CRISPR-Cas-9 has revolutionized the scientific community. Synonymous with the groundbreaking discovery of the double helix in 1957, the field of genetics and medicine have elaborately shifted. CRISPR is acronymous for Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR- associated protein 9 (cas9). Working as a “molecular knife,” CRISPR-Cas-9 endonucleases are able to recognize specific base pairs to perform a double-stranded break and allow natural repair processes to recombine the strand of DNA (Smith et al. 2014). While CRISPR is prominent for the ability to delete strands of DNA, the large risk associated with this technology comes with the consequential repair method (Brinkman et al. 2018). In a recent study conducted in 2014, researchers from Sun-Yatsen University investigated the reliability and specificity of CRISPR-CAS-9 genome editing techniques in model embryos. Although the implications of this technology are relatively endless, there are risks and reservations that are imperative to understand.
While CRISPR technology may only apply to genetics, the potential benefits of this technology span widely throughout research communities. In addition to performing genome editing, CRISPR has the potential to further analyze a gene’s function and assess gene therapy in humans (Liang et al. 2015). Prior research has suggested that CRISPR technology may show higher rates of off-site targets in cancer cells— further proving the exceptional need for efficacy studies (Smith et al. 2014). Although CRISPR technology offers an appealing option for gene therapy and will adequately cleave directed strands of DNA, it does not offer an efficient repair method that is both ethical and reliable for human implications (Foulkes et al. 2019). Published in April 2015 by Protein and Cell, “CRISPR-Cas9-mediated gene editing in human tripronuclear zygotes” was primarily written by Puping Liang, Yanwen Xu, and Xiya Zhang. Specifically researching the efficacy of this technology, the study aimed to understand the repair structure of CRISPR, and the potential to incorrectly target strands of DNA or recombine DNA into point mutations. The researchers found that in order for CRISPR techniques to be used in human trials, the technology needs to be improved to decrease the risks of possible mutations forming from its application (Liang et al. 2015). While this research could have possible medical uses, it runs the great risk that comes with editing the human genome. This research is exciting yet cautionary because if a mutation arises from a germline edit, the future DNA of the human race could be altered (Foulkes et al. 2019). From evolutionary biology to medicinal practices, this research has vast implications, and in order to fully understand genome editing, the public should both understand its great risks and opportunities.
Moving into the methods of this study, in order to construct the CRISPR plasmids, both pX330 and pDR274 were transformed and amplified in vectors. Because of the ethical drawbacks of human embryo applications, the researchers artificially inseminated mature oocytes. After a period of four hours, three abnormal pronuclear embryos were preserved through cryostasis. In order to ensure the reliable preservation of the embryos, a thorough, uniform procedure was followed maintaining their efficacy.
After being taken from cryopreservation, four groups of three pronuclear zygotes were injected, in vivo, with GFP (Green Fluorescent Protein) mRNA and CAS9/gRNA/ssDNA. In order to gain further insights into CRISPR technology, each group was injected with different concentrations of CAS9/gRNA/ssDNA (ng/ul), one hundred to two hundred ng/ul, twenty to forty 40 ng/ul, and two to two hundred ng/ul respectively (Liang et al. 2015). The embryos exposed successfully to GFP were then purified and amplified on the target sequence and converted to a vector to be genetically sequenced. Using Polymerase Chain Reaction (PCR) analysis and chromatograms, embryos on and off the target sequence were analyzed for efficiency and point mutations. After exposure to the CRISPR plasmid, a cleavage assay was performed to analyze the mutations and indels of the manipulated DNA. Further, to identify potential mutations and other target sites, the researchers used an online tool to predict strands of DNA that may be affected as a byproduct of the exposure. In addition to looking only at certain strands and mutations in the DNA, the researchers performed full genome amplification by briefly incubating the embryos in diluted solutions, preparing for amplification and PCR analysis.
Before moving on, identifying two technical terms are important to understand the methods of this study: Polymerase Chain Reaction and Green Fluorescent Protein. PCR is an experimental method of replicating millions of strands of DNA in order to analyze a certain strand of DNA (Liang et al. 2015). PCR is a common resource in the scientific community to study DNA and to identify the base pairs that incorporate a certain gene. With the information about PCR, GFP is a technology used by researchers to identify specific areas of DNA (Liang et al. 2015). Attaching GFP to a specific target allows scientists to use ultraviolet light and identify its location. Both of these practices are vital to microbiology and are attributed to many scientific discoveries.
While this study was meant to further understand CRISPR technology and to understand its risks, the findings of this study were rather uneventful and unquantifiable. Much of the results of this study were attributed to further research and qualitative reasoning. After identifying potential offsite targets and point mutations, the researchers found that further evidence and experimentation is needed before a determination into the efficiency of CRISPR, especially as it relates to human trials. Overall, the study did cite the overall efficiency, from their findings, to be twenty-five percent. Although that rate is striking, it highlights the obvious need for further research and analysis of CRISPR.
The question of ethics is imperative for this research. While research is constantly being conducted on CRISPR, none of the scientific articles are able to escape the ethical conversations surrounding this topic. The full potential of CRISPR-Cas-9 lies at the midpoint between the ethical discussions and the scientific discoveries, and while genetic editing could revolutionize the field of medicine, not all uses of CRISPR are both practical and ethical. While CRISPR is precise in the deletion of certain base pairs, the shortcomings of this technology fall in the repair of DNA. CRISPR involves the precise breakage of DNA, and a mere repair that connects a “perfect storm” of base pairs could alter the germline of the human race with unforeseen mutations (Foulkes et al. 2019).
With the complex nature of the experimental design, the authors did cite two specific limitations of their study: tightness in scope and underestimated mutations. While the authors used a computer program to find potential off-site targets, they likely underestimated all the error targets. Because the embryos were mosaic, the study design is not capable of fully estimating the challenges implicated in human trials. Although this study was meant to further investigate the broad scope of CRISPR gene editing, the authors cite that further investigations, even broader studies, are critical before human trials can occur.
Although the authors did list the limitations of their study, I found that the authors did not deeply discuss their methods. Because of the complex nature of this experimental design, I figured it would be imperative for the researchers to deeply elaborate on their materials and methods; however, the researchers only included one method’s figure and, in my opinion, did not adequately discuss their methods. With the sheer complexities that this experiment involved, it is critical for their audience to understand the process of genetic editing— especially as it relates to Crisper-CAS-9. Although the methods figure was detailed in describing results, the early stages of the experiment were hardly explained.
The full implications of CRISPR-Cas-9 technology are, like the human genome, endless. With the findings of this article, any current implications on the human genome feel distant and obscure; however, the study did positively highlight possible improvements. Although this study only tackled this subject from a research perspective, it ensured ethics will consistently be a part of CRISPR research. As improvements are constantly being made to this technology, the ethical and social questions will undoubtedly arise, and those inquiries will strengthen the efficacy of CRISPR. Although the current efficiency of gene editing needs to be further enhanced, at the common ground of ethics and science lies the boundless potential of this research. At the horizon of CRISPR research could be the cure to any genetic disease, curing millions worldwide. This technology has the potential to impact the genetic makeup of any organism- connecting to every living soul on Earth. In other words, although this technology may operate in nanometers, it has the potential to impact billions of lives.
Brinkman, E. K., Chen, T., de Haas, M., Holland, H. A., Akhtar, W., & van Steensel, B. 2018. Kinetics and Fidelity of the Repair of Cas9-Induced Double-Strand DNA Breaks. Molecular cell. 70(5), 801–813. 10.1016/j.molcel.2018.04.016
Foulkes AL, Soda T, Farrell M, Giusti-Rodríguez P, Lázaro-Muñoz G. 2019. Legal and ethical implications of CRISPR applications in psychiatry. North Carolina law review. 97(5):1359–1398.
Liang P, Xu Y, Zhang X, Ding C, Huang R, Zhang Z, Lv J, Xie X, Chen Y, Li Y, et al. 2015. CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes. Protein & Cell. 6(5):363–372. doi:10.1007/s13238-015-0153-5.
Smith C, Gore A, Yan W, Abalde-Atristain L, Li Z, He C, Wang Y, Brodsky RA, Zhang K, Cheng L, et al. 2014. Whole-genome sequencing analysis reveals high specificity of CRISPR/Cas9 and TALEN-Based Genome Editing in Human iPSCs. Cell Stem Cell. 15:12–13.
Featured Image Citation:
Heidt A. 2020. The Scientists Magazine. https://media.istockphoto.com/vectors/and-genome-editing-vector-id1189916097