Genome engineering technology started since the 1970s and this technology has developed quickly. It is now a more efficient and sturdy tool for genetic perturbations. Genome engineering is a process of altering a genetic layout of an organism in a specific and targeted fashion, and surrounds the techniques or strategies to accomplish the modification process as well. This technology has enabled researchers to expand our knowledge of what we know about the gene function. The possibilities to alter DNA allows researchers to imitate human diseases in animal models. Hence, this can be exploited for gene therapy and drug development. (Geurts, et al. 2009)
There are currently four major classes of genome editing, zinc finger nucleases (ZFNs), transcription activator-like effectors (TALENs), meganucleases and the latest addition, the clustered regularly interspaced short palindromic repeats (CRISPR)(Mali, et al. 2013). By inducing site-specific DNA double-strand breaks (DSBs), these four technologies can manipulate genetic material. This would result in genome editing through homologous recombination (HR) or non-homologous end joining (NHEJ) (Niu, et al. 2013). Even though they are categorized under the same category; programmable nucleus, the mechanism of each genome editing technologies are different from each other. Generally, specific DNA sequences are targeted by nucleases such as TALENs, meganucleases and ZFNs via protein-DNA interactions (Stranneheim, 2012). The homing endonucleases, also recognized as meganuclease are highly specific according to nature, whereby its DNA binding domains and nuclease are merged into one sole domain. Whereas, TALENS and ZFNs are nucleases that are artificially engineered with a non-specific nuclease domain of Fok1. Hence, ZFNs and TALENs are more efficient than meganucleases because they are not limited in their capacity to bind to new DNA sequences with specificity. With that being said, ZFNs and TALENs have some drawbacks. The difficulty of context-dependent binding preference between individual finger domains of ZFNs make designing of programmable ZFNs difficult even though solutions have been drawn up to address this limitation as extensive screening is necessary ( Sander, et al. 2011). TALENs, on the other hand, express lesser context-dependent binding preference and their modular assembly makes it possible to target any possible DNA sequence (Maeder, et al. 2013). But, Biological cloning methods are required for the assembly of DNA encoding the repetitive domains of TALENs which can be costly. But now with the arrival of CRISPR system, genome engineering technology has shown great results in tackling issues relevant to modular DNA-binding protein construction. The ease of customization to target any desired DNA sequence in a genome simply through customized sgRNA is the reason why the CRISPR system has been used in variety of studies.(Niu, et al. 2013). This essay will talk about the implications of CRISPR towards medical and research
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Firstly, this how CRISPR works : the cas9 enzyme acts as the scissors and the single guided RNA (sgRNA) acts as the The native Cas9-mediated genome is completed with two steps. In the begining, Cas9 induces a DSB at on one targeted site on the genomic DNA which is guided by a 20-nt guide sequence in the crRNA. Next, the DSBs either undergo HR or NHEJ pathway.
Cas9 can be utilized to ease a broad diversity of targeted genome engineering applications. Using traditional manipulation genetic techniques, the Cas9 nuclease has enabled efficient and targeted genetic manipulation strategies. CRISPRs ease of simply designing a short RNA sequence to retarget Cas9 enables a large- scale of unbiased genome perturbation experiments to elucidate cause genetic variants or to probe gene function.
Cas9-mediated genome editing has allowed rapid generation of transgenic model and widens biological research over classic, genetically tractable animal model organisms (Sander, Joung, 2014).
