Genetic Modification: CRISPR

Introduction

Over the past few decades, genetic engineering has progressed significantly, moving from basic lab techniques in the mid-20th century to real world applications in medicine, agriculture and research. The most prominent factor of this advancement was the development of the revolutionary gene editing tool CRISPR that offers simplicity, efficiency and versatility compared to former methods. This paper explores the mechanism and development of CRISPR, its applications, its limitations, case studies, the ethical concerns that come up and the opinions and responses of international bodies and stakeholders.

Genetic modification (GM) is any deliberate change made to an organism’s DNA sequence or its regulation to give new traits, i.e. pest resistance, altered metabolism, therapeutic changes. GM spans older methods such as selective breeding, mutagenesis, transgenics, and modern precision tools that edit specific DNA bases.
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a family of bacterial immune-system tools adapted for targeted genome editing. Unlike older transgenic (artificial introduction of DNA) or random-mutagenesis (introduction of random mutations throughout a DNA sequence) methods, CRISPR enables precise, programmable cuts at chosen DNA sites using a guide RNA—making edits faster, cheaper, and far more specific.

Discovery and Development

CRISPR and its existence were first identified in 1987 by Ishino et al. at Osaka University, Japan, during studies on the DNA sequences of Escherichia coli bacteria by Yoshizumi Ishino 1987. Although the researchers successfully recorded these unusual DNA repeats, their biological role remained unknown at the time.

In the years that followed, scientists proposed that CRISPR loci could be used for bacterial genotyping to differentiate between strains such as Mycobacterium tuberculosis and Streptococcus pyogenes due to the high degree of polymorphism (presence of two or more variant forms of a specific DNA sequence) in CRISPR sequences (by Groenen et al., 1993; Pourcel et al., 2005).

A breakthrough occurred in 2007 when French scientists Rodolphe Barrangou and Philippe Horvath, working for the Danish food company Danisco, discovered the mechanism behind the CRISPR system while studying Streptococcus thermophilus, a bacterium used in yogurt production (Barrangou et al., 2007). They demonstrated that CRISPR sequences act as a form of adaptive immunity, allowing bacteria to acquire resistance to viral infections (bacteriophages) by incorporating fragments of viral DNA—called “spacers”—into their genome. This discovery led to the development of CRISPR-based bacterial “vaccination” techniques and one of the first patents in CRISPR technology (Barrangou & Horvath, 2017). (Sources used: PMC, BMC)

Mechanism

A CRISPR gene-editing process involves several key steps that allow scientists to precisely modify DNA within a cell. These steps can vary depending on the organism or purpose, but the basic procedure goes like this:

1. Target Identification:
   Researchers first identify the specific gene or DNA sequence they want to modify. This target sequence must be located next to a short DNA motif known as PAM (protospacer adjacent motif), which is required for CRISPR to recognize and cut the DNA correctly.
2. Guide RNA (gRNA) Design:
   A short RNA molecule called guide RNA is designed to match the target DNA sequence. The gRNA directs the Cas9 enzyme ( CRISPR associated protein) to the exact spot in the genome that needs to be edited.
3. CRISPR-Cas9 Complex Formation:
   The guide RNA and Cas9 enzymes are combined to form a complex. Cas9 acts as molecular scissors, while the gRNA serves as a GPS that guides it to the correct DNA location.
4. Delivery into Cells:
   The CRISPR-Cas9 complex is introduced into the target cells using various delivery methods, such as plasmid vectors, viral carriers, or lipid nanoparticles.
5. DNA Cutting/Double-Strand Break:
   Once inside the cell, the Cas9 enzyme, guided by the gRNA, binds to the target DNA and cuts both strands at the specified site.
6. DNA Repair and Editing:
   The cell naturally tries to repair this break. Scientists can use this process to either disrupt a gene (by letting the cell repair the break imperfectly) or to insert a new DNA sequence. Two main repair pathways are used:
   ◦ Non-Homologous End Joining: Leads to small insertions or deletions that can “knock out” a gene.
   ◦ Homology-Directed Repair: Uses a supplied DNA template to precisely insert or replace a gene.
7. Screening and Verification:
   After editing, scientists check the cells to confirm that the desired genetic change has occurred. This is done through DNA sequencing or other molecular biology techniques.

Applications of CRISPR

Medicine: Since scientists now have the power to edit, remove, or replace defective genes with great accuracy, genetic diseases caused by gene mutations, such as sickle cell anemia, can be prevented. From gaining insights into the underlying causes for genetic diseases to the potential of correcting disease-causing mutations directly in the genomes of patient cells, CRISPR-Cas9 also holds promise for the development of gene therapies. It makes it possible to correct a mutation associated with a genetic blood disorder, β-thalassemia, in patient-derived stem cells. CRISPR-Cas9 technology has opened up new avenues for cancer research and therapy through targeted disruption of genes involved in cancer progression and enhancing the effectiveness of the treatment in cancer cells.

Agriculture: CRISPR helps by creating crops with improved traits, such as higher yields, better nutritional content, disease resistance, pest resistance, and tolerance to drought or salinity. In livestock, CRISPR enables breeding of animals with disease resistance (e.g., pigs resistant to viral infections) or better growth efficiency, which supports sustainable farming.

Biotech industry: CRISPR-Cas9 technology also plays a role in gene regulation and functional genomics (deals with gene functions and interactions). It allows for the precise manipulation of gene expression, enabling researchers to investigate gene function and unravel the complexities of biological systems.CRISPR-Cas9 technology has enabled the rapid and cost-effective generation of animal models for studying human diseases. It allows for the introduction of specific mutations associated with human genetic disorders, providing valuable tools for understanding disease mechanisms and developing therapeutic interventions. In a study, CRISPR-Cas9 was used to create a pig model of cardiovascular disease, demonstrating the potential of CRISPR-Cas9 in modeling complex human diseases in animals.

Case Studies

1. CTX001 (sickle cell / β-thalassemia trials): Casgevy, a CRISPR-based therapy for sickle cell disease, cures most individuals with a patient’s own corrected cells. But it is very expensive and requires an immense amount of medical care.
2. He Jiankui (2018 human embryo edits): The He Jiankui Incident, where a Chinese scientist created the world’s first gene-edited babies in 2018, sparked major controversy and the whole experiment was widely criticised due to ethical concerns, scientific malpractice, and risk of unintended, heritable mutation-causing stricter regulations worldwide.

Limitations

The CRISPR system is not fully reliable. The Cas 9 enzyme can make cuts in inaccurate regions within the DNA, which is deadly. Getting the CRISPR components to the right cells inside an organism is also very tricky with current methods . Normal CRISPR will create breaks in DNA, so it is not possible to perform minor, accurate adjustments. The editing process might not be completed in embryos, leading to a situation called mosaicism, which is a condition where an individual has two or more different sets of cells in their body.
There are risks of unintended consequences:
Cutting DNA in the wrong places could cause cancer. Changes passed down could have hidden effects over many generations. Using “gene drives" to spread changes through a population can lead to accidental species extinction or ecological disruption.

Ethical Concerns

1. Germline Editing: Through germline editing, changes made to embryos or reproductive cells can be passed down to future generations. This raises questions about consent, as future individuals cannot agree to modifications made before their existence along with the potential for unforeseen long term effects.
2. Autonomy and Consent: The ability to edit genes in unborn individuals removes their autonomy to choose whether they wanted those changes in the first place. This also creates the ethical dilemma of what constitutes a ‘desirable’ trait.
3. Genetic Discrimination and Inequality: There is a risk that advanced genetic enhancements may only be accessible to the wealthy, exacerbating existing social inequalities. This could lead to a society where genetic modifications create new forms of discrimination and stigmatization against those who cannot afford such enhancements.
4. Misuse and “Designer Babies”: The potential for CRISPR technology to be used for non-therapeutic enhancements, such as selecting traits for perceived advantages, produces significant ethical dilemmas. The concept of ‘designer babies’ raises moral questions about the extent to which humans should interfere with natural genetic processes. Some people believe altering DNA interferes with natural or divine creation, raising philosophical and spiritual objections.

Legal Response and General Opinions

The US looks at the final product and does not dig deep into the technique and process itself. The EU is stricter. China became more serious after a scientist’s scandal in 2018 so unauthorised gene editing is now a crime. The World Health Organization (WHO) says we should not edit human genes in a way that can be inherited until it is proven safe and everyone is on board.
Scientists’ views on CRISPR depend heavily on how it’s used. Overall, the scientific community is excited by its potential but cautious about its risks. The policymakers aim to regulate CRISPR to prevent misuse while allowing responsible innovation. The general public are mostly happy with the advancement in treatments and agriculture but some are skeptical of GM crops.
CRISPR technology is rapidly evolving beyond just cutting genes. Ongoing research includes:

1. Base and Prime Editing: These newer methods can change a single DNA letter without breaking the DNA, making them safer and more precise.
2. Epigenome Editing: This technique focuses on regulating gene activity by adjusting molecular "switches."
3. Improved Delivery: Scientists are also working on better and safer ways to deliver CRISPR tools to the right cells.
4. Diagnostics: CRISPR systems like Cas12/13 are being used for cheap, rapid diagnostic tests for diseases.

Conclusion

CRISPR has transformed genetic engineering by making gene editing faster, cheaper and more precise. While its applications are crucial in the development of advanced healthcare and production of sustainable crops, the ethical concerns and limitations should not be overlooked. Strict regulations and responsible usage are needed as the future impact of CRISPR will depend on how it is developed and handled now.