Essay About Genome Modification And Similar Dna

Genome Modification by Crispr-Cas9: Uses and Ethics
Genome modification by CRISPR-Cas9: Uses and Ethics        In general, genome modification or also called genome engineering refers to making a targeted change to the genome, including its context or outputs. Context refers to epigenetic marks while outputs include transcripts. Figure 1 shows the application of genome engineering in different fields 1.[pic 1]Figure 1. Applications of Genome Engineering 1.Introducing a change in the cell’s genome is essentially easy to accomplish. What is hard to do is to target specific parts of the chromosome for the intended effect to be obtained. Historically, this was done using homologous recombination-based (HR) strategies, usually in mice. In HR, the desired mutation is introduced to mouse embryonic stem (ES) cells by electroporation or microinjection of a targeting vector. The mutation is introduced by exchanging the similar DNA to the target sequence (homologous recombination). HR is naturally-found in nature and used by organisms to repair double-strand (DSB) breaks introduced in their genome by mutagens, like UV.  The ES cells will then be screened for those that incorporated the mutation. These will be propagated as pure population. Next, these will be injected into the blastocoel cavity of preimplantation embryo; blastocyst is then surgically transferred into the uterus of a foster mother. The resulting animal would be chimeric, containing the introduced mutation and wild-type. To generate a progeny that only contains the intended mutation, the chimeric mouse would be cross-breed albino mice 2. It can be inferred in here, that genome modification naturally do occur and this particular HR strategy cannot be ethically applied to humans. HR, however, has been used in human cell lines but with only moderate success indicating low efficiency. The process could also take a year to generate the desired progeny 3. Scientists have to go around by going fundamentally, introducing DSB into the genome. This could easily be accomplished by introducing restriction enzymes and nucleases. However, restriction enzymes could potentially digest the entire genome. The prominent solution then, is to design and engineer nucleases that could generate site-specific DSBs.         To overcome the challenges presented by HR, nuclease-based genome editing technologies have been developed namely, zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). ZFNs are a class of engineered proteins with an array of site-specific DNA-binding domains fused to cleavage domain of the bacterial Fokl restriction enzyme. They can be used to create highly targeted DSB within the genome with unprecedented ease and precision. The DNA-binding domain recognizes 3-4 DNA base pairs; array of which can potentially bind to 9 to 18 base pairs of target DNA. The cleaved DNA can be repaired by either [1] nonhomologous end joining (NHEJ), occurs when template DNA is absent and often results in erroneous repair or [2] homology directed repair (HDR), is preferred when there is a high concentration of donor DNA and thus genome modification is achieved (Figure 2). TALENS, on the other hand, are a novel class of sequence-specific nucleases. They are fusion proteins made up from the fusion of transcription activator-like effector (TALE) DNA binding domain (originally found in plant pathogens) and endonuclease domain of Fokl. The TALE repeats comprise 10 to 30 repeats, each 33 to 35 amino acids in length, specific to the target DNA (Figure 3). Custom TALEs can be designed to recognize specific DNA sequences. Since the cleavage domain is similar to ZFN, the cleavage and repair process are essentially the same. Comparing the two, TALENs are definitely bigger in size and has off-target issues. However, it could be an advantage to extend the length to whatever is desired. Also, TALENs require less time to be developed. ZFNs, on the other hand, are smaller and well-proven in different systems. Though in ZFN design, site selection has constraints and products longer to develop 4. There were successes reported using ZFN and TALENS in cells of hematopoietic in origin and human pluripotent cells but in general, engineering nucleases could be costly and require significant expertise 3.