Team:AFCM-Egypt/Description

Project Description



Introduction



Liver cancer is a leading cause of cancer deaths worldwide, accounting for more than 600,000 deaths each year The American Cancer Society’s estimates -of primary liver cancer and intrahepatic bile duct cancer in the United States for 2016 are about 39,230 of newly diagnosed cases - and 27,170 died people of these cancers(ⅰ) .In Egypt; Liver cancer is a serious if not the most serious cancer problem. It is ranked the first among cancers in males (33.6%) and next to breast cancer among females based upon results of National Cancer Registry Program (NCRP 2008-2011)(ⅱ) .The rising rates of HCC in Egypt are due to the high prevalence of hepatitis B virus (HBV) and hepatitis C virus infection (HCV) among Egyptian population ( ⅲ). Therefore; we need effective strategies for early detection and better management of HCC which will be of great value in developing countries with limited resources and high incidence rates of HCC, such as Egypt.

It is well known that the human genome is actively transcribed; however, there are only about 20, 000 protein-coding genes, accounting for about 2% of the genome, and the rest of the transcripts are non-coding RNAs including microRNAs and long non-coding RNAs (lncRNAs). For instance, over 56, 000 human lncRNAs have been identified to date ; however, the biological function for most of them is not known. Therefore, there is a critical need for functional studies of lncRNAs. A most commonly used approach for gene functional study is knockdown by RNA inference (RNAi) which is mainly functional in the cytoplasm where RISC complexes are located. However, many lncRNAs are localized to the nucleus ( ⅳ), which can make it difficult to achieve robust knockdown. Thus, genetic editing at the genomic level provides a better alternative because it targets the genomic DNA. There are several genetic tools available for this purpose, including zinc finger nuclease (ZFN) and transcription activation-like element nuclease (TALEN) (( ⅴ), ). For example, type II restriction enzyme FokI is often used as a cleavage domain in ZFN ; similarly, engineered TAL effectors can also be fused to the cleavage domain of FokI to create TALENs for genome editing (ⅵ ). Recently, a novel genetic engineering tool called clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated (Cas) system is more advanced because of easy generation and high efficiency of gene targeting . Importantly, it only requires changing the sequence of the guide RNA (gRNA); and it can be directly delivered into embryos, to generate sequence-modified animals (ⅶ ). Furthermore, multiplexing capability of CRISPR/Cas makes it possible to target multiple genes simultaneously.

CRISPR/Cas9 has rapidly gained popularity due to its superior simplicity ( ⅷ ). In this system, a single guide RNA (sgRNA) complexes with Cas9 nuclease, which can recognize a variable 20-nucleotide target sequence adjacent to a 5′-NGG-3′ protospacer adjacent motif (PAM) and introduce a DSB in the target DNA (ⅸ ). The induced DSB then triggers DNA repair process mainly via two distinct mechanisms, namely, the non-homologous end joining (NHEJ) and the homology-directed repair (HDR) pathways. Clustered regularly interspaced short palindromic repeats (CRISPR) loci and their associated cas (CRISPR-associated) genes provide adaptive immunity against viruses (phages) and other mobile genetic elements in bacteria and archaea. While most of the early work has largely been dominated by examples of CRISPR-Cas systems directing the cleavage of phage or plasmid DNA, recent studies have revealed a more complex landscape where CRISPR-Cas loci might be involved in gene regulation.

In human cells, efficient knock-in of foreign DNA into a selected genomic locus has been long awaited. It is anticipated to facilitate various applications, ranging from gene function study to therapeutic genome editing. Currently, most studies have focused on HDR-based strategies, and the rate of targeted integration was reported to be low (ⅹ ). This is because HDR in human cells is intrinsically inefficient, whereas NHEJ-mediated DNA repair is prevalent ( ⅺ). These properties result in generation of few target clones amid a large number of random integrations. Notably, in human embryonic stem cells (ESCs) ( ⅻ) and induced pluripotent stem cells (iPSCs) (), which are pluripotent and possess unprecedented potentials for basic research and cell-based therapies (), gene targeting via HDR is found to be particularly difficult and has imped the application of these cells (). Even in the presence of ZFN, TALEN or CRISPR/Cas9, the efficiency of HDR-based gene targeting in human pluripotent stem cells is found to be consistently low (ⅼ ). In a recent study by Merkle et al., the efficiency of CRISPR/Cas9-induced HDR-mediated knock-in was estimated to be around 1 × 10−5 without pre-selection (&#8569ⅳ ). Hence, technical expertise for sophisticated selections and cumbersome screening of a large number of clones are required to obtain genetically modified cells (&#8569ⅴ ). To date, it still remains unclear whether the extremely low efficiency of HDR is a feature unique to human pluripotent stem cells. Furthermore, it has not been investigated whether the prevalent NHEJ repair can be employed to mediate high-efficiency knock-in in a wide range of human cells, especially in ESCs . In order to address these questions, we constructed a universal reporter system, by targeting the GAPDH locus in human genome with a promoterless fluorescent reporter. Through systematic investigation into the potentials of both HDR and NHEJ repair in mediating CRISPR/Cas9-induced reporter integration, we demonstrated that CRISPR/Cas9-induced NHEJ can mediate reporter knock-in more efficiently than HDR-based strategy, in various human cells types including human ESCs. This finding paves a new path for efficient genome editing in human ESCs and somatic cells, and it offers a great potential in their subsequent applications.



Our Goal

We will develop a CRISPR-Cas9 system-based continual genome editing strategy, including gene insertions and knock-in of both single and multiple (up to three) targets, including circular RNA associated competing endogenous RNA as a new therapeutic strategy in management of HCC.



Aim of the Project

  • To analyze circRNA and disease databases to select significantly relevant circRNA for HCC.
  • To analyze circRNA-miRNA interaction databases to retrieve competing endogenous RNA specific for HCC.
  • 3- To characterize the expression of the serum cirRNA-associated ceRNA genes in HepG2 cell line to evaluate their role in pathogenesis of HCC.
  • To characterize the efficacy of a CRISPR-Cas9 on modulating cirRNA-associated ceRNA related HCC expression using HepG2 cell line.

We the iGEM team of Egypt took it on our shoulders in a mission to find a new method to detect and cure one of the most endemic diseases in our country. We feel it's a must since we are the connection between the medical field and research department never to forget mentioning that the honour of replying to your homeland's call for help is one of the greatest signs of patriotism. We started to by tracing the problem to a much higher level to the epigenitic level, where the sequence of the DNA is mis-formed and got the idea to modulate a certain circRNA related to Hepatocelluar Carcinoma and adjust its gene expression, this is best described with the use of CRISPR.



References

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