Cancer Therapy with CRISPR/Cas9: Prospects and Challenges

Globally, Cancer is believed to be second biggest reason of mortality and one of the significant social as well as economic liabilities. Despite our advance at molecular level in comprehension of cancer, more therapeutic tools and tactics are needed to exploit this advance. The CRISPER/Cas9 genome modifying approach has lately appeared as an effective cancer therapy method due to its high accuracy and efficiency. CRISPER/Cas9 has enormous clinical potential in discovering new targets for cancer treatment and also to dismember genetic-chemical interaction thus helping us to understand the response of tumor to the treatment by drugs. Additionally, Cas9/CRISPER can also be used in cancer immunotherapeutic applications by engineering immune cells and oncolytic viruses. Perhaps the most important therapeutic application of Cas9/CRISPER is its ability to edit genes with great precision both in animal models and humans. In this review, we will debate and explore some important concerns of using CRISPER/Cas9 in remedial settings and some vital hurdles that are needed to overcome before it is used for a clinical trial for a polygenic and complex ailment like cancer. Keywords— Advance therapy; CRISPER; Cas9; Genome engineering; Cancer models. I. BENEFITS AND TECHNIQUES OF CRISPER

ISSN: 2456-1878 https://dx.doi.org/10.22161/ijeab. 63. 16 144 cost-effectiveness, adaptibility, and convenient for use, thus innovating the work of genome related engineering. Cas9 technology is based on a bacterial and archaeal immune defence mechanism that protects the host from viruses and phages that target nucleic acids (Barrangou et al., 2007). According to their most popular classification, CRISPER/Cas system is classified into three main types, all with various sub classification(Makarova et al., 2011). The most widely used gene editing system is the type II CRISPR/Cas system, which consists of three components: an endonuclease (Cas9), a CRISPR RNA (crRNA), and a transactivatingcrRNA (tracrRNA) (Jinek et al., 2012). The guide RNA (gRNA) is a duplex structure formed by the crRNA and tracrRNA molecules which can be substituted using a fused synthetic chimeric single gRNA (sgRNA), making CRISPR/Cas9 easier to use in genome engineering (Jinek et al., 2012).The sgRNA carries a special sequence of approximately 20 base-pairs (bp) and is intended to complement the DNA site that is targeted, and it should be accompanied by a small length DNA sequence known as the "protospacer-adjacent motif" (PAM), that is required for affinity of Cas9 protien. The expression of sgRNA as well as Cas9 nuclease in the cell create a ribonucleoprotein (RNP) complex, which is directed to a target DNA site by the sgRNA. Cas9 specifically cleaves the DNA to generate a DSB after the sgRNA binds to the target sequence using Watson-Crick base-pairing. The cleavage takes place inside the protospacer, three nucleotides upstream of the PAM, resulting in blunt ends. Cas9 active-site motifs RuvC and HNH, which acts on the (-) and (+) strands respectively, are liable for splitting of opposite DNA strands ( Relevant sequences or mutations may be introduced into a target area of the genome using homology-directed repair. A more common NHEJ pathway is error-prone mechanism that generates frameshift mutations at the DSB siteby randomly inserting or deleting nucleotides (indels). It may thus be used to cause specific gene knockouts (Fig. 1).
Cas9/CRISPER techniques permit for accurate as well as effective splitting of desired targeted DNA sequences, and it has greatly enabled genome editing due to the relative ease along with clarity of constructing sgRNAs. The use of diverse sgRNAs allows this technology to be multiplexed, which is an additional benefit. Only the CRISPR/Cas9 method, among genome editing nucleases, can edit several loci at the same time by adding sgRNAs to different locations (Jakočinas et al., 2015;Li, Teng, Li, & Zhou, 2013).Where two sgRNAs are used in the same cell, minor deletions (Wyman et al., 2013), complex rearrangements(P. S. Choi & Meyerson, 2014;Torres, Martin, et al., 2014), and even wholechromosome suppression can occur(Adikusuma, Williams, Grutzner, Hughes, & Thomas, 2017). One more significant benefit of Cas9/CRISPR is its adaptability: amendments and personalization of Cas9/CRISPR modules and also interactors have upgraded the system's precision and efficacy while also broadening its scope of applications beyond editing (Dominguez, Lim, & Qi, 2016). CRISPR technology's DNA precision has become a main focus in the work field and the existence of offtarget activity is shown by many experiments (Tsai & Joung, 2016). As a result, a number of tactics have been devised to reduce the products that are off-target. Out of various methods, one method uses a cellular delivery of in vitro-assembled RNP complexes instead of plasmid delivery, which generates longer-lived Cas9 and sgRNA expression, as well as a higher ratio of on-target:off-target editing of genes in the cells of mammals and vastly effective editing ( (Oakes et al., 2016).The Cas9 was modified to induce break just one strands of DNA, researchers were able to use pair of Cas9 nickases directed by two different gRNAs aiming at the same locus but on opposing DNA strands. This technique creates highly precise DNA splitting with efficacy similar to traditional Cas9/CRISPR but with less far-off incidents (Ran et al., 2013). A similar strategy uses two Cas9 which are catalytically inactivated mutants fused to (fCas9) FokI nuclease (directed by two opposing gRNAs projecting at the same position), in which the only dimer (fCas9) is functional. In human cells, Fokl nucleases were found to alter a specified location with >150-folds greater accuracy than Cas9 which are wild-type nucleases (Guilinger, Thompson, & Liu, 2014 technology has a number of benefits over prior genome editing programmable nucleases, but it also has some disadvantages. CRISPR/Cas9 performance and sequence specificity requires to be developed beyond. Effects that are off-target must also be minimized, and developing a CRISPR/Cas9 delivery system that is reliable, stable, and cell-specific remains a major challenge. In the absence of homologous repair template, NHEJ usually results in deletions or insertions of arbitrary base pairs disturbing the selected sequence. By supplying a donor DNA template and using the homology-driven repair pathway, specific genome edition can also be achieved.

II. DRUG EXPLORATION WITH CRISPER
Drug discovery and production is a lengthy and complicated procedure that involves recognizing novel products and presenting them in the market. Typically, the procedure starts with the proposition that disrupting certain biological target will result in one useful outcome that will alter disease progression. These targets should be confirmed in physiologically pertinent models of animals prior to clinical models whose pharmacological adjustment may lead to the required therapeutic impact.
In cancer studies, drug formulation aims to recognize molecules against genetic defects in tumor suppressor gene as well as oncogenes that lead to tumor formation.

III. DRUG TARGET EXPLORATION USING CRISPER/CAS9 LIBRARY SCREENS
The discovery of genes that are not known and the finding out about their role are normally done using highthroughput genetic screening platforms. Screening of mutations has been utilized to recognize fundamental biotic procedures and pathways for signaling, and it can also be used to establish genes which are accountable for a specified phenotype. The major restraint of mutagenesis screening for targeted discovery of drug with the mutations that are not known is the propagation of heterozygous mutants. Targeted

IV. DRUG RESISTANCE AND CRISPER/CAS9
Finding of genes that play a part in resistance of drugs is a crucial implementation of CRISPER/Cas9 in drug finding. Conventionally, worldwide mutagenesis across a cell population is used to evaluate the mechanism of resistance of anticancer agents. Only cells carrying the mutations that spoil the action of drug would survive in ensuing usage of drug to be tested. However, generation of significant number of false positives is the drawback of this approach (Guichard, 2017

V. DRUG EFFECTIVENESS MODELS OF DISEASES
In drug development, cells and animal models are of substantial importance. Before clinical testing on humans, experiments must be performed in models to test drug effectiveness and toxicity. Most subjects, encompassing cell as well as animal models, are not able to, however, accurately represent the condition observed in patients. Moreover, to generate subjects that precisely summarize the variety and complexity of disease are very expensive and time consuming process. Cancer cell line can be modified to accurately mimic the deviations seen in patients by using CRISPER/Cas9 and it is cheap as compared to standard protocol. Model of ovarian cancer of mice ID8 was altered to hinder TP53 and BRCA2 which ultimately resulted in increased sensitivity to inhibit PARP, is a good example (Walton et al., 2016).
The example above demonstrates clearly that CRISPER/Cas9 platform has become vital element of drug discovery in oncology. This mechanization has enhanced the finding and authentication of novel drug targets, as well as providing more accurate models of human diseases for evaluating safety of drug in a more prognostic way as well as reducing and combating drug resistance.

VI. CRISPER AS A CANCER-FIGHTING TOOL
Although there is some advancement in past decade but significant number of people still die due to cancer which demonstrate the dire need for novel and more effective therapeutic options. CRISPER/Cas9 genome editing has a great potential in cancer therapeutic besides its use as a research tool. The regulation of endogenous gene expression is a probable application of CRISPER/Cas9 system in Cancer therapy. As discussed above, gRNAs can be used to recruit catalytically inactive dCas9 to specific target DNA sites (Friedland et al., 2013) and can also be utilized to activate or suppress particular target genes by fusing it with transcriptional activation or inhibition domain (Chen et al., 2013). Epigenome editing could be another therapeutic application based on linking dCas9 to histone modifiers and proteins involved in altering DNA methylation (Klann et al., 2017). Finally, by specifically targeting tumor markers in cancer cells, it allows for the elimination of genetic changes that can contribute to tumor proliferation and/or metastatic capacity (Shachaf et al., 2004). However, the effectual delivery of CRISPER component in all the cancer cells is still a challenge.
Elaborate interaction between tumor, host and environment is needed for effective immunity against cancer cells because cancer is a complex disease. Immunotherapy, which targets PD-1 or increases immune action to cancer cells that has chimeric antigen therapy (CAR) therapy, has recently emerged as a promising treatment choice for cancer (Shachaf et al., 2004). Unlike chemotherapy or radiotherapy, Cancer immunotherapy has many benefits like durable activity, favorable benefits, and low risk ratio. The wild type of adenovirus, in case of DNA tumor viruses, encodes protein (E1A) that can bind to pRb, and therefore capturing the cycle of cell by release of transcription factor E2F. The release of this transcriptional factor E2F also causes an orderly activation of genes which are viral, resulting in the propagation of new viruses, which then cause the infected cell to lyse and release novel virus. Because cancerous cells usually contain genetic changes in the pathway named Rb, the EIA gene has been knocked out of oncolytic adenoviruses to avoid replication. Production of the next generation CAR T-cells that are genetically altered to present tumor-targeting receptor is another impressive anti-cancer immune therapy that has a great potential for the treatment of hematological and solid cancers (Maus, Grupp, Porter, & June, 2014). Intracellular chimeric signaling domain that is capable of activating Tcells and an extracellular binding domain that recognize a highly specific antigen for and strongly expressed on tumor cells, together constitute a chimeric antigen receptor (CAR) and both domains work in combination to reprogram T-cell facilitated killing of Tumor cells. In 2016, an oncologist team led by LU You at Sichuan University in China became the first to inject T-cells modified by CRISPER/Cas9 to disable PD-1 into patients with aggressive lung cancer (Cyranoski, 2016). Although ACT therapies are of great potential in the treatment of leukemia and lymphoma, but some individuals died while conducting trial phases due to neurotoxicity and cytokine release syndrome (Gauthier & Turtle, 2018). At this moment, FDA has approved CAR T-cell therapy only for the treatment of relapsed and refractory B-cell acute lymphoblastic leukemia in paediatric and young adults (Kansagra & Litzow, 2017).  (Sun et al., 2015). Moreover, Cas9 RNP complexes with donor DNA was shown to be delivered by gold nanoparticle combined with DNA a well as further added with disruptive polymers that are endosomal and cationic, could induce homology DNA repair (HDR) to fix DNA mutant variations of Duchenne muscular dystrophy in the mice(Lee et al., 2017). Goldnanoparticle is an excellent carrier that is not toxic for gene and drug delivery application because gold core of the particle provides solidity to the assemblage, meanwhile the monolayer permits surface tuning of properties like hydrophobicity and charge. There is still need for testing the safety and efficiency of this method, but it is expected CRISPER components delivery mechanism. As nanoparticles that are inorganic including bare mesoporus or dense silica nanoparticles and carbon nanotubes have already been used for various purposes, they are the natural and potential carriers of CRISPER components (Xu, Zeng, Lu, & Yu, 2006).Furthermore, inorganic nanoparticles have some other benefits including their reproducible composition, size and stability over time as well as simplicity to generate them.

VIII. CONCLUSION
Although CRISPER/Cas9 based technology is still in development but it has already displayed its potential in research and hold great therapeutic promise but for favorable clinical application of this technology, secure and efficient transport into selected tissue is required. There are high expectations for this technology, which necessitated careful planning, such as allowing regulatory processes for its development. However, the technology still requires optimization mainly with respects to safety, specificity and efficacy before its widespread translation into clinics. Despite the many obstacles that must be met, we expect that the continued development of genetic science will significantly contribute to existing cancer therapies.