Cancer being one of the major causes of disease associated mortality along with being increased with time worldwide. The main pillar in developing therapeutic and medicine for cancer involves improved understanding of tumor biology. The studying of tumor biology led to understanding of the key proteins of oncogenic signalling pathway. This led to development of small molecules and antibiotics which targeted specific cancer but not all. Thus in order to treat those with unknown treatment or resistance mechanisms we had to undertake efforts to characterize genetic blueprints of cancer and tumor biology and get to the depth of carcinogenesis and treatment response. There’s where CRISPR/Cas9 comes into play. This is an important genetic tool that has capabilities to edit genome of several organisms. It has its several modified versions which help in engineering genomes and also to activate and repress expression of genes. So this article focuses on how CRISPR/Cas9 is used for development in therapeutic for cancer. This article further progress towards introduction towards CRISPR/Cas9 and how it is developed and modified which in turn help towards cancer therapeutics.
CRISPR basically stands for Clustered regularly interspersed short palindromic repeats. This was discovered first from E. coli in 1987. This wasn’t earlier clear but the beginning of this century it was discovered that they play role in adaptive immunity in bacteria against viruses. Their major function is to guide and activate Cas proteins to bind viral DNA sequences which are subsequently cleaved. Thus DNA cleavage is what is done by these proteins. If this system is modified a little, we are able to cleave as well as replace that DNA strand with desired strand of DNA. Holding these advantages CRISPR, cas9 stands as a major gene editing tool. This system is also developed in a manner that it can perform other functions like CRISPRi (CRISPR interference) and CRISPRa (CRISR activator), both f this exploits fused transcription regulators to repress or induce gene transcription when Dcas9 is directed t the transcription start site of target gene. Both mentioned systems lack nucleases. Some examples for CRISPRa are VPR, SAM and SunTag. These are one of the most effective systems. Also recently Cas9 variants have been engineered that induce specific single nucleotide base change. In its mechanism DBS (double strand break) are not made, instead this system relies on Cas9 nickase coupled to cystidine deaminases domains to induce targeted transition from C to T or G to A.
All this described CRISPR systems can be used by scientists in several places which could help them understand the genetic blueprint of tumor biology and develop respective therapeutics. With help of these CRISPR Cas9 systems the scientist produce CRISPR pooled screens. This is done by talking a population of small oligonucleotide which can target specific gene in tumor cell and this can induce a knockout in that cells which change its genotypic characteristic and so its respective phenotype can be studied. This allows us to study the role of genes in tumor cells. There are certain libraries that help us design this tool which contains information about guide RNA and their efficacy and effectiveness. For a scientist it is important to understand so called `genotype specific vulnerabilities`. These essential genes can be potential drug target as their functional depletion leads to reduces viabilities.
Let’s take an example and understand it better. To identify essential genes for acute myeloid leukaemia (AML) Tezlepis et al. focused on eight AML cell lines and identified 924 candidate among which KAT2A was characterized and leukaemia specific target (1). One more similar approach was seen in AML where its cell lines harbouring a mutation in the NPM1 gene specifically depend on the menin binding site of the MLL1 protein(2). Supporting this observational, pharmacological, inhibition of this binding site has a pronounced anti leukemic effect. Keep to mind that sometimes this essential gene have also showed lethal if they are disrupted by any means, thus not every gene qualify to be druggable or be potential site for repressing a tumour.
One of the most popular in vitro model in adult stem cell derived organoid which are used for several in vitro studies. We can derive stem cells from healthy tissue and cultivate them in media, they can be developed into 3D cultures with help of tissue specific growth factor. We can develop such cancerous tissue and compare it with normal tissue organoid and infer many things about development of that cancerous organoid. In such studies CRISPR Cas9 plays its important role in converting that normal organoid to cancerous one.
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