 and to decrease it in human ovarian illness RMG-1 cells. 103 The CRISPR/Cas9 innovation can be utilized to study the genetic reason for chemoresistance in epithelial ovarian disease. Chemoresistance was reversed when the ovarian malignant growth biomarker HE4 was altered. 104 In-vitro , taking out LY75 diminished cancer cell rearrangement and obtrusiveness, and in-vivo , taking out LY75 brought down the metastatic capability of EOC cell lines. 105 The ovarian carcinoma immuno-reactive antigen area containing 1 ( OCIAD1 ) gene was additionally taken out using CRISPR/Cas9 in a BJNhem20-OCIAD1-CRISPR-39 line. 106 This ex-vivo study, as well as other CRISPR/Cas9-based research, prepares for future ovarian disease treatment. 107 More generally, CRISPR/Cas9 has been broadly utilized in disease research, for certain promising discoveries.

Lung Cancer

Cellular breakdown in the lungs is the most terrible cancer among different tumors occurring in both industrialized and underdeveloped countries, including China, United States, and countries in Europe. 108 , 109 Cellular breakdown in the lungs is brought about by various genes and signaling pathways. 110 It has been a subject of broad clinical exploration and dynamic gene modifications therapy is considered as disease quality treatment. 111 It has been a subject of broad clinical exploration. Compared with other gene editing technologies like ZFNs 112 and transcription activator-like effector nucleases (TALENs), 113 CRISPR/Cas9 framework is a much more desirable method with its high target specificity, ease of use, and thus is being used more recently to study lung cancer, its prognosis, and treatment. 35 – 114 CRISPR-based approach can be used to investigate lung cancer in individual patients, obstacles in anti-cancer medication efficacy, and/or to treat genetic causes of cancer before it progresses any further. 115 The applications of CRISPR/Cas9 in studies on the treatment of lung cancer is given in Figure 4 .

Bone Cancer

In children and adolescents, osteosarcoma (OS) is a highly vascular and particularly destructive tumor. 117 , 118 Distant metastases (25%–30%) 119 and pathologic fracture due to bone loss 120 are the most common consequences. The most common metastatic location of OS is the lung. 121 Surgery in combination with chemotherapy is the standard treatment for OS. 117–121, 122 Patients with OS who have metastasis, on the other hand, have a much worse prognosis 123 , 124 As a result, developing novel therapeutic techniques for these people is critical. The angiogenic agent VEGFA (vascular endothelial growth factor A) is an inducer of angiogenesis and lymphangiogenesis. 125 Studies reveal that VEGF's effect on cancer development is not restricted to angiogenesis. 126 In light of hypoxia, VEGFA is delivered to the cancer microenvironment. 117 From one perspective, malignant growth cells’ paracrine VEGFA advances angiogenesis through endothelial cells, which convey oxygen and supplements. VEGFA, then again, goes about as an autocrine growth cell endurance factor. 127 VEGFA is presently known to be profoundly transformed in OS cells and to be firmly related to an unfortunate disease in patients. 128 VEGFA suppression repressed OS development, metastasis, and 72 angiogenesis, 129 – 131 inferring that VEGFA is a possible therapeutic option for OS.

Prostate Cancer

Prostate cancer (PCa) is treated primarily by inhibiting androgen receptor (AR) signaling, although the disease eventually advances to castration-resistant prostate cancer (CRPC). Patients with CRPC have seen a significant improvement in survival thanks to next-generation AR signaling inhibitors, but resistance is still an issue. 132 – 134 Regardless of the way, genomic studies have recognized hereditary causes (ETS combinations, CDKN2A misfortune, PTEN, RB1, and SPOP changes, among others) and subatomic subtypes of PCa for designated treatment, 135 – 137 some of the inherited anomalies found in patients’ of prostate cancer have shown promise as suitable drug targets. Thus, getting a more profound understanding of PCa's crucial conditions might prompt more sensible restorative methodologies. In contrast to shRNA-or siRNA-based hereditary reliance testing, CRISPR/Cas9 innovation limits adverse consequences, is efficient, and recognizes more positive benefits. 138 , 139 CRISPR/Cas9 framework has been effective in recognizing genes important for malignant tumor cell endurance as forthcoming targets, 140 , 141 yet benefits of combining it with existing therapeutics have gotten less consideration. A summary of the genes targeted for the treatment of different cells or/and tissues in various human cancers is provided in Table 4 .

Challenges of Using CRISPR in Cancer Treatment

CRISPR/Cas9 has in no time evolved as a productive genome editing technology in only a couple of years. It has enormous potential in cancer therapy; however, there are still many challenges in its full clinical applications.

Off-Target Effects

The first challenge in CRISPR-based cancer therapy is off-target effects. In therapeutics, every small and low off-target editing is detrimental thus they should be controlled and identified accurately. Off-target DSB can cause small- and large-scale indels, inversions, and translocations at the nontarget sites. 102 Through whole genome sequencing, large-scale off the target mutations can be detected. However, it is very difficult both technically and economically to do the same for small-scale off target mutations as whole genome sequencing is unable to distinguish between SNP and small genetic alterations. A better, practical and cost-effective option for detection of small scale indels is through whole exome sequencing which focuses on off-target mutations occurring in coding region of genes. 102 , 103 Various strategies have been developed to avoid or minimize off-target mutations such as (1) identification of a unique target sequence with very few homologous sequences in then whole genome; (2) high GC content up to 75% in target sequence; (3) use of more potent Cas9 variants to avoid off-target effects; (4) designing of more suitable, and powerful sgRNA, i.e., truncated, 17 to 18 nt long, stem loop 2 of sgRNA, etc. A combination of these approaches can be used to completely avoid off-target effects. 103,106, 107 ,157,161

Editing Efficiency

Editing efficiency is another challenge in CRISPR Cas9 therapy in cancer patients. Unmodified cells tend to proliferate more quickly than modified cells and can lead to relapse, thus CRISPR-based therapies need very high editing efficiencies. Editing efficiency is based on (1) careful selection of target sites; (2) efficient and reliable delivery vectors; (3) potent Cas9 selection; (iv) careful selection and designing of sgRNA. Further research insights are needed to get a higher genome editing efficiency. 35

Efficient Delivery of CRISPR/Cas Components

Other than off-target effects and editing efficiency, another challenge in CRISPR-based cancer treatment is the efficient delivery of CRISPR/Cas components at target sites. The most widely used vehicles for delivery are viral vectors and in particular adeno-associated virus vectors (AAVs). AAV is the most preferable choice because of its transient expression, impressive efficiency, and low cytotoxicity. AAV has a packaging obstacle that has just been solved with the help of Staphylococcus aureus (SaCas9) based split-Cas9 system or Streptococcus thermophiles (St1Cas9) based smaller Cas9 orthologs. There are some other nonviral delivery methods aimed to enhance cell membrane permeability and target specificity of CRISPR/Cas9, i.e., cationic lipids, electroporation-mediated gene transfer, nanoparticles, and cell-penetrating peptides (CPPs). 35 In a study on adult mice, a combination of AAV encoding sgRNA, homologous template, and lipid nanoparticle-mediated delivery of Cas9 mRNA was successfully applied to activate gene repair in adult mice. This suggests that a careful combination of viral and nonviral delivery methods may be an alternative approach for cancer treatment in patients. 116

Low Efficiency of HDR

In CRISPR-based cancer therapies, the low efficiency of HDR is another issue on which further work is needed. Both NHEJ and HDR repair DSB however, NHEJ is more preferable than HDR. 162 In some cases, HDR-based DSB repair is needed where low efficiency of HDR becomes a problem. One way to increase HDR efficiency is by inhibition of NHEJ pathway. Recently, in a study paired Cas9 nickase has been found to increase HDR efficiency by producing single-strand nicks. 116

Future Perspectives and Conclusion

CRISPR/Cas9 is a novel and effective way of genome editing that has lately outperformed other approaches due to its significant characteristics. This multifaceted tool, which is often described as an umbrella terminology, has transformed life sciences by enabling advancements in basic research for a wide range of applications. The CRISPR/Cas9 genome altering apparatus has colossal research and clinical possibilities in disease treatment. This straightforward and scalable approach has the potential to aid in the comprehension of cancer predisposition and metastatic pathways, along with the prediction of therapy response and drug tolerance. CRISPR is expected to be implemented eventually in clinics, permitting an extensive range of treatment opportunities for human diseases, particularly cancer. The ongoing effort in developing and revolutionizing new ways to deliver genome manipulating tools into cells, as well as refining their modification capabilities, will enable these technologies to be utilized in a number of therapeutic applications. The production of chimeric antigen receptor (CAR) T-cells that can identify specific antigens on cancer cells is a prominent application of the CRISPR technique. CRISPR/Cas9 can play a critical role in CAR-T cell research, which has surged in recent years. However, certain concerns including off-target effects, in-vivo delivery, immunogenicity, and ethical considerations must be handled in order for it to be implemented successfully. Nevertheless, many clinical trials are being conducted to overcome these issues, and we anticipate gene-editing technology to play an important role in the future. 76

Acknowledgements

This work was carried out with the help of prestigious material of the institute's libraries.

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding: The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD: Fatima Akram https://orcid.org/0000-0001-8438-706X

References

1 Katti A Diaz BJ Caragine CM , et al. CRISPR In cancer biology and therapy . Nat. Rev. Cancer . 2020 ; 22 ( 5 ): 1 ‐ 21 . 10.1038/s41568-022-00441-w 35194172

2 Pon JR Marra MA . Driver and passenger mutations in cancer . Annu. Rev. Pathol.: Mech. Dis. 2015 ; 10 : 25 ‐ 50 . 10.1146/annurev-pathol-012414-040312 25340638

3 Shen P Jing Y Zhang R , et al. Comprehensive genomic profiling of neuroendocrine bladder cancer pinpoints molecular origin and potential therapeutics . Oncogene . 2018 ; 37 ( 22 ): 3039 ‐ 3044 . 29535424 10.1038/s41388-018-0192-5

4 Stupp R Hegi ME Mason P , et al. Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial . Lancet Oncol . 2009 ; 10 ( 5 ): 459 ‐ 466 . 19269895 10.1016/S1470-2045(09)70025-7

5 Sung H Ferlay J Siegel RL . 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 . 33538338 10.3322/caac.21660

6 Mirza Z Karim S . Advancements in CRISPR/Cas9 technology—focusing on cancer therapeutics and beyond . Semin. Cell Dev. Biol . 2019 ; 96 : 13 ‐ 21 . 31150758 10.1016/j.semcdb.2019.05.026

7 Mehravar M Shirazi A Nazari M , et al. Mosaicism in CRISPR/Cas9-mediated genome editing . Dev. Biol . 2019 ; 445 ( 2 ): 156 ‐ 162 . 30359560 10.1016/j.ydbio.2018.10.008

8 Li H Yang Y Hong W , et al. Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects . Signal Tansduct. Targeted Ther . 2020 ; 5 ( 1 ): 1 ‐ 23 . 10.1038/s41392-019-0089-y PMC6946647 32296011

9 Carroll D . Genome engineering with zinc-finger nucleases . Genetics . 2011 ; 188 ( 4 ): 773 ‐ 782 . 21828278 10.1534/genetics.111.131433 PMC3176093

10 Rupp LJ Schumann K Roybal KT , et al. CRISPR/Cas9-mediated PD-1 disruption enhances anti-tumor efficacy of human chimeric antigen receptor T cells . Sci. Rep . 2017 ; 7 ( 1 ): 1 ‐ 10 . 28389661 10.1038/s41598-017-00462-8 PMC5428439

11 Martinez-Lage M Puig-Serra P Menendez P , et al. CRISPR/Cas9 for cancer therapy: hopes and challenges . Biomedicines . 2018 ; 6 ( 4 ): 105 . 10.3390/biomedicines6040105 PMC6315587 30424477

12 Tavakoli K Pour-Aboughadareh A Kianersi F , et al. Applications of CRISPR-Cas9 as an advanced genome editing system in life sciences . BioTech . 2021 ; 10 ( 3 ): 14 . 35822768 10.3390/biotech10030014 PMC9245484

13 Ishino Y Krupovic M Forterre P . History of CRISPR-Cas from encounter with a mysterious repeated sequence to genome editing technology . J. Bacteriol . 2018 ; 200 ( 7 ): e00580 ‐ 17 . 29358495 10.1128/JB.00580-17 PMC5847661

14 Jansen R Embden JDV Gaastra W , et al. Identification of genes that are associated with DNA repeats in prokaryotes . Mol. Microbiol . 2002 ; 43 ( 6 ): 1565 ‐ 1575 . 11952905 10.1046/j.1365-2958.2002.02839.x

15 Haft DH Selengut J Mongodin EF , et al. A guild of 45 CRISPR-associated (Cas) protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes . PLoS Comput. Biol . 2015 ; 1 ( 6 ): e60 . 10.1371/journal.pcbi.0010060 PMC1282333 16292354

16 Shabbir MAB Hao H Shabbir MZ , et al. Survival and evolution of CRiSPR–Cas system in prokaryotes and its applications . Front Immunol . 2016 ; 2016 ( 7 ): 375 . 10.3389/fimmu.2016.00375 PMC5035730 27725818

17 Garneau JE Dupuis MÈ Villion M , et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA . Nature . 2010 ; 468 ( 7320 ): 67 ‐ 71 . 21048762 10.1038/nature09523

18 Horvath P Barrangou R . CRISPR/Cas, the immune system of bacteria and archaea . Science . 2010 ; 327 ( 5962 ): 167 ‐ 170 . 20056882 10.1126/science.1179555

19 Karginov FV Hannon GJ . The CRISPR system: small RNA-guided defense in bacteria and archaea . Mol Cell . 2010 ; 37 ( 1 ): 7 ‐ 19 . 20129051 10.1016/j.molcel.2009.12.033 PMC2819186

20 Kunin V Sorek R Hugenholtz P . Evolutionary conservation of sequence and secondary structures in CRISPR repeats . Genome Biol . 2007 ; 8 ( 4 ): 1 ‐ 7 . 10.1186/gb-2007-8-4-r61 PMC1896005 17442114

21 Jinek M Chylinski K Fonfara I , et al. A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity . Science . 2012 ; 337 ( 6096 ): 816 ‐ 821 . 22745249 10.1126/science.1225829 PMC6286148

22 Wu X Kriz AJ Sharp PA . Target specificity of the CRISPR-Cas9 system . Quant Biol . 2014 ; 2 ( 2 ): 59 ‐ 70 . 25722925 10.1007/s40484-014-0030-x PMC4338555

23 Naeem M Majeed S Hoque MZ , et al. Latest developed strategies to minimize the off-target effects in CRISPR-Cas-mediated genome editing . Cells . 2020 ; 9 ( 7 ): 1608 . 10.3390/cells9071608 PMC7407193 32630835

24 Montague TG Cruz JM Gagnon JA , et al. CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing . Nucleic Acids Res . 2014 ; 42 ( W1 ): W401 ‐ W407 . 24861617 10.1093/nar/gku410 PMC4086086

25 Ceasar SA. Rajan V Prykhozhij SV , et al . Insert, remove or replace: a highly advanced genome editing system using CRISPR/Cas9 . Biochim. Biophys. Acta – Mol. Cell. Res . 2016 ; 1863 ( 9 ): 2333 ‐ 2344 10.1016/j.bbamcr.2016.06.009 27350235

26 Liu H Wei Z Dominguez A , et al. CRISPR-ERA: a comprehensive design tool for CRISPR-mediated gene editing, repression and activation . Bioinformatics . 2015 ; 31 ( 22 ): 3676 ‐ 3678 . 26209430 10.1093/bioinformatics/btv423 PMC4757951

27 Hwang WY Fu Y Reyon D , et al. Efficient genome editing in zebrafish using a CRISPR-Cas system . Nat. Biotechnol . 2013 ; 31 ( 3 ): 227 ‐ 229 . 23360964 10.1038/nbt.2501 PMC3686313

28 Wong N Liu W Wang X . WU-CRISPR: characteristics of functional guide RNAs for the CRISPR/Cas9 system . Genome. Biol . 2015 ; 16 ( 1 ): 1 ‐ 8 . 26521937 10.1186/s13059-015-0784-0 PMC4629399

29 Stemmer M Thumberger T del Sol Keyer M , et al. CCTop: an intuitive, flexible and reliable CRISPR/Cas9 target prediction tool . PloS One . 2015 ; 10 ( 4 ): e0124633 . 25909470 10.1371/journal.pone.0124633 PMC4409221

30 Heigwer F Kerr G Boutros M . E-CRISP: fast CRISPR target site identification . Nat. Methods . 2014 ; 11 ( 2 ): 122 ‐ 123 . 24481216 10.1038/nmeth.2812

31 Xiao A Cheng Z Kong L , et al. CasOT: a genome-wide Cas9/gRNA off-target searching tool . Bioinformatics . 2014 ; 30 ( 8 ): 1180 ‐ 1182 . 24389662 10.1093/bioinformatics/btt764

32 Zhu LJ Holmes BR Aronin N , et al. CRISPRseek: a bioconductor package to identify target-specific guide RNAs for CRISPR-Cas9 genome-editing systems . PloS One 2014 ; 9 ( 9 ): e108424 . 25247697 10.1371/journal.pone.0108424 PMC4172692

33 Bae S Park J Kim JS . Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases . Bioinformatics . 2014 ; 30 ( 10 ): 1473 ‐ 1475 . 24463181 10.1093/bioinformatics/btu048 PMC4016707

34 Ma M Ye AY Zheng W , et al. A guide RNA sequence design platform for the CRISPR/Cas9 system for model organism genomes . BioMed Res. Int . 2013 ; 2013 . 10.1155/2013/270805 PMC3809372 24199189

35 Yi L Li J . CRISPR-Cas9 therapeutics in cancer: promising strategies and present challenges . Biochim. Biophys. Acta – Rev. Cancer . 2016 ; 1866 ( 2 ): 197 ‐ 207 . 10.1016/j.bbcan.2016.09.002 27641687

36 Zuckermann M Hovestadt V Knobbe-Thomsen CB , et al. Somatic CRISPR/Cas9-mediated tumour suppressor disruption enables versatile brain tumour modelling . Nat. Commun . 2015 ; 6 ( 1 ): 1 ‐ 9 . 10.1038/ncomms8391 PMC4467376 26067104

37 Ratan ZA Son YJ Haidere MF , et al. CRISPR-Cas9: a promising genetic engineering approach in cancer research . Ther. Adv. Med. Oncol . 2018 ; 10 : 1758834018755089 . 29434679 10.1177/1758834018755089 PMC5802696

38 Guedan S Calderon H Posey AD Jr Maus MV . Engineering and design of chimeric antigen receptors . Mol. Ther. Methods Clin. Dev . 2019 ; 12 : 145 ‐ 156 . 30666307 10.1016/j.omtm.2018.12.009 PMC6330382

39 Kulemzin SV Kuznetsova VV Mamonkin M Taranin AV . Engineering chimeric antigen receptors . Acta Nat . 2017 ; 9 ( 1 (32) ): 6 ‐ 14 . PMC5406655 28461969

40 Gilham DE Debets R Pule M , et al. CAR–T cells and solid tumors: tuning T cells to challenge an inveterate foe . Trends Mol. Med . 2012 ; 18 ( 7 ): 377 ‐ 384 . 22613370 10.1016/j.molmed.2012.04.009

41 Sterner RC Sterner RM . CAR-T cell therapy: current limitations and potential strategies . Blood Cancer J . 2021 ; 11 ( 4 ): 1 ‐ 11 . 33824268 10.1038/s41408-021-00459-7 PMC8024391

42 Rafiq S Hackett CS Brentjens RJ . Engineering strategies to overcome the current roadblocks in CAR T cell therapy . Nat. Rev. Clin. Oncol . 2020 ; 17 ( 3 ): 147 ‐ 167 . 31848460 10.1038/s41571-019-0297-y PMC7223338

43 Fry TJ Shah NN Orentas RJ , et al. CD22-targeted CAR T cells induce remission in B-ALL that is naive or resistant to CD19-targeted CAR immunotherapy . Nat. Med . 2018 ; 24 ( 1 ): 20 ‐ 28 . 29155426 10.1038/nm.4441 PMC5774642

44 Susanibar Adaniya SP Cohen AD Garfall AL . Chimeric antigen receptor T cell immunotherapy for multiple myeloma: a review of current data and potential clinical applications . Am. J. Hematol . 2019 ; 94 ( S1 ): S28 ‐ S33 . 30730071 10.1002/ajh.25428

45 Raje N Berdeja J Lin YI , et al. Anti-BCMA CAR T-cell therapy bb2121 in relapsed or refractory multiple myeloma . N. Engl. J. Med . 2019 ; 380 ( 18 ): 1726 ‐ 1737 . 31042825 10.1056/NEJMoa1817226 PMC8202968

46 Holstein SA Lunning MA . CAR T-cell therapy in hematologic malignancies: a voyage in progress . Clin. Pharmacol. Ther . 2020 ; 107 ( 1 ): 112 ‐ 122 . 31622496 10.1002/cpt.1674

47 Boyiadzis MM Dhodapkar MV Brentjens RJ , et al. Chimeric antigen receptor (CAR) T therapies for the treatment of hematologic malignancies: clinical perspective and significance . J. Immunother. Cancer . 2018 ; 6 ( 1 ): 1 ‐ 12 . 30514386 10.1186/s40425-018-0460-5 PMC6278156

48 Wagner J Wickman E DeRenzo C Gottschalk S . CAR T cell therapy for solid tumors: bright future or dark reality? Mol. Ther . 2020 ; 28 ( 11 ): 2320 ‐ 2339 . 32979309 10.1016/j.ymthe.2020.09.015 PMC7647674

49 Sadeqi Nezhad M Yazdanifar M Abdollahpour-Alitappeh M , et al. Strengthening the CAR-T cell therapeutic application using CRISPR/Cas9 technology . Biotechnol Bioeng . 2021 ; 118 ( 10 ): 3691 ‐ 3705 . 34241908 10.1002/bit.27882

50 Razeghian E Nasution MKM Rahman HS , et al. A deep insight into CRISPR/Cas9 application in CAR-T cell-based tumor immunotherapies . Stem Cell Res. Ther . 2021 ; 12 ( 1 ): 428 . 34321099 10.1186/s13287-021-02510-7 PMC8317439

51 Shifrut E Carnevale J Tobin V , et al. Genome-wide CRISPR screens in primary human T cells reveal key regulators of immune function . Cell . 2018 ; 175 ( 7 ): 1958 ‐ 1971 . 30449619 10.1016/j.cell.2018.10.024 PMC6689405

52 Liu X Zhang Y Cheng C , et al. CRISPR-Cas9-mediated multiplex gene editing in CAR-T cells . Cell Res . 2017 ; 27 ( 1 ): 154 ‐ 157 . 27910851 10.1038/cr.2016.142 PMC5223227

53 Ghaffari S Khalili N Rezaei N . CRISPR/Cas9 revitalizes adoptive T-cell therapy for cancer immunotherapy . J. Exp. Clin. Cancer Res . 2021 ; 40 ( 1 ): 1 ‐ 18 . 34446084 10.1186/s13046-021-02076-5 PMC8390258

54 Puig-Saus C Ribas A . Gene editing: towards the third generation of adoptive T-cell transfer therapies . Immuno-Oncol Technol . 2019 ; 1 : 19 ‐ 26 . 10.1016/j.iotech.2019.06.001 PMC9216344 35755321

55 Liu Q . World-first phase I clinical trial for CRISPR-Cas9 PD-1-edited T-cells in advanced nonsmall cell lung cancer . Glob. Med. Genet . 2020 ; 7 ( 03 ): 073 ‐ 074 . 10.1055/s-0040-1721451 PMC7772009 33392608

56 Rohaan MW van den Berg JH Kvistborg P Haanen JB . Adoptive transfer of tumor-infiltrating lymphocytes in melanoma: a viable treatment option . J. Immunother Cancer . 2018 ; 6 ( 1 ): 1 ‐ 16 . 30285902 10.1186/s40425-018-0391-1 PMC6171186

57 Palmer DC Guittard GC Franco Z , et al. Cish actively silences TCR signaling in CD8 + T cells to maintain tumor tolerance . J. Exp. Med . 2015 ; 212 ( 12 ): 2095 ‐ 2113 . 26527801 10.1084/jem.20150304 PMC4647263

58 Hammerl D Rieder D Martens JW , et al. Adoptive T cell therapy: new avenues leading to safe targets and powerful allies . Trends Immunol . 2018 ; 39 ( 11 ): 921 ‐ 936 . 30309702 10.1016/j.it.2018.09.004

59 Rahman N . Mainstreaming genetic testing of cancer predisposition genes . Clin. Med . 2014 ; 14 ( 4 ): 436 . 10.7861/clinmedicine.14-4-436 PMC4312836 25099850

60 Zhang M Eshraghian EA Al Jammal O , et al. CRISPR Technology: the engine that drives cancer therapy . Biomed. Pharmacother . 2021 ; 133 : 11007 . 10.1016/j.biopha.2020.111007 33227699

61 Harrington LB Burstein D Chen JS , et al. Programmed DNA destruction by miniature CRISPR-Cas14 enzymes . Sci . 2018 ; 362 ( 6416 ): 839 ‐ 842 . 10.1126/science.aav4294 PMC6659742 30337455

62 Dranoff G . Cytokines in cancer pathogenesis and cancer therapy . Nat. Rev. Cancer . 2004 ; 4 ( 1 ): 11 ‐ 22 . 14708024 10.1038/nrc1252

63 Chen JS Ma E Harrington LB . CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity . Science . 2018 ; 360 ( 6387 ): 436 ‐ 439 . 29449511 10.1126/science.aar6245 PMC6628903

64 Aquino-Jarquin G . Emerging role of CRISPR/Cas9 technology for MicroRNAs editing in cancer ResearchCRISPR/Cas9-mediated MicroRNAs editing . Canc Res . 2017 ; 77 ( 24 ): 6812 ‐ 6817 . 10.1158/0008-5472.CAN-17-2142 29208606

65 Bester AC Lee JD Chavez A , et al. An integrated genome-wide CRISPRa approach to functionalize lncRNAs in drug resistance . Cell . 2018 ; 173 ( 3 ): 649 ‐ 664 . 29677511 10.1016/j.cell.2018.03.052 PMC6061940

66 Chen S Sanjana NE Zheng K , et al. Genome-wide CRISPR screen in a mouse model of tumor growth and metastasis . Cell . 2018 ; 160 ( 6 ): 1246 ‐ 1260 . 10.1016/j.cell.2015.02.038 PMC4380877 25748654

67 McFadden DG Papagiannakopoulos T Taylor-Weiner A , et al. Genetic and clonal dissection of murine small cell lung carcinoma progression by genome sequencing . Cell . 2014 ; 156 ( 6 ): 1298 ‐ 1311 . 24630729 10.1016/j.cell.2014.02.031 PMC4040459

68 Cheng Z Ma R Tan W Zhang LI . MiR-152 suppresses the proliferation and invasion of NSCLC cells by inhibiting FGF2 . Exp Mol Med . 2014 ; 46 ( 9 ): e112 ‐ e112 . 25190353 10.1038/emm.2014.51 PMC4150934

69 Tang JT Wang JL Du W , et al. MicroRNA 345, a methylation-sensitive microRNA is involved in cell proliferation and invasion in human colorectal cancer . Carcinogenesis . 2011 ; 32 ( 8 ): 1207 ‐ 1215 . 21665895 10.1093/carcin/bgr114

70 Sudhakar A . History of cancer, ancient and modern treatment methods . J. Canc. Sci. Ther . 2009 ; 1 ( 2 ): 1 . 10.4172/1948-5956.100000e2 20740081

71 Vogelstein B Papadopoulos N Velculescu VE , et al. Cancer genome landscapes . Science . 2013 ; 339 ( 6127 ): 1546 ‐ 1558 . 23539594 10.1126/science.1235122 PMC3749880

72 Neelapu SS Locke FL Bartlett NL , et al. Axicabtagene ciloleucel CAR T-cell therapy in refractory large B-cell lymphoma . N. Engl. J. Med . 2017 ; 377 ( 26 ): 2531 ‐ 2544 . 29226797 10.1056/NEJMoa1707447 PMC5882485

73 Eyquem J Mansilla-Soto J Giavridis T , et al. Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection . Nature . 2017 ; 543 ( 7643 ): 113 ‐ 117 . 28225754 10.1038/nature21405 PMC5558614

74 https://www.cancer.gov/news-events/cancer-currents blog/2020/crispr-cancer

75 Stadtmauer EA Fraietta JA Davis MM , et al. CRISPR-engineered T cells in patients with refractory cancer . Science . 2020 ; 367 ( 6481 ): eaba7365 . 32029687 10.1126/science.aba7365 PMC11249135

76 U.S. National Library of Medicine URL: https://clinicaltrials.gov/. Accessed on 25th July, 2022

77 Cyranoski D . CRISPR gene-editing tested in a person for the first time . Nature . 2016 ; 539 ( 7630 ). 10.1038/nature.2016.20988 27882996

78 Li-Kuo S Kinzler KW . Multiple intestinal neoplasia caused by a mutation in the murine homolog of the APC gene . Science . 1992 ; 256 ( 5057 ): 668 ‐ 670 . 1350108 10.1126/science.1350108

79 Roper J Tammela T Cetinbas NM , et al. In vivo genome editing and organoid transplantation models of colorectal cancer and metastasis . Nat. Biotechnol . 2017 ; 35 ( 6 ): 569 ‐ 576 . 28459449 10.1038/nbt.3836 PMC5462879

80 Bakshi R Hassan MQ Pratap J , et al. The human SWI/SNF complex associates with RUNX1 to control transcription of hematopoietic target genes . J. Cell. Physiol . 2010 ; 225 ( 2 ): 569 ‐ 576 . 20506188 10.1002/jcp.22240 PMC3045090

81 Ciriello G Gatza ML Beck AH , et al. Comprehensive molecular portraits of invasive lobular breast cancer . Cell . 2015 ; 163 ( 2 ): 506 ‐ 519 . 26451490 10.1016/j.cell.2015.09.033 PMC4603750

82 Annunziato S Kas SM Nethe M , 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 . 27340177 10.1101/gad.279190.116 PMC4926868

83 Raoof S Mulford IJ Frisco-Cabanos H , et al. Targeting FGFR overcomes EMT-mediated resistance in EGFR mutant non-small cell lung cancer . Oncogene . 2019 ; 38 ( 37 ): 6399 ‐ 6413 . 31324888 10.1038/s41388-019-0887-2 PMC6742540

84 Cui CH Liu Y Gerloff D , et al. NOP10 Predicts lung cancer prognosis and its associated small nucleolar RNAs drive proliferation and migration . Oncogene . 2021 ; 40 : 909 ‐ 921 . 33288886 10.1038/s41388-020-01570-y PMC7862062

85 Wohlhieter CA Richards AL Uddin F , et al. Concurrent mutations in STK11 and KEAP1 promote ferroptosis protection and SCD1 dependence in lung cancer . Cell Rep . 2020 ; 33 ( 9 ): 108444 . 33264619 10.1016/j.celrep.2020.108444 PMC7722473

86 Hussmann D Madsen AT Jakobsen KR , et al. IGF1R Depletion facilitates MET-amplification as mechanism of acquired resistance to erlotinib in HCC827 NSCLC cells . Oncotarget . 2017 ; 8 ( 20 ): 33300 . 28418902 10.18632/oncotarget.16350 PMC5464869

87 Van Treuren T Vishwanatha JK . CRISPR Deletion of MIEN1 in breast cancer cells . PLoS One . 2018 ; 13 ( 10 ): e0204976 . 30286132 10.1371/journal.pone.0204976 PMC6171864

88 Carlisle SM Trainor PJ Hong KU . CRISPR/Cas9 knockout of human arylamine N-acetyltransferase 1 in MDA-MB-231 breast cancer cells suggests a role in cellular metabolism . Sci Rep . 2020 ; 10 ( 1 ): 1 ‐ 15 . 32555504 10.1038/s41598-020-66863-4 PMC7299936

89 Mintz RL Lao YH Chi CW , et al. CRISPR/Cas9-mediated mutagenesis to validate the synergy between PARP1 inhibition and chemotherapy in BRCA1-mutated breast cancer cells . Bioeng. Transl. Med . 2020 ; 5(1):e10152 . 10.1002/btm2.10152 PMC6971465 31989039

90 Wu B Jiang WG Zhou DS Cui YX . Knockdown of EPHA1 by CRISPR/CAS9 promotes adhesion and motility of HRT18 colorectal carcinoma cells . Anticancer Res . 2016 ; 36 ( 3 ): 1211 ‐ 1219 . 26977017

91 Li H Zhao L Lau YS , et al. Genome-wide CRISPR screen identifies LGALS2 as an oxidative stress-responsive gene with an inhibitory function on colon tumor growth . Oncogene . 2021 ; 40 ( 1 ): 177 ‐ 188 . 33110234 10.1038/s41388-020-01523-5 PMC7790754

92 Yan F Ying L Li 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 . 28402947 10.18632/oncotarget.16638 PMC5564541

93 Ouyang Q Liu Y Tan J , et al. Loss of ZNF587B and SULF1 contributed to cisplatin resistance in ovarian cancer cell lines based on genome-scale CRISPR/Cas9 screening . Am J Cancer Res . 2019 ; 9 ( 5 ): 988 . 31218106 PMC6556596

94 Zhao GN Wang QH Gu QQ , et al. Lentiviral CRISPR/Cas9 nickase vector mediated BIRC5 editing inhibits epithelial to mesenchymal transition in ovarian cancer cells . Oncotarget . 2018 ; 8 : 94666 ‐ 94680 . 10.18632/oncotarget.21863 PMC5706903 29212257

95 Zhang YQ Pei JH Shi SS , et al. CRISPR/Cas9-mediated knockout of the PDEF gene inhibits migration and invasion of human gastric cancer AGS cells . Biomed. Pharmacother . 2019 ; 111 : 76 ‐ 85 . 30576937 10.1016/j.biopha.2018.12.048

96 Chen W Ding R Tang J , et al. Knocking out SST gene of BGC823 gastric cancer cell by CRISPR/Cas9 enhances migration, invasion and expression of SEMA5A and KLF2 . Cancer Manage Res . 2020 ; 12 : 1313 . 10.2147/CMAR.S236374 PMC7040191 32110105

97 Kim H Jeong H Cho Y , et al. Disruption of the Tff1 gene in mice using CRISPR/Cas9 promotes body weight reduction and gastric tumorigenesis . Lab Anim Res . 2018 ; 34 ( 4 ): 257 ‐ 263 . 30671113 10.5625/lar.2018.34.4.257 PMC6333602

98 Li M Xie H Liu Y , et al. Knockdown of hypoxia-inducible factor-1 alpha by tumor targeted delivery of CRISPR/Cas9 system suppressed the metastasis of pancreatic cancer . J Control Release . 2019 ; 304 : 204 ‐ 215 . 31100311 10.1016/j.jconrel.2019.05.019

99 Bakke J Wright WC Zamora AE , et al. Genome-wide CRISPR screen reveals PSMA6 to be an essential gene in pancreatic cancer cells . BMC Cancer . 2019 ; 19 ( 1 ): 1 ‐ 12 . 30898113 10.1186/s12885-019-5455-1 PMC6429770

100 Hou C Zhuang Z Deng X , et al. Knockdown of trio by CRISPR/Cas9 suppresses migration and invasion of cervical cancer cells . Oncol Rep . 2018 ; 39 ( 2 ): 795 ‐ 801 . 29207149 10.3892/or.2017.6117

101 Slotta C Schlüter T Ruiz-Perera LM , et al. CRISPR/Cas9-mediated knockout of c-REL in HeLa cells results in profound defects of the cell cycle . PloS One . 2017 ; 12 ( 8 ): e0182373 . 28767691 10.1371/journal.pone.0182373 PMC5540532

102 Lamouille S Xu J Derynck R . Molecular mechanisms of epithelial–mesenchymal transition . Nat. Rev. Mol. Cell Biol . 2014 ; 15 : 178 ‐ 196 . 24556840 10.1038/nrm3758 PMC4240281

103 Haraguchi M Sato M Ozawa M . CRISPR/ Cas9n-mediated deletion of the snail1 gene (SNAI1) reveals its role in regulating cell morphology, cell–cell interactions, and gene expression in ovarian cancer (RMG-1) cells . PLoS One . 2015 ; 10 ( 7 ): e0132260 . 26161782 10.1371/journal.pone.0132260 PMC4498756

104 Ribeiro JR Schorl C Yano N , et al. HE4 Promotes collateral resistance to cisplatin and paclitaxel in ovarian cancer cells . J Ovarian Res . 2016 ; 9 ( 1 ): 1 ‐ 18 . 27184254 10.1186/s13048-016-0240-0 PMC4869286

105 Faddaoui A Bachvarova M Plante M , et al. The mannose receptor LY75 (DEC205/CD205) modulates cellular phenotype and metastatic potential of ovarian cancer cells . Oncotarget . 2016 ; 7 ( 12 ): 14125 . 26871602 10.18632/oncotarget.7288 PMC4924702

106 Shetty DK Inamdar MS . Generation of a heterozygous knockout human embryonic stem cell line for the OCIAD1 locus using CRISPR/ CAS9 mediated targeting: BJNhem20-OCIAD1CRISPR-39 . Stem Cell Res . 2016 ; 16 ( 2 ): 308 ‐ 310 . 27345991 10.1016/j.scr.2015.12.037

107 Liu Y Hu X Han C . Targeting tumor suppressor genes for cancer therapy . Bioessays . 2015 ; 37 ( 12 ): 1277 ‐ 1286 . 26445307 10.1002/bies.201500093 PMC8638220

108 Chen W Zheng R Baade PD , et al. Cancer statistics in China, 2015 . CA Cancer J Clin . 2016 ; 66 ( 2 ): 115 ‐ 132 . 26808342 10.3322/caac.21338

109 Islami F Sauer AG Miller KD , et al. Proportion and number of cancer cases and deaths attributable to potentially modifiable risk factors in the United States . CA Cancer J. Clin . 2018 ; 68 ( 1 ): 31 ‐ 54 . 29160902 10.3322/caac.21440

110 Vigneswaran N Wu J Sacks P , et al. Microarray gene expression profiling of cell lines from primary and metastatic tongue squamous cell carcinoma: possible insights from emerging technology . J. Oral. Pathol. Med . 2005 ; 34 ( 2 ): 77 ‐ 86 . 15641986 10.1111/j.1600-0714.2004.00258.x

111 Alexandrov LB Nik-Zainal S Wedge DC , et al. Signatures of mutational processes in human cancer . Nature . 2013 ; 500 ( 7463 ): 415 ‐ 421 . 23945592 10.1038/nature12477 PMC3776390

112 Bhakta MS Henry IM Ousterout DG , et al. Highly active zinc-finger nucleases by extended modular assembly . Genome Res . 2013 ; 23 ( 3 ): 530 ‐ 538 . 23222846 10.1101/gr.143693.112 PMC3589541

113 Moscou MJ Bogdanove AJ . A simple cipher governs DNA recognition by TAL effectors . Science . 2019 ; 326 ( 5959 ): 1501 . 10.1126/science.1178817 19933106

114 Moses C Kaur P . Applications of CRISPR systems in respiratory health: entering a new ‘red pen’ era in genome editing . Respirology . 2019 ; 24 ( 7 ): 628 ‐ 637 . 30883991 10.1111/resp.13527

115 Sanchez-Rivera FJ Jacks T . Applications of the CRISPRCas9 system in cancer biology . Nat Rev Cancer . 2015 ; 15 ( 7 ): 387 ‐ 395 . 26040603 10.1038/nrc3950 PMC4530801

116 Liu W Li L Jiang J , et al. Applications and challenges of CRISPR-Cas gene-editing to disease treatment in clinics . Precis Clin Med . 2021 ; 4 ( 3 ): 179 ‐ 191 . 34541453 10.1093/pcmedi/pbab014 PMC8444435

117 Gill J Ahluwalia MK Geller D , et al. New targets and approaches in osteosarcoma . Pharmacol Ther . 2013 ; 137 ( 1 ): 89 ‐ 99 . 22983152 10.1016/j.pharmthera.2012.09.003

118 Botter SM Neri D Fuchs B . Recent advances in osteosarcoma . Curr. Opin. Pharmacol . 2014 ; 16 : 15 ‐ 23 . 24632219 10.1016/j.coph.2014.02.002

119 Saha D Saha K Banerjee A , et al. Osteosarcoma relapse as pleural metastasis . South Asian J. Cancer . 2013 ; 2 ( 2 ): 56 . 10.4103/2278-330X.110483 PMC3876644 24455551

120 Kundu ZS . Classification, imaging, biopsy and staging of osteosarcoma . Indian J. Orthop . 2014 ; 48 ( 3 ): 238 ‐ 246 . 24932027 10.4103/0019-5413.132491 PMC4052020

121 Staddon AP Lackman R Robinson K , et al. Osteogenic sarcoma presenting with lung metastasis . J. Oncol . 2002 ; 7 ( 2 ): 144 ‐ 153 . 10.1634/theoncologist.7-2-144 11961198

122 Kansara M Teng MW Smyth MJ , et al. Translational biology of osteosarcoma . Nat. Rev. Cancer . 2014 ; 14 ( 11 ): 722 ‐ 735 . 25319867 10.1038/nrc3838

123 Link MP Goorin AM Miser AW , et al. The effect of adjuvant chemotherapy on relapse-free survival in patients with osteosarcoma of the extremity . N. Eng. J. Med . 1986 ; 314 ( 25 ): 1600 ‐ 1606 . 10.1056/NEJM198606193142502 3520317

124 Meyers PA Healey JH Chou AJ , et al. Addition of pamidronate to chemotherapy for the treatment of osteosarcoma . Cancer . 2011 ; 117 ( 8 ): 736 ‐ 1744 . 10.1002/cncr.25744 PMC3059356 21472721

125 Hoeben A Landuyt B Highley MS , et al. Vascular endothelial growth factor and angiogenesis . Pharmacol. Rev . 2004 ; 56 ( 4 ): 549 ‐ 580 . 15602010 10.1124/pr.56.4.3

126 Goel HL Mercurio AM . VEGF Targets the tumour cell . Nat. Rev. Cancer . 2013 ; 13 ( 12 ): 871 ‐ 882 . 24263190 10.1038/nrc3627 PMC4011842

127 Niu G Chen X . Vascular endothelial growth factor as an anti-angiogenic target for cancer therapy . Curr. Drug Targets . 2010 ; 11 ( 8 ): 1000 ‐ 1017 . 20426765 10.2174/138945010791591395 PMC3617502

128 Yang J Yang D Sun Y , et al. Genetic amplification of the vascular endothelial growth factor (VEGF) pathway genes, including VEGFA, in human osteosarcoma . Cancer . 2011 ; 117 ( 21 ): 4925 ‐ 4938 . 21495021 10.1002/cncr.26116 PMC3465081

129 Tanaka T Yui Y Naka N , et al. Dynamic analysis of lung metastasis by mouse osteosarcoma LM8: VEGF is a candidate for anti-metastasis therapy . Clin. Exp. Metastasis . 2013 ; 30 ( 4 ): 369 ‐ 379 . 23076771 10.1007/s10585-012-9543-8 PMC3616224

130 Daft PG Yang Y Napierala D , et al. The growth and aggressive behavior of human osteosarcoma is regulated by a CaMKII-controlled autocrine VEGF signaling mechanism . PloS One . 2015 ; 10 ( 4 ): e0121568 . 25860662 10.1371/journal.pone.0121568 PMC4393114

131 Zhao J Zhang ZR Zhao N , et al. VEGF Silencing inhibits human osteosarcoma angiogenesis and promotes cell apoptosis via PI3K/AKT signaling pathway . Int. J. Clin. Exp. Med . 2015 ; 8 ( 2 ): 12411 ‐ 7 . 26550152 PMC4612837

132 Ryan CJ Smith MR De Bono JS , et al. Abiraterone In metastatic prostate cancer without previous chemotherapy . N. Engl. J. Med . 2013 ; 368 ( 2 ): 138 ‐ 148 . 23228172 10.1056/NEJMoa1209096 PMC3683570

133 Beer TM Armstrong AJ Rathkopf DE , et al. Enzalutamide in metastatic prostate cancer before chemotherapy . N. Engl. J. Med . 2014 ; 371 ( 5 ): 424 ‐ 433 . 24881730 10.1056/NEJMoa1405095 PMC4418931

134 Smith MR Saad F Chowdhury S , et al. Apalutamide treatment and metastasis-free survival in prostate cancer . N. Engl. J. Med . 2018 ; 378 ( 15 ): 1408 ‐ 1418 . 29420164 10.1056/NEJMoa1715546

135 Abeshouse A Ahn J Akbani R , et al. The molecular taxonomy of primary prostate cancer . Cell . 2015 ; 163 ( 4 ): 1011 ‐ 1025 . 26544944 10.1016/j.cell.2015.10.025 PMC4695400

136 Robinson D Van Allen EM Wu YM , et al. Integrative clinical genomics of advanced prostate cancer . Cell . 2015 ; 161 ( 5 ): 1215 ‐ 1228 . 26000489 10.1016/j.cell.2015.05.001 PMC4484602

137 Abida W Cyrta J Heller G , et al. Genomic correlates of clinical outcome in advanced prostate cancer . Proc. Natl. Acad. Sci . 2019 ; 116 ( 23 ): 11428 ‐ 11436 . 31061129 10.1073/pnas.1902651116 PMC6561293

138 Shalem O Sanjana NE Hartenian E , et al. Genome-scale CRISPR-Cas9 knockout screening in human cells . Science . 2014 ; 343 ( 6166 ): 84 ‐ 87 . 24336571 10.1126/science.1247005 PMC4089965

139 Wang T Wei JJ Sabatini DM , et al. Genetic screens in human cells using the CRISPR-Cas9 system . Science . 2014 ; 343 ( 6166 ): 80 ‐ 84 . 24336569 10.1126/science.1246981 PMC3972032

140 Hart T Chandrashekhar M Aregger M , et al. High-resolution CRISPR screens reveal fitness genes and genotype-specific cancer liabilities . Cell . 2015 ; 163 ( 6 ): 1515 ‐ 1526 . 26627737 10.1016/j.cell.2015.11.015

141 Behan FM Iorio F Picco G , et al. Prioritization of cancer therapeutic targets using CRISPR–Cas9 screens . Nature . 2019 ; 568 ( 7753 ): 511 ‐ 516 . 30971826 10.1038/s41586-019-1103-9

142 Takei N Yoneda A Sakai-Sawada K , et al. Hypoxia-inducible ERO1alpha promotes cancer progression through modulation of integrin-beta1 modification and signalling in HCT116 colorectal cancer cells . Sci. Rep . 2017 ; 7 ( 1 ): 9389 . 28839225 10.1038/s41598-017-09976-7 PMC5571208

143 Novellasdemunt L Foglizzo V Cuadrado L , et al. USP7 Is a tumor-specific WNT activator for APC-mutated colorectal cancer by mediating β-catenin deubiquitination . Cell Rep . 2017 ; 21 ( 3 ): 612 ‐ 627 . 29045831 10.1016/j.celrep.2017.09.072 PMC5656747

144 Oh S You E Ko P , et al. Genetic disruption of tubulin acetyltransferase, αTAT1, inhibits proliferation and invasion of colon cancer cells through decreases in Wnt1/β-catenin signaling . Biochem. Biophys. Res. Commun. 2017 ; 482 ( 1 ): 8 ‐ 14 . 27836544 10.1016/j.bbrc.2016.11.039

145 Drost J van Jaarsveld RH Ponsioen B , et al. Sequential cancer mutations in cultured human intestinal stem cells . Nature . 2015 ; 521 ( 7550 ): 43 ‐ 47 . 25924068 10.1038/nature14415

146 Hannafon BN Cai A Calloway CL , et al. miR-23b and miR-27b are oncogenic microRNAs in breast cancer: evidence from a CRISPR/Cas9 deletion study . BMC Cancer . 2019 ; 19 ( 1 ): 642 . 31253120 10.1186/s12885-019-5839-2 PMC6599331

147 Luo S Chen J Mo X . The association of PTEN hypermethylation and breast cancer: a meta-analysis . Onco Targets Ther . 2016 ; 9 : 5643 ‐ 5650 . 27672335 10.2147/OTT.S111684 PMC5026181

148 Yang L Yu SJ Shao ZM . Abstract P2-01-16: CRISPR-Cas9 screen identifies TMEM106A as a suppressor of breast cancer metastasis . Can. Res. 2019 ; 79 : P2 ‐ 01 .

149 Behbahani RG Danyaei A Teimoori A , et al. Breast cancer radioresistance may be overcome by osteopontin gene knocking out with CRISPR/Cas9 technique . Cancer/Radiothér . 2021 ; 25 ( 3 ): 222 ‐ 228 . 10.1016/j.canrad.2020.08.048 33422414

150 Yang M Zeng C Li P , et al. Impact of CXCR4 and CXCR7 knockout by CRISPR/Cas9 on the function of triple-negative breast cancer cells . Onco Targets Ther . 2019 ; 12 : 3849 ‐ 3858 . 31190884 10.2147/OTT.S195661 PMC6527053

151 Zhu W Liu C Lu T , et al. Knockout of EGFL6 by CRISPR/Cas9 mediated inhibition of tumor angiogenesis in ovarian cancer . Front Oncol . 2020 ; 10 : 1451 . 32983976 10.3389/fonc.2020.01451 PMC7477343

152 Huo W Zhao G Yin J , 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 ‐ 64 . 28123598 10.7150/jca.16723 PMC5264040

153 Xu M Huang S Chen J , et al. Cytotoxic lymphocytes-related gene ITK from a systematic CRISPR screen could predict prognosis of ovarian cancer patients with distant metastasis . J. Transl. Med . 2021 ; 19 ( 1 ): 447 . 34702300 10.1186/s12967-021-03119-3 PMC8549276

154 Bu X Kato J Hong JA , et al. CD38 Knockout suppresses tumorigenesis in mice and clonogenic growth of human lung cancer cells . Carcinogenesis . 2018 ; 39 ( 2 ): 242 ‐ 251 . 29228209 10.1093/carcin/bgx137 PMC5862338

155 Tang KJ Constanzo JD Venkateswaran N , et al. Focal adhesion kinase regulates the DNA damage response and its inhibition radiosensitizes mutant KRAS lung cancer . Clin. Cancer Res . 2016 ; 22 ( 23 ): 5851 ‐ 5863 . 27220963 10.1158/1078-0432.CCR-15-2603 PMC5122471

156 Chen X Sun X Guan J , et al. Rsf-1 influences the sensitivity of non-small cell lung cancer to paclitaxel by regulating NF-κB pathway and its downstream proteins . Cell. Physiol. Biochem . 2017 ; 44 ( 6 ): 2322 ‐ 2336 . 29258089 10.1159/000486116

157 Cheung AHK Chow C Zhang J , et al. Specific targeting of point mutations in EGFR L858R-positive lung cancer by CRISPR/Cas9 . Lab Invest . 2018 ; 98 ( 7 ): 968 ‐ 976 . 29748615 10.1038/s41374-018-0056-1

158 Zhou X Curbo S Li F , et al. Inhibition of glutamate oxaloacetate transaminase 1 in cancer cell lines results in altered metabolism with increased dependency of glucose . BMC Cancer . 2018 ; 18 ( 1 ): 1 ‐ 14 . 29751795 10.1186/s12885-018-4443-1 PMC5948873

159 Romero R Sayin VI Davidson SM , et al. Keap1 loss promotes KRAS-driven lung cancer and results in dependence on glutaminolysis . Nat Med . 2017 ; 33 ( 11 ): 1362 ‐ 1368 . 10.1038/nm.4407 PMC5677540 28967920

160 Li CG Pu MF Li CZ , et al. MicroRNA-1304 suppresses human non-small cell lung cancer cell growth in vitro by targeting heme oxygenase-1 . Acta Pharmacol Sin . 2017 ; 38 ( 1 ): 110 ‐ 119 . 27641735 10.1038/aps.2016.92 PMC5220548

161 Yang D Shi Y Tang Y , et al. Effect of HPV infection on the occurrence and development of laryngeal cancer: a review . J Cancer . 2019 ; 10 ( 19 ): 4455 . 31528209 10.7150/jca.34016 PMC6746124

162 Matano M Date S Shimokawa M , et al. Modeling colorectal cancer using CRISPR-Cas9–mediated engineering of human intestinal organoids . Nat. Med . 2015 ; 21 ( 3 ): 256 ‐ 262 . 25706875 10.1038/nm.3802