GMO Research Paper Topic 8: CRISPR Technology
Introduction:
Gene editing is method that allows scientists to modify the DNA of organisms by inserting, deleting, or replacing its genetic material. For the last 30 years, the products of GMOs (genetic modified organisms) have predominantly made an appearance in supermarkets, agriculture, and healthcare; because of its widespread development, consumers are voicing their concerns and questioning the benefits of genome engineering. The National Human Genome Research Institute reports that the discovering of genome editing technologies happened in late 1900s, and its latest advancement is the CRISPR technology (2). CRISPR is an acronym for Clustered Regularly Interspaced Short Palindromic Repeat that is derived from the genomes of prokaryotes as a component of their immune system. CRISPR and the Cas proteins that are produced by the CRISPR system forms the CRISPR/Cas complex present in prokaryotes’ immune system. Harvard Medical School published an article briefly explaining the mechanism of CRISPR in bacteria in three steps: adaptation, production of CRISPR RNA, and targeting. (7) The bacteria captures the viral DNA and inserts the segments into the spacers, that allows the bacteria to recognize viral genome and produce CRISPR RNA to cut viral DNA and kill the virus. Researchers Jennifer Doudna and Emmanuelle Charpentier studied further the CRISPR-Cas system in streptococcus pyogenes in specifically the Cas9 gene that encodes for the Cas9 protein (4). The Cas9 protein has nucleases and helicase domains, which can become useful in genome editing because of its ability to break the double stranded DNA, recognize the complementary sites of the guide RNA and cleaves it. In figure 1, the process is illustrated. The development and applications of the CRISPR-associated protein Cas9 allowing site-specific double stranded break in the DNA is a game changer because it allows gene editing to be simple, affordable, and efficient. The future of this technology can enhance the biomedicine field as well change the course of agricultural research. On the other hand, specificity can pose complications, as well as unwanted mutations. Due to these reasons, CRISPR technology used on organisms remains a controversial topic.
a. Gene editing using CRISPR is a game changer for GMO technology
i. Benefits of CRISPR
CRISPR uses ‘cut-and-paste’ technology that differentiates itself from other gene-editing tools because it increases precision in correcting DNA fragments without introducing insertion or deletions mutations to DNA during the correcting process within reasonable cost and time (1). In efforts of comparing the efficiency and potential of site-specific nucleases, researchers published Plant genome editing with TALEN and CRISPR to compare the methods relative to each other. Both TALEN and Cas9 are use as molecular scissors and an experiment done both using TALEN and CRISPR to analyze its effectiveness in plant genome editing. Although one cannot conclusively state that CRISPR technology is more suitable in activating or repressing gene expression than TALEN technology, CRISPR is easier to engineer and its ability to multiplex in comparison to TALEN makes it unique in comparison to other genome editing tools. Cas9’s ability to be paired with multiple guided RNAs allows it to target multiple sites in a cell and therefore, makes the process less time consuming (3). The benefits of the CRISPR-Cas9 system can also be supported by utilizing the tool to further research in the biomedical field, specifically using the technology for human gene therapy to possibly treat genetic disorders; the ability to analyze gene functions and be able to study specific sites of organism’s genomes will further human’s understanding of diseases and make a major impact on the studied of genomic mutations (4). An example mentioned in Doudna and Charpentier’s The new frontier of genome engineering with CRISPR-Cas9 states the simple two component system can correct genetic mutations such as those with cystic fibrosis by targeting the stem cells to correct the mutated CTFR genes. A study was done and data was published in Functional Gene Correction for Cystic Fibrosis in Lung Epithelial Cells Generated From Patient iPSCs to show the efficiency of CRISPR to correct the ΔF508 mutation in patients that have the disease, and leaving a footprint free approach (9). By designing a unique gRNA to specifically target the desired DNA region of the ΔF508 mutation and delivering it to induced pluripotent stem cells, scientists can gain insight on the efficiency and off target mutations that could occur using the CRISPR mechanism. When using the excision-specific pBac transposase to remove the selection markers and then doing PCR, data showed nearly 90% efficiency and no mutations occurred within 300 bps at the vicinity of the target site. This evidently shows that CRISPR/Cas9 functionality is useful in the research of diseases and would benefit medical science.
Moreover, the Cas9 functionality can also be applied to agriculture. As mentioned earlier, when a research was conducted to see the effectiveness of gene editing among plants, it showed CRISPR is a promising tool to expose desirable traits in crops such as soybean, rice, wheat, corn, and cotton (3). Not only will it assist with better crop yield, it will also improve the crop quality and revolutionize the agricultural industry. Gene edited crops can be produced in one generation with desired characteristics such as higher yield, herbicide resistant, and more nutritious (9). Overall, Cas9 delivery methods provides significant advancement for GMO technology.
ii. Drawbacks of CRISPR
Although genome editing using CRISPR is a useful tool when studying genomes, development, and diseases, there are still risks with error in DNA manipulation as well as ethical concerns. First, in the event that scientists use the CRISPR/Cas9 technique to modify species, there are risks in modifications such as targeting insertions and deletions in the DNA that could potentially introduce off-target sequences. This disadvantage would cause undesired mutations or unwanted
transformations in the GMOs, thus may disrupt the ecosystem equilibrium (5). For example, it could diminish genetic diversity by introducing a gene into an organism allowing it to gain fitness advantage. Another drawback of CRISPR technology is that could intervene with the human species because it can ultimately customize the living organism to express desirable traits which raises morality and ethical concerns. Using the CRISPR-Cas9 technology to alter the human genomes somatic cells raises ethical concerns as well as bring to question on how scientists would be able to facilitate appropriate research and development of this technology. The possibility of paying for this technology for enhancement purposes such as intelligence and physical advantages is controversial because it can worsen inequality for people who cannot afford it. This problem will greatly impact human population. Moreover, when editing the DNA of plants or animals in the interest of commercial application, there are huge risks of mutations and unpredictable changes being made that could introduce health hazards to consumers such as affecting nutritional value and introducing food allergens. Finally, while CRISPR-Cas9 is efficient in modifying DNA sequences, researches still show the specificity of CRISPR/Cas9 is still questionable and unintended mutations as well as efficiency in homologous directed repair is low, therefore, hinders its application in biomedical studies. The frequencies of this happening is more prevalent in cancer cell lines when studying off-target effects of CRISPR on model organisms based on the journal published in 2015 that calls for further improvement in exploring the capabilities of the CRISPR/Cas9 system before extending its usage in medical industry (8). Overall, the drawbacks of this gene-editing tool is common among the existing genome editing technology. However, the CRISPR/Cas9 technology proves itself to be beneficial in studying molecular biology, human gene therapy, agriculture, and medicine.
Summary:
The discovery of CRISPR/Cas9 protein mechanism that is cost efficient, and accessible to researchers allows them to expand their knowledge in the field of agriculture, biomedicine, and microbiology. Scientists identified the function of Cas9 protein as the enzyme to cleave desired DNA when paired with the guided RNA that is complementary to the target sequence along with the trans-activating RNA. This enables innovative research towards DNA modifications and transcription regulation.