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
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 can be used to facilitate a wide variety of targeted genome engineering applications. The wild-type Cas9 nuclease has enabled efficient and targeted genome modification in many species that have been intractable using traditional genetic manipulation techniques. The ease of retargeting Cas9 by simply designing a short RNA sequence also enables large-scale unbiased genome perturbation experiments to probe gene function or elucidate causal genetic variants. In addition to facilitating co-valent genome modifications, the wild-type Cas9 nuclease can also be converted into a generic RNA-guided homing device (dCas9) by inactivating the catalytic domains. The use of effector fusions can greatly expand the repertoire of genome engineering modalities achievable using Cas9. For example, a variety of proteins or RNAs can be tethered to Cas9 or sgRNA to alter transcription states of specific genomic loci, monitor chromatin states, or even rearrange the three-dimensional organization of the genome.
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). CRISPR-based editing
By recapitulating genetic mutations found in patient populations, CRISPR-based editing could be used to rapidly model the causal roles of specific genetic variations instead of relying on disease models that only phenocopy a particular disorder
Secondly, with the efficient genome editing tool Cas9, it is possible to change various targets in parallel. Therefore, allowing an unbiased genome-wide functional screens to recognize genes that play an important role in a phenotype of interest. Lentiviral delivery of sgRNAs directed against all genes ( together with Cas9 or to cells already expressing Cas9) are able to be used to perturb ten thousands of genomic elements in parallel. The ability to perform robust negative and positive selection screens in human cells have been indicated in recent papers ( (Wang, et al., 2013) by introducing loss-of-function mutations into early, constitutive coding exons of a different gene in each cell. Moreover, RNAi was previously used for genome-wide loss-of-function. But this approach leads to incomplete knockdown, large-scale of target effects, and is restricted to transcribed genes. In contrast, increased screening sensitivity, consistency and the ability to be designed to target almost any DNA sequence is shown to be provided by the Cas9-mediated pooled sgRNA screens(Shalem, 2014).