Cutting Edge CRISPR Cas9 Applications

In 2015, the journal Science chose CRISPR-Cas9 technology as one of the most important technological advances in science in the last few years.

Gene editing is the new era of biotechnology, which makes it possible to edit, correct and modify the genetic information of any cell in a precise, fast and cheap way. CRISPR-Cas and genome engineering research fields are two fields which merged in 2012 with the discovery that Cas9 is an RNA-programmable DNA endonuclease, leading to many scientific papers beginning in 2013 in which Cas9 has been used to modify genes in human cells as well as many other cell types and organisms [1].

The genome-editing system based on CRISPR-Cas is becoming a valuable tool for different applications in biomedical research, drug discovery and human gene therapy by gene repair and gene disruption, gene disruption of viral sequences, and programmable RNA targeting [1,2].

Genome editing is the most efficient technique in terms of manipulating the gene expression by using programmable DNA nuclease comparing to gene transfer approaches. Nowadays, the four genome-editing platforms mostly used are: meganucleases, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR–Cas system [1,3-5]. Currently, it is possible to correct many genetic diseases with genome-editing technologies but there are some obstacles and challenges to overcome such as the genome-editing biomacromolecules. Hao Yin et al., has given a very interesting overview about the different programmable nucleases and mechanisms of genome editing, by focusing on the principles of biomacromolecules delivery, relevant delivery methods and associated delivery challenges. 

Furthermore, cancer is one of the leading causes of morbidity and mortality worldwide, with approximately 14 million new cases in 2012 [6]. I strongly believe in the importance of exploring innovative approaches to study cancer on the genomic, epigenetic and transcriptional level at highest-possible resolution.

Nowadays, many diseases such as cancer have risen to the forefront of genomic profiling, which are being used to identify actionable driver mutations and other markers. These investigations can help clinicians design therapies and monitor patient responses. In addition, new technologies are being established such as induced pluripotent stem cells (iPS) models that are very well established, can be used for regenerative medicine. In addition to the iPS cells, one of the revolutionary genetic tools is the CRISPR technique that facilitates the genomics studies in all model organisms. Scientists are engineering the CRISPR-Cas9 into a screening tool, they can modify sites by knockout at a genome-wide scale, or they use other options including loss-of-function or gain-of-function screens that use transcriptional activation (CRISPRa), transcriptional repression (CRISPRi), base editing, directed mutagenesis, epigenetic editing, RNA interference (RNAi) or combinatorial methods. MIT–Harvard team has built Combi-GEM-CRISPR for high-throughput combinations of genetic perturbations to explore, in parallel, how different gene networks or epigenetic regulators shape cancer cell phenotypes [7]. Generally speaking, pharmaceutical researchers screen in cell lines, but comprehensive genetic validation after that step has been challenging said Dr Johannes Zuber, a researcher at The Research Institute of Molecular Pathology in Vienna. New tools are changing this, which might alter the approximately 90% failure rate of cancer drug candidates. With CRISPR- and RNAi-based techniques labs might more readily identify and validate new candidate targets in much greater depth, he says, and generate animal models that better reflect the genetic complexity of human tumors.

Let’s go further with the use of genome editing technique for cellular improvements. Dr Marc Tessier-Lavigne, a neuroscientist at the Rockefeller University in New York City, started to work with iPS cells made from people with early-onset Alzheimer’s disease and frontotemporal dementia. He realized that comparing a patient’s iPS cells with those from a healthy control didn’t work because the cells behaved too differently in culture, possibly the result of disparities in genetic background or gene expression.  The CRISPR–Cas9 gene-editing tool opened doors for researchers to introduce disease-associated mutations into a sample of iPS cells and then compare them with the original, unedited cell lines. For instance, Dominik Paquet and Dylan Kwart in Tessier-Lavigne’s lab have introduced specific point mutations into iPS cells using CRISPR and editing just one copy of the gene instead of both genes. They could on the one hand, generate cells with a specific combination of Alzheimer’s-associated mutations and on other hand study their effects [8].

Another interesting and different use of the gene-editing tool would be in food and industrial biotechnologies such as the application of CRISPR in bacteria including genotyping, vaccinating industrial cultures against viruses, controlling uptake and dissemination of antibiotic resistance genes by bacteria, and engineering probiotic cultures [9]. Let’s take the example of one of the first start of CRISPRs in food with the commercial success of native CRISPR–Cas immune systems for the vaccination of Streptococcus thermophilus starter cultures used in dairy fermentations (yogurt and cheese) [10-11]. Besides, gene-editing application does not integrate transgenic modifications and is better use comparing to conventional chemical mutagenesis. Moreover, crop developers, prefer using the genome-editing tool because they can introduce mutations such as specific alterations of target-gene functions at a precise site in the genome to either obtain gene silencing or enhancement of gene expression.

Actually, the CRISPR/Cas9 and related systems have moved to the forefront of the Gene-editing technology for plants as well as other organisms [12].

In conclusion, we have seen in this article some examples of gene-editing application in different field such as human gene therapy, screening for drug target ID and food/agriculture field (crops, animals). The field of application is wider as of the ecology field (for example, ecological vector control in the malaria disease by sterilizing the mosquito), viral gene disruption, programmable RNA targeting and synthetic biology by engineering pathways. CRISPR-Cas9 system can be used in different cell types i.e. iPS cells for biomedicine and different model organisms in biology/biotechnologies i.e. mice, Arabidopsis thaliana, crop plants, yeasts and fungi. Genome editing is a rapidly advancing technology in a highly specific manner and great precision. Some studies have examined the effectiveness of CRISPR/Cas9 in treating single gene disorders such as Duchenne muscular dystrophy, as well as eye conditions like retinitis pigmentosa and Leber congenital amaurosis (LCA) [13, 14]. Editas, Medicine Company, suggests that the first human clinical trials using CRISPR/Cas9 will aim to treat LCA. Apparently, the eye presents an ideal testing location, as it is immunologically isolated from the rest of the body, easily monitored externally, and can be measured using established standards of function [15]. What can we ethically do /or not do with the CRISPR-Cas9 technique? Today, there are ethical considerations using CRISPR/Cas9 on germline cells same as changing the germline. Some scientists believe that without knowing the downstream effects for future generations, it is unethical and too risky to proceed [15]. Some scientists believe that CRISPR/Cas9-based germline editing is currently far from being efficient or safe enough to warrant clinical applications, this technology could be extremely useful for basic research into early human development. Which other ethical issues could be considered apart from the safety and efficiency aspects that are not as relevant for basic research?

There are some arguments against the use of germline genome editing in basic research such as the moral status of the embryo itself prohibits germline modifications. This view is fully understandable but is not only limited to germline modifications and would therefore prevent any type of experimentation on supernumerary embryos. Besides, it might open up for a possibility of a misuse of the technology [16]. Ongoing studies are in progress in order to perform a better use of the gene-editing technique in different fields. As Rabelais said:” Science without conscience is but the ruin of the soul”.


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