Introduction: What is CRISPR/Cas9 and How Does it Work?
Clusters of regularly interspaced short palindromic repeats, commonly known as CRISPR, is a simple yet formidable implement allowing researchers to effectively edit genomes. Some of its potential applications include treatment and prevention of diseases, amelioration of genetic defects, and removal of certain gene types. The CRISPR/Cas9 technology can be deconstructed into four components (CRISPR, spacer, crRNA, and Cas9), each serving its own purpose. The CRISPR is a unique sequence of deoxyribonucleic acids (DNA) consisting of two distinct attributes: recurrent nucleotide progressions and spacers, which are short-variable sequences interspersed between nucleotides [1]. These spacers are derived from the DNA of viruses that have previously attacked the host CRISPR [2]. Once the CRISPR has been fashioned and the virus attacks again, the CRISPR is transcribed and processed into crRNA. The crRNA integrates with a secondary RNA string, the trans-activating crRNA, to provide the Cas9 enzyme with a passage to its target site, where the Cas9 enzyme will then execute what is recognized as a double-stranded break to remove the superfluous module of the genome sequence. One theory as to why CRISPR/Cas9 technology is so attractive compared to other genome editing techniques to researchers and consumers alike is its meticulous accuracy and efficiency in deriving results. It is predicted that the rationale behind this lies in the technology’s prototype adjacent motifs or PAMs. These PAMs serve as a tag, sitting adjacent to the Cas9 enzyme’s target site. Should there be no PAM next to the target site, the Cas9 enzyme will avoid “cutting” the particular genome sequence [1]. Once the cut has been finalized, programmed DNA may take its place.
Why CRISPR: A Deeper Understanding
Over the past decade, four major classes of engineered nucleases have been used for genome editing: meganucleases, zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and CRISPR [6.] Meganucleases are endonucleases that can recognize extended DNA progressions of 14-40 bp via extensive non-modular protein-DNA connectors, but their target specifications have proven to be difficult to re-engineer [6]. Both ZFNs and TALENs are “fusions between arrays of ZF or TALE DNA-binding domains and the non-specific, dimerization-dependent FokI nuclease domain” [6]. Put simply, these three classes of nucleases all rely exclusively on protein-DNA connections to recognize their target sites.
In contrast, the CRISPR/Cas9 endonuclease relies on RNA strands to guide the Cas9 protein to its target site. This process allows researchers to easily modify and reprogram gene sequences. Additionally, due to the Cas9 enzyme’s complex nature of being intersected with a secondary RNA string, it can be re-engineered to recognize a target site based off of a protospacer and a PAM progression [5]. The PAM progression serves as a mediator, ensuring the Cas9 enzyme does not lacerate a necessary fibril of the DNA sequence. Thus, engineers can easily facilitate gene modification by simply editing the composition of the RNA strand conjoined with the Cas9 enzyme. They are not to worry about accidentally fabricating unwanted modifications to the RNA progression because the Cas9 enzyme will not act on it should a PAM not be present. These distinctive aspects of the CRISPR/Cas9 technology promote accuracy by minimizing off-site targeting by the Cas9 enzyme, leaving little to no space for error. Despite the expeditious advances in the field of genetic engineering/modification, bioethical questions are still raised regarding human trials.
Bioethical Concerns
Currently, the majority of all genome editing technologies are being tested in the interest of treatment and prevention of human disease. Genome editing shows considerable promise in treating and preventing both single-gene diseases such as muscular dystrophy and sickle cell disease and more complex disorders such as cancers and human immunodeficiency virus (HIV) [4]. A significant amount of research conducted on genome editing directly affects somatic cells (cells that are not egg and sperm cells). However, at the alarming pace that this field of study is evolving, there is no doubt that gene alteration technology to edit germline cells (egg and sperm cells) will be readily available soon, which is where bioethical challenges are raised. The ability to edit germline cells will enable consumers to enhance normal human characteristics, such as height or intelligence. It will also allow consumers to edit their offspring’s appearance [4].
The second major debacle that is brought into question is human autonomy. When germline editing becomes readily available, it will allow consumers to change normal human attributes in their offspring. This will be a non-consensual act on the parents’ behalf which will impact their offspring. Further, genetic editing will allow consumers to model the perfect human, which in turn, will infringe on a diverse number of societal rights [7]. These societal rights include but are not limited to the right to morality, the right given to doctors to advise patients, the rights scientists are given to conduct research, and the right children should have to consent or not consent to decisions their parents will make that affect their livelihoods.
Based on these ethical challenges, many countries have made it illegal to further study genome editing, more specifically germline and embryo genome editing. However, many more countries are persistent in advancing research and trials into the field.
Conclusion
Genome editing presents an attractive approach and considerable promise to treatment and prevention of human disease. Out of the four common classes of engineered nucleases used in the past decade, CRISPR/Cas9 has shown the most promise regarding accuracy. Its distinct features that make it a favorable tool are its negligible margin of error for targeting off-site DNA progressions via PAMs, its reliance on intertwined Cas9 enzymes and RNA progressions which allow for efficient reprogramming, and its unique PAM system which enables engineers to directly trim an RNA sequence without effectuating complications. However, amidst the steady progress of the genetic engineering field and its universal appeal to researchers, one can make a point to further analyze bioethical inquiries that have arisen before implementing further studies, trials, and inevitably, introducing this technology to the healthcare market.
References
- E. P. (2014, July 31). CRISPR: A game-changing genetic engineering technique. Retrieved August 29, 2020, from http://sitn.hms.harvard.edu/flash/2014/crispr-a-game-changing-genetic-engineering-technique/
- Barrangou, R., Fremaux, C., Deveau, H., Richards, M., Boyaval, P., Moineau, S., Romero, D.A., and Horvath, P. (2007). CRISPR provides acquired resistance against viruses in prokaryotes. Science 315, 1709–1712
- Doudna, J. A., & Charpentier, E. (2014). The new frontier of genome engineering with CRISPR-Cas9. Science, 346(6213), 1258096. doi:10.1126/science.1258096*, E. P. (2014, July
- Jiang, W., Bikard, D., Cox, D. et al. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol 31, 233–239 (2013). https://doi.org/10.1038/nbt.2508
- What are genome editing and CRISPR-Cas9? – Genetics Home Reference – NIH. (n.d.). Retrieved August 29, 2020, from https://ghr.nlm.nih.gov/primer/genomicresearch/genomeediting
- Tsai, S., & Joung, K. (2020). Defining and improving the genome-wide specificities of CRISPR-Cas9 nucleases. Nat Rev. Genet, 1-25. doi:10.1038/nrg.2016.28.
- Kersch, L. (n.d.). GENETIC MODIFICATION: THE ETHICAL AND SOCIETAL IMPLICATIONS OF CRISPR TECHNOLOGY. Retrieved August 29, 2020, from https://med.nyu.edu/departments-institutes/population-health/divisions-sections-centers/medical-ethics/sites/default/files/medical-ethics-high-school-bioethics-crispr.pdf
Sabriyah Morshed, Youth Medical Journal, 2020
Youth Medical Journal 