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 .CRISPR/Cas9-mediated genome editing depends on the generation of double-strand break (DSB) and subsequent cellular DNA repair process. In endogenous CRISPR/Cas9 system, mature crRNA is combined with transactivating crRNA (tracrRNA) to form a tracrRNA:crRNA complex that guides Cas9 to a target site.
TracrRNA is partially complementary to crRNA and contributes to crRNA maturation. At the target site, CRISPR/Cas9-mediated sequence-specific cleavage requires a DNA sequence protospacer matching crRNA and a short protospacer adjacent motif (PAM). After binding to the target site, the DNA single-strand matching crRNA and opposite strand are cleaved, respectively, by the HNH nuclease domain and RuvC-like nuclease domain of Cas9, generating a DSB at the target site (Fig.
2). For easy application in genome editing, researchers designed a delicate guide RNA (gRNA), which was a chimeric RNA containing all essential crRNA and tracrRNA components (21). Multiple CRISPR/Cas9 variants have been developed, recognizing 20 or 24 nt sequences matching engineered gRNA and 2–4 nt PAM sequences at target sites. Therefore, CRISPR/Cas9 can theoretically target a specific DNA sequence with 22–29 nt, which is unique in most genomes.
However, recent studies observed that CRISPR/Cas9 had high tolerance to base pair mismatches between gRNA and its complementary target sequence, which was sensitive to the numbers, positions and distribution of mismatches (21,26–29). For instance, the CRISPR/Cas9 of Streptococcus pyogenes appeared to tolerate up to six base pair mismatches at target sites (21).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. Other than, facilitationg to co-valent genome modifications, the wild-type Cas9 nuclease can also be converted into a generic RNA-guided homing device.
A variety of proteins or RNAs can be tethered to sgRNA or Cas9 to alter transcription states of specific genomic loci, monitor chromatin states, or rearrange three-dimensional organization of the genome. Firstly, 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 could be used to quickly model the causal roles of particular genetic deviation alternatively of depending on disease models that only phenocopy a specific disorder. By applying this, it could expand novel transgenic animal models ( Wang, et al.
, 2013) to engineer isogenic embryonic stem cells (ES) and induced pluripotent stem cells (iPS) cell disease models with particular mutations corrected or introduced(Schwank, et al., 2013). For all these years of cellular models, Cas9 can be effortlessly commenced into the target cells using transient transfection of plasmids carrying Cas9 and suitable designed sgRNA. In addition to that, common diseases—like heart disease, schizophrenia, diabetes and autism— that are usually polygenic can have a promising approach for studying them with the multiplexing capabilities of Cas9. Besides that, heritable gene modification at one or multiple alleles in models such as monkeys and rodents are achievable by injecting transcribed sgRNA and Cas9 protein directly into fertilized zygotes( Li et al.
, 2013). 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, et al., 2014).Furthermore, the functional output of genomes, which can be intensified or suppressed dynamically are contributed by the spatial organization of functional and structural elements within the cell. But the way that the genomes are altered and how their structural organization in vivo modulates functional output remains unclear. Changing in chromatin states would require a robust method to visualize DNA in living cells when studying the interactions of specific genes. Fluorescence in situ hybridization (FISH), which is a traditional strategy for labeling DNA, needs sample fixation and are thus unable to capture live processes.
Cas9 labeling of specific DNA loci that are fluorescently tagged was recently developed as a robust live-cell-imaging alternative to DNA-FISH (Chen et al., 2013). Multi-color and multi-locus capabilities to enhance the utility of CRISPR-based imaging for studying complex chromosomal architecture and nuclear organization with the advancement in orthogonal Cas9 proteins or modified sgRNAs.In conclusion, CRISPR genome editing system is an ideal genome editing tool because it is simple, efficient and the cost of assembly of nucleases that can target any site without any off-target mutations in genomes is low.
The huge benefits of CRISPR which is the adaptability and simplicity, opens many door for revealing gene function in biology and repairing gene defects in diseases.