CRISPR-Cas9

What is CRISPR-Cas9? 

CRISPR-Cas9, or Clustered Regularly Interspaced Short Palindromic Repeats,

is a system of DNA modifying technology. CRISPR itself is a group of DNA sequences that evolved from the defense mechanism of a bacterial genome. This mechanism is designed to fight off foreign pathogens and phages that it deems as a threat. The gene editing system utilizes two principal biological tools in order to implement alterations to genetic information, including a guide RNA complex composed of a series of proteins, as well as an endonuclease enzyme Cas9. The process of making edits occurs in stages: targeting, binding, cleaving, and DNA repair (Biointeractive 2018). These intricate steps can be simplified into the target DNA being identified, its double helix being unwinded by the Cas9 enzyme, and the now single helical structure having cuts, insertions, and deletions of nucleotide sequences occurring following its binding to the guide RNA complex.  The guide RNA is carefully programmed to contain only bases that correspond to the nucleotide sequences of the target DNA. Theoretically, this implies that the RNA complex is unable to bind to any other sites and instigate a wrongful RNA-DNA formation; however, this does not exclude the fact that unintended mutations can still transpire as a result of CRISPR-Cas9 use (Redman 2016). The strand breaks elicited by these steps are finally fixed with the help of the cells’ natural polymerase enzymes. CRISPR-Cas9 has been used and has the potential to be wielded in a variety of contexts, such as in genetically modified and drought-resistant crops as well as enhanced pharmaceutical drugs, but no application of this biotechnological innovation has sparked as much controversy as interference with live humans genes using CRISPR-Cas9. 

The Dual Nature of Gene Editing- Does the fate of future generations lie in our hands?

The two polarizing variations of CRISPR-Cas9’s gene editing are somatic and germline. Gene editing is commonly used within a clinical context, such as gene therapy, where redesigned DNA is inserted into the cells of a patient in order to instigate the desired effect achieved through manipulating gene expression. Somatic gene therapy deals with inducing mutations in all forms of bodily cells excluding gametes, so the DNA modifications and any unexpected genetic changes solely impact the patient undergoing treatment. In its opposition, the highly disputed germline editing technique involves producing changes within the reproductive cells of the patient which would cause a change within their descending bloodline (Baylis 2020). Generally speaking, germline gene editing is considered contentious as 75 countries have either directly prohibited heritable genome editing entirely, placed strict regulations on the continuity of this research, or established a moratorium to resolve some of the ethical implications. Reputable biomedical organizations like the National Institute of Health are strongly aligned with implementing a concrete moratorium that stalls all progress of germline gene editing (Wolinetz 2019). Some argue that tampering with genetic information that has the potential to influence countless generations of people is an automatic violation of human rights and threatens a bloodline’s natural heritage. A consistent argument against medical germline editing is that it is still in its early stages of development, and the likelihood of the embryo experiencing off-target gene edits or undergoing mosaicism, where they possess multiple sets of DNA, is unpredictable. Some might even say that germline editing is akin to gambling with human lives. Consider the example of He Jiankui, who modified the genome of two children to be HIV-resistant, deeming these risks as worth the potential elimination of countless heritable diseases (Bergman 2019).

Gene Editing in Cancer Treatment

Recently CRISPR has been developing a new form of immunotherapy called CAR T-cell treatments that boost the immune system to fight off cancer cells in three main ways: eliminating the malignant tumor(s), alerting other components of the immune system about the presence and location of the cancerous mass, and distinguishing cancerous cells from healthy ones in order to avoid excessive damage. Immunotherapy within the context of cancer is defined as a type of treatment that uses the body’s own substances (either produced in vivo or in a lab)  to support the immune system in identifying and eliminating cancerous cells.

              The procedure itself can be reduced into three integral steps: extracting the T-cells from the cancer patient, adding a protein called chimeric antigen receptors, or CAR, to the surface of the T-cells in a laboratory, and re-injecting these modified lymphocytes back into the patient’s body.  This protein allows T-cells to accurately target cancerous cells by directing them towards spotting antigens, which are indicators of harmful substances, on top of cancer cells. The individual protein-based constituents of CAR are responsible for the CAR T-cells’ efficient recognition of abnormalities as well as activation response time. As soon as a CAR T-cell interacts with the antigen on a cancer cell, it becomes activated, rapidly multiplies itself, and sends out cytokines which signal activated killer T-cells and other leukocytes of the humoral immune response to concentrate themselves at the infected site. Inflammation soon follows thereby killing off the harmful cells and typically sending the cancer into remission. This specialized breed of T-cell is programmed to continuously free-roam the body, so it also serves as an effective treatment for cancer that has metastasized. This immunotherapy medium can attack multiple tumors at once from all angles (Wang 2022). Its specificity in instigating inflammation only when coming in contact with antigens originating from cancer typically hinders healthy cells from being hurt, as false alarms don’t occur.

Overall, CAR T-cell immunotherapy proves to be a revolutionary tool towards reducing the number of deaths caused by various manifestations of cancer each year. The immunotherapy has a 40% success rate for lasting remission, according to UChicago Medicine, and is evidently both more efficient and precise at eliminating cancerous cells, relative to the unnecessary healthy tissue deterioration caused by standard treatments, such as external radiation (Bartosch 2021). Its precision in preserving the vitality of normal cells is clearly communicated via its success in treating blood cancer, large B-cell lymphoma, multiple myeloma, follicular lymphoma, and many other variants. 

Final Thoughts

Since its conception in the early 2000s, CRISPR-Cas9 has proven itself to be a pivotal discovery that has changed the future direction of our understanding of gene editing research and the innovations that will develop from this ever-expanding field. The unique DNA-altering mechanism has demonstrated its invaluableness, versatility, and potential to grow via its numerous applications in cancer treatment, gene therapy, and the agricultural industry. CRISPR-Cas9 is a target for ambivalent feelings from both the scientific community and the general population alike, so it invariably provokes ethical debates due to its innately incalculable and presently unreliable disposition; however, new developments in nanotechnology, such as nanocarriers, have determined ways to alleviate the concerns of the public. Nanocarriers are inorganic nanoparticles, such as cationic polymers, that serve as non-viral vectors which aid the process of the intracellular delivery of Cas9 enzymes alongside the guide RNA (Hejabi 2022). These minuscule molecules facilitate the journey of CRISPR-Cas9’s systems from their first introduction into the cell to their final binding process with the target DNA. This new system of co-delivery ensures that CRISPR only tampers with the desired sections of genetic information, thereby reducing the likelihood of accidental modifications made to the double helix. Fine-tuning the genome-influencing apparatus’s accuracy opens up a realm of new possibilities in the gene-editing world, including the elimination of deadly genetic blood disorders, neurodegenerative diseases, and ailments causing organ dysfunction. Regardless of the exciting prospects that CRISPR-Cas9 presents, people must stay cognizant of the fact that there will never be a guarantee for completely safe gene-altering procedures, and that the value of human lives will always trump the proclaimed urgency for novel innovations in the field of heritable germline editing. The practice of exercising caution must be prioritized over society’s insatiable desire to test the limits of our capacity to alter our  DNA. 

References

 1.    Redman, Melody, et al. “What Is CRISPR/Cas9?” Archives of Disease in Childhood - Education & Practice Edition, vol. 101, no. 4, Apr. 2016, pp. 213–15, ep.bmj.com/content/101/4/213.short.

2. Synthego. “The Ultimate Guide to CRISPR: Mechanism, Applications, Methods & More.” Synthego.com, 2019, www.synthego.com/learn/crispr.

3. MedlinePlus. “What Are Genome Editing and CRISPR-Cas9?” Medlineplus.gov, Medlineplus, 22 Mar. 2022, medlineplus.gov/genetics/underst  anding/genomicresearch/genomeediting/.

4. Biointeractive, Team. “CRISPR-Cas9 Mechanism & Applications.” HHMI BioInteractive, 11 Apr. 2018, www.biointeractive.org/classroom-resources/crispr-cas9-mechanism-applications?playlist=181755. 

5. ingenious. “CRISPR Gene Targeting and Genome Editing: Increased Accuracy.” Genetargeting.com, 12 Apr. 2018, genetargeting.com/crispr/crispr-gene-targeting/. Accessed 4 Aug. 2023.

6. Bergman, Mary Todd. “Harvard Researchers Share Views on Future, Ethics of Gene Editing.” Harvard Gazette, 9 Jan. 2019, news.harvard.edu/gazette/story/2019/01/perspectives-on-gene-editing/#:~:text=While%20 somatic%20 gene%20edition%20 affects . 

7. “Ethical Issues: Germline Gene Editing | ASGCT - American Society of Gene & Cell Therapy |.” Patienteducation.asgct.org, patienteducation.asgct.org/patient-journey/ethical-issues-germline-gene-editing#:~:text=Ethical%20Concerns%20for%20Germline%20Gene%20Editing&text=Because%20genetic%20diseases%20are%20inherited . 

8. Wolinetz, Carrie D., and Francis S. Collins. “NIH Supports Call for Moratorium on Clinical Uses of Germline Gene Editing.” Nature, vol. 567, no. 7747, Mar. 2019, pp. 175–75, https://doi.org/10.1038/d41586-019-00814-6.

9. Baylis, Françoise, et al. “Human Germline and Heritable Genome Editing: The Global Policy Landscape.” The CRISPR Journal, vol. 3, no. 5, Oct. 2020, pp. 365–77, https://doi.org/10.1089/crispr.2020.0082 .

‌10. Bartosch, Jamie. “Three Years after CAR T-Cell Therapy for Lymphoma, Patient Still Cancer-Free.” Www.uchicagomedicine.org, 28 Dec. 2021, www.uchicagomedicine.org/forefront/cancer-articles/a-walking-miracle-car-t-cell-therapy. 

11. Wang, Si-Wei, et al. “Current Applications and Future Perspective of CRISPR/Cas9 Gene Editing in Cancer.” Molecular Cancer, vol. 21, no. 1, Feb. 2022, https://doi.org/10.1186/s12943-022-01518-8.

12. Hejabi, Faranak, et al. “Nanocarriers: A Novel Strategy for the Delivery of CRISPR/Cas Systems.” Frontiers in Chemistry, vol. 10, July 2022, https://doi.org/10.3389/fchem.2022.957572.