CRISPR-Cas9 Applications and Ethical Issues Explained
Learn how CRISPR-Cas9 is used in gene editing, cancer research, and agriculture, and understand the ethical concerns and regulatory challenges it raises.
CRISPR-Cas9 gene-editing system
CRISPR-Cas9 is one of the gene editing tools that can modify many different genes in plants, animals, or cell lines.
Clustered Regularly Interspaced Short Palindromic Repeat, or CRISPR, is a specific sequence of DNA that is found in bacteria and archaea as a defense mechanism. This CRISPR sequence protects bacteria from various viruses. After bacteriophage infection, these specific sequences bind with CRISPR-associated (Cas) proteins, detect, and cleave the bacteriophage DNA.
CRISPR sequences use Cas9 as a guide and detect specific sequences of DNA. After recognition of a DNA fragment that is complementary to the CRISPR sequence, Cas9 opens up the DNA fragment and cleaves at a specific site. CRISPR and Cas9 endonuclease are both known as CRISPR-Cas9, which is advanced technology for gene editing in animals and plants.
Nowadays the CRISPR-Cas9 plays various roles in the modern gene editing technology field, such as genetic disease, viral infections, antibiotic-resistant bacteria mechanisms, and cancer therapy.
In 1987, Japanese researchers observed this unique pattern of DNA sequence in gut Escherichia coli for the first time. They observed five short repeated DNA fragments separated by short non-repeated ‘spacer’ DNA sequences.
In 2008, researchers from Maryland first demonstrated the mechanism of CRISPR/Cas9. When a virus attacks bacteria, the bacteria copy and insert viral DNA fragments as a ‘spacer’ into their own genome between the CRISPR. These spacers are stored in the bacterial genome as a genetic memory and protect from future viral infection. These spacers allow the Cas9 enzyme to cut the invading viral DNA and protect the bacteria from infection.
In 2012, many researchers worldwide recognized the efficiency of CRISPR-Cas9 and found out how this technology could be used in genome editing in plant and animal systems.
Now researchers have modified and improved the efficiency of the CRISPR-Cas9 system. They introduce a novel system of genome editing; they understand the mechanism of CRISPR-Cas9 editing and analyze how it evolved as a bacterial immune system and how modification, adaptation, and application in humans, animals, or plants will make CRISPR-Cas9 a promising tool for genome editing.
CRISPR: Immune System for Bacteria and Archaea
Bacteria and Archaea have their own defense system that protects them from viral and plasmid cellular invaders such as viruses. Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR), along with the CRISPR-associated Proteins (Cas proteins), is one of the adaptive immune systems found in bacteria and archaea.
When DNA sequences from viral invaders enter into bacterial or archaeal genomes, they generate cellular memory from past viral invaders. After integration of foreign DNA sequences, bacteria and archaea defense mechanisms are activated. They recognize the foreign DNA from viral or plasmid invaders as non-self and start cleaving and degrading the foreign DNA sequence using the CRISPR-Cas9 mechanism and work as an adaptive immune system for prokaryotes.
The CRISPR–Cas immune response follows three steps, and these are adaptation, expression, and interference.
During the adaptation stage, Cas proteins recognize a short DNA sequence called the protospacer-adjacent motif (PAM) present in invading DNA. Cas protein binds to invading DNA and further creates two double-strand breaks in it. This cleaved and released short fragment of DNA from an invading phage or plasmid is known as a protospacer. These release protospacers inserted between two repeat sequences of the CRISPR array and create a new spacer.
During the expression stage, the cas genes are expressed and the CRISPR locus is transcribed into precursor CRISPR RNA (pre-crRNA). This pre-crRNA is then processed by Cas proteins and accessory factors into short and mature CRISPR RNAs (crRNAs).
During the interference stage, crRNA combines with Cas protein and recognizes the invader DNA. After recognition, both the combined crRNA and Cas protein cause cleavage of invader DNA and protect the host species from infection. The expression and interference steps are different for each and every CRISPR system.
Application of CRISPR Cas-9
Cancer Research
Cancer develops and progresses due to mutations and abnormal expression of multiple genes, including oncogenes, tumor suppressor genes, chemoresistance genes, metabolism-related genes, and cancer stem cell-related genes. The objective behind cancer treatment is to restore normal expression of dysregulated genes by suppressing tumor growth and reducing cancer growth by correcting lethal mutations. The CRISPR/Cas9 gene editing tool has become a promising system and has been used in cancer research.
In poultry industry
Significant progress has been made in the poultry industry by using CRISPR-Cas9 technology, especially in two poultry species, chicken and quail. The purpose is not to replace the traditional breeding system but to improve the system by introducing genetic variation in chickens and quail. This is possible by the CRISPR-Cas9 system, which introduces genetic variation and enhances the growth and productivity of the poultry. These growths are increased egg production, increased bird immunity and disease resistance, production of birds that contain less or no fat, better fatty acid composition, and better nutrient profiles.
Genetic Diseases
Leber congenital amaurosis (LCA) is a rare genetic eye disease that causes severe vision loss during birth. It is caused by a loss-of-function mutation in the CEP290 gene. Using CRISPR-Cas9, researchers treated the eyes in a humanized CEP290IVS26 knock-in mouse model, and mouse vision significantly improved to 94%.
Duchenne muscular dystrophy (DMD) is a genetic musculoskeletal disease that causes progressive muscle weakness in the early and later stages of the disease with symptoms of fibrosis and fat replacement. Researchers successfully found the treatment of DMD using CRISPR-Cas9 technology in the mouse model that excised the exon from the dystrophin gene responsible for the disease. The mouse model showed significant recovery of the dystrophin protein, and myofibers expressed dystrophin.
Antibiotic Resistant Bacteria
In the healthcare and agriculture industries, the overuse of antibiotics in the past 70 years has raised a serious issue that has caused the development of antibiotic resistance in bacteria. Bacteria become resistant to antibiotics due to the mutations and addition of new resistance genes. Various approaches have been implemented, such as new antibiotic production and the use of bacteriophages or peptides or synthetic or natural enzymes to target the bacterial genomes. The researcher reveals that the CRISPR–Cas system could be the potential approach to prevent, control, and treat antibiotic-resistant bacteria.
Ethical implications in human and agricultural genetics
CRISPR-Cas9 is promising editing tool but there is certain limitation that need to investigated.
Off-target effects
In CRISPR-Cas9 technology, off-target effects are the main drawback that limit the application in animals and plants. These off-target mutations are always present in the population, and due to genetic drift, these mutations can transfer from generation to generation. Over time the impact and number of mutations can increase as generations progress, which could further lead to abnormalities in living systems. Transfer of mutation from generation to generation results in dispersion of gene drive that is difficult to regulate. Gene drive can eliminate a complete population, which can have a huge impact on the ecosystem and cause severe negative effects and imbalances in the system.
Gene Transfer
Another limitation of CRISPR-Cas9 is the transfer of modified genes to other species. This gene transfer to other species may impact the environment and can change the species at the genetic level. Genetic mosaicism in the founder organism is one of the consequences of the CRISPR-Cas9 gene editing tool. Genetic mosaicism happens when the CRISPR/Cas9 system is used in embryos, causing the presence of genetically different cell populations within the same organism. It is more common in transgenic and knockout animal model generation. For example, the founder mouse has a homozygous deletion, but that deletion allele never transmits to the offspring.
Genome editing drawback
The purpose of CRISPR-Cas9 is to create different mutations in various species, including humans. The main objective for this purpose is to treat the human species for lethal diseases by DNA modification before the baby is born. In China, some scientists tried to cure ß-thalassaemia. ß-thalassaemia is a blood-related disorder in which blood is unable to clot and becomes lethal for patients. This research was showing promising results and creating new hope for β-thalassemia patients, but the gene editing of human embryos that were further implanted into the uterus raises serious questions, concerns, and ethical issues for humans. Further, the research group used “non-viable triploid embryos,” but these embryos are unable to develop normally because they were developed from two sperm fertilizing one egg. Due to this precaution and ethical concerns, scientific journals refuse to publish their research work.
Ethical impact
Though CRISPR-Cas9 is used in all domains, including gene therapy, agricultural improvement, drug discovery and screening, and treatment of genetic diseases, advancements in CRISPR-Cas9 have raised questions related to safety and ethical regulations.
Worldwide, several researchers used CRISPR-Cas9 to edit genes in human embryos, although these experiments were done in non-viable, triploid zygotes. The objective of this type of research is to observe the specificity and accuracy of the CRISPR–Cas9 system. These types of studies raise serious concern in the scientific community about what the exact ethical regulation is for gene editing methods without affecting the research, which is for research benefit and scientific discovery.
The objective of CRISPR-Cas9 is to develop precise genetically modified species, but it becomes difficult to identify genetically modified species once they leave the lab and come into the environment. There are no ethics or regulations on how regulatory agencies will manage those CRISPR-Cas9-modified species. Controversies and ethical issues have become the main problem among biotechnology companies over patents related to the use of CRISPR-Cas9 in human and plant species.
References:
- Janik, E., Niemcewicz, M., Ceremuga, M., Krzowski, L., Saluk-Bijak, J. and Bijak, M., 2020. Various aspects of a gene editing system—crispr–cas9. International journal of molecular sciences, 21(24), p.9604.
- Thurtle‐Schmidt, D.M. and Lo, T.W., 2018. Molecular biology at the cutting edge: a review on CRISPR/CAS9 gene editing for undergraduates. Biochemistry and molecular biology education, 46(2), pp.195-205.
- Zhang, H., Qin, C., An, C., Zheng, X., Wen, S., Chen, W., Liu, X., Lv, Z., Yang, P., Xu, W. and Gao, W., 2021. Application of the CRISPR/Cas9-based gene editing technique in basic research, diagnosis, and therapy of cancer. Molecular cancer, 20(1), p.126.
- Lorenzo, D., Esquerda, M., Palau, F., Cambra, F.J. and en Bioética, G.I., 2022. Ethics and Genomic Editing Using the Crispr-Cas9 Technique: Challenges and Conflicts. NanoEthics 16: 313–321 [online]
