Genetic Engineering | Science & Technology for UPSC CSE PDF Download

Introduction

• Genetic engineering, also known as genetic modification, employs laboratory-based technologies to modify an organism's DNA. This can involve altering a single base pair (A-T or C-G), removing specific DNA regions, or adding new segments of DNA.
• Gene editing, a subset of genetic engineering, entails the insertion, deletion, modification, or replacement of DNA within a living organism's genome. In contrast to earlier genetic engineering methods that randomly incorporated genetic material into a host's genome, genome editing allows for targeted insertions at specific locations.
• CRISPR is widely recognized as the most precise, cost-effective, and rapid approach to gene editing.
• Genetic engineering techniques, including the creation of recombinant DNA, gene cloning, and gene transfer, have overcome previous limitations. They enable scientists to isolate and introduce only desired genes or sets of genes into a target organism, without introducing undesirable genetic material.
• Genetically modifying an organism involves three fundamental steps:
  • Identifying DNA containing desirable genes.
  • Introducing the identified DNA into the host organism.
  • Ensuring the introduced DNA is maintained in the host and passed on to its offspring.

Techniques of Genetic Engineering

• DNA/RNA Extraction: This involves isolating and extracting DNA/RNA from cells, typically by breaking open the cells with enzymes to eliminate unwanted macromolecules.
• PCR (Polymerase Chain Reaction): PCR techniques amplify a specific DNA segment into thousands of copies quickly. Desired DNA is replicated through repetitive cycles of amplification.
• Enzymes: Enzymes used in genetic engineering include Restriction Endonucleases, DNA Ligase, and DNA Polymerase.
• Gel Electrophoresis: Gel electrophoresis is a method that separates molecules based on their size using an electric field's charge.
• Hybridization, Southern and Northern Blotting: These techniques are used to detect and analyze specific DNA or RNA sequences.
• Molecular Cloning: Molecular cloning involves replicating DNA fragments and inserting them into host organisms.
• Three T’s (Transduction, Transfection, Transformation): These are methods used to introduce foreign DNA into host cells for genetic modification.

Benefits of genetic engineering

• Genetic modification offers a faster and more efficient approach compared to selective breeding to achieve similar outcomes.
• It can enhance crop yields and quality, which is particularly beneficial in developing nations, contributing to global efforts to reduce hunger.
• Herbicide resistance can be introduced, resulting in reduced herbicide usage as weeds can be more effectively controlled.
• Plants can be engineered for insect and pest resistance by producing toxins that deter these pests from consuming the crop.
• Sterile insects like mosquitoes can be created, leading to infertile offspring and potentially aiding in the control of diseases like malaria, dengue fever, and the Zika virus.

Risks of genetic engineering

• Transfer of the selected gene into other species. What benefits one plant may harm another.
• Some people believe it is not ethical to interfere with nature in this way. Also, GM crop seeds are often more expensive and so people in developing countries cannot afford them.
• GM crops could be harmful, for example toxins from the crops have been detected in some people’s blood.
• GM crops could cause allergic reactions in people.
• Pollen produced by the plants could be toxic and harm insects that transfer it between plants.

Difference between Gene therapy and Gene editing

Gene Editing

• Purpose: Gene editing is the process of directly modifying or altering the DNA sequence within an organism's genome. It can be used for various purposes, including treating genetic disorders, creating genetically modified organisms, and advancing medical research.
• Modification: In gene editing, specific changes are made to the DNA sequence, such as revising, removing, or replacing a mutated gene.
• Outcome: The primary aim of gene editing is to make precise changes to the DNA sequence, either to correct genetic mutations or to introduce specific genetic modifications.

Gene Therapy

• Purpose: Gene therapy is a broader concept that encompasses various techniques used to treat or cure diseases by modifying an individual's genes. It can involve both gene editing and other approaches.
• Modification: In gene therapy, the focus is on treating or mitigating diseases. It may involve replacing a disease-causing gene with a healthy one, inactivating a malfunctioning gene, or introducing a new gene to address a specific medical condition.
• Outcome: The goal of gene therapy is to alleviate or cure diseases by modifying the patient's genetic material, and the changes may or may not be heritable.

Types of Gene Therapy

• Somatic Gene Therapy: This type of gene therapy involves modifying the genes in somatic (body) cells. The effects are not passed on to the next generation because they do not affect the germline (reproductive cells).
• Germline Gene Therapy: Germline gene therapy aims to make genetic modifications in reproductive cells, affecting the genes that can be passed on to future generations.

Mitochondrial Gene Therapy (MGT)

• Mitochondria and mtDNA: Mitochondria are small structures found within cells that play a crucial role in generating cellular energy. They contain their own unique DNA, known as mitochondrial DNA (mtDNA), separate from the DNA in the cell nucleus.
• Proportion of mtDNA: MtDNA makes up a small fraction, approximately 0.1%, of a cell's total DNA content. It does not influence an individual's physical characteristics, such as appearance or personality, but it is vital for energy production within cells.
• Purpose of MGT: Mitochondrial diseases can result from mutations or defects in mtDNA, leading to severe and sometimes life-threatening conditions. MGT is designed to address these conditions by replacing the defective mtDNA with healthy mtDNA from a donor.
• Technique: In MGT, a woman's eggs with defective mtDNA are subjected to a procedure similar to in vitro fertilization (IVF). The process involves transferring the nucleus of the woman's egg into a donor egg with healthy mtDNA after the original donor egg's nucleus has been removed. Subsequently, this reconstructed egg, now containing the woman's nucleus and the donor's healthy mtDNA, can be fertilized with sperm to create an embryo.
• Inheritance of mtDNA: Unlike nuclear DNA, which combines genetic material from both parents, mtDNA is inherited exclusively from the mother. Therefore, MGT allows for the replacement of the maternal mtDNA with that of a healthy donor, potentially preventing the transmission of mitochondrial diseases to future generations.
• Rare Mitochondrial Diseases: Mitochondrial diseases caused by mtDNA mutations are relatively rare but can result in severe health issues. MGT offers a potential treatment option for individuals at risk of passing on such diseases to their offspring.

Why is it so controversial?

• Three Biological Parents: One of the key ethical concerns surrounding MGT is the concept of a child having genetic material from three different individuals: the mother (nuclear DNA), the father (nuclear DNA), and the mitochondrial donor (mtDNA). Some people find this idea ethically uncomfortable or unnatural, even though mtDNA mainly plays a role in energy production and does not significantly influence personal characteristics.
• Uncertainty of Outcomes: Critics argue that altering the genetic makeup of an embryo through MGT may introduce unpredictable long-term consequences. Since the technique involves modifying the genetic material passed down through generations, there is concern that unforeseen genetic changes could have unintended effects on future generations.
• Slippery Slope: There is a fear that the development of MGT and related genetic technologies may pave the way for broader genetic modifications, potentially leading to the concept of "designer babies." The ability to manipulate genes for specific traits or characteristics raises ethical questions about the limits of genetic engineering.
• Efficacy and Necessity: Some critics question the necessity of MGT, especially for individuals who have already been born with mitochondrial diseases. They argue that parents who are aware of their carrier status for such diseases have alternative options, such as using donor eggs or adopting, rather than undergoing genetic modification procedures.
• Ethical and Regulatory Oversight: The ethical implications of MGT and related genetic technologies require careful consideration and regulatory oversight to ensure that they are used for legitimate medical purposes and do not lead to unethical or unintended outcomes.

CRISPR-Cas9

• CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats - CRISPR-associated protein 9) is a revolutionary technology that empowers geneticists and medical researchers to modify specific segments of the genome, allowing them to delete, insert, or modify DNA sequences with a high degree of precision.
• This groundbreaking technique is celebrated for its simplicity, versatility, and accuracy, making it a topic of significant interest and excitement in the scientific community.

How does it work?

The CRISPR-Cas9 system operates through a precise sequence of molecular events:

• Cas9 Enzyme: The Cas9 enzyme, often referred to as "molecular scissors," is a key component. It is capable of cutting both strands of the DNA at a specific location in the genome.
• Guide RNA (gRNA): A piece of RNA known as guide RNA (gRNA) plays a crucial role. This gRNA comprises a short, pre-designed RNA sequence (about 20 bases long) embedded within a longer RNA scaffold. The scaffold part binds to the DNA, while the pre-designed sequence serves as a guide for Cas9 to the intended genomic location.
• Target Recognition: The guide RNA is designed to recognize and bind to a specific DNA sequence within the genome. The sequence of the guide RNA is complementary to the target DNA sequence, ensuring it selectively binds to the intended genomic location.
• DNA Cleavage: Cas9, guided by the RNA sequence, moves to the same spot in the DNA sequence and cleaves both strands of the DNA. This generates a break in the DNA molecule.
• DNA Repair: Upon detecting the DNA damage, the cell initiates a repair process. Scientists can utilize this repair machinery to introduce modifications to one or more genes within the genome of a particular cell.

Base Editing

Base editing is a revolutionary technique in genetic engineering that enables scientists to make precise changes to the genetic code by targeting and altering individual bases within DNA. Here's a breakdown of how it works:

• Importance of Bases: In the genetic code, there are four types of bases: adenine (A), cytosine (C), guanine (G), and thymine (T). These bases are the fundamental building blocks of DNA and carry the instructions for the body's functioning.
• Genetic Code: The sequence of these bases in DNA is akin to letters in an alphabet, spelling out the genetic instructions that govern various biological processes. Any misarrangement or mutation in this sequence can lead to genetic diseases, including cancer.
• Base Editing Precision: Base editing technology allows scientists to pinpoint a specific location within the genetic code and modify the molecular structure of a single base. This precise alteration involves converting one base into another, effectively changing the genetic instructions encoded in that sequence.
• Targeting Cancer Cells: Base editing has been applied in various fields, including medicine. Scientists have used this technique to engineer specialized T-cells that can selectively hunt down and eliminate cancerous T-cells, offering new prospects for cancer treatment.
• CRISPR-Cas9 Adaptation: The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology, known for its gene-editing capabilities, has been adapted to perform base editing. This innovation allows researchers to directly change specific bases, such as converting a C into a G or a T into an A, with a high degree of precision.

Chimeric Antigen Receptor T-cell (CAR-T) therapy

Chimeric Antigen Receptor T-cell (CAR-T) therapy is an innovative approach to harness the body's immune system, specifically T cells, for fighting cancer. Here's how it works:

• Isolating T Cells: T cells, a type of white blood cell crucial for the immune response, are extracted from the patient's blood.
• Laboratory Modification: In the laboratory, these T cells are genetically modified by introducing a man-made receptor known as a Chimeric Antigen Receptor (CAR). This engineered receptor enhances the T cells' ability to recognize and target specific antigens present on cancer cells.
• Improved Cancer Cell Identification: The addition of CARs equips the T cells with the capability to better identify and bind to cancer cell antigens.
• Patient-Specific Treatment: The modified CAR T cells, now armed to attack cancer cells more effectively, are then infused back into the patient's body.
• Cell-Based Gene Editing: CAR-T therapy is often discussed as a form of cell-based gene editing because it involves the genetic modification of T cells to empower them to combat cancer.
• Support for CAR-T Technology: Various initiatives, such as those by the Biotechnology Industry Research Assistance Council (BIRAC) and the Department of Biotechnology (DBT), have been undertaken in recent years to promote and advance CAR-T cell technology. These efforts have focused on its development for treating diseases like acute lymphocytic leukemia, multiple myeloma, glioblastoma, hepatocellular carcinoma, and type-2 diabetes.
• Cost and Availability: Currently, CAR-T cell therapy is not available in India, primarily due to its high cost, with each patient's treatment costing 3-4 crore INR. One of the major challenges is to develop a cost-effective manufacturing process to make this technology accessible to patients.

Orphan Drug

• An orphan drug refers to a biological product or medicine designed to treat diseases that are exceptionally rare, to the extent that pharmaceutical companies are hesitant to develop them under typical market conditions.
• In 1983, the United States government introduced the Orphan Drugs Act with the aim of encouraging research into treatments for diseases that have historically received minimal attention from the pharmaceutical industry. Similar legislations have been adopted in other countries, including Japan, Australia, and the European Union. These laws provide various incentives to pharmaceutical companies, such as streamlined clinical trials, extended periods of exclusivity, tax benefits, and a higher likelihood of regulatory approval. 
• These incentives make it financially viable for pharmaceutical firms to invest in the research and development (R&D) necessary to discover cures for these rare diseases. However, it's important to note that India does not have a nationwide Orphan Drug policy.
• In 2016, the state of Karnataka in India took a pioneering step by introducing a Rare Diseases and Orphan Drugs Policy. This policy recommended the implementation of preventive and carrier testing as a strategy to reduce morbidity and mortality associated with rare diseases. Since a significant portion of rare diseases have a genetic basis, the policy also proposed the use of genetic testing to expedite the identification of critical genes involved in rare diseases.
• Furthermore, the court offered several suggestions to the government, emphasizing the relevance of corporate social responsibility (CSR) provisions outlined in the Companies Act of 2013. The court affirmed that sponsoring the treatment of rare diseases would qualify as a CSR activity, encouraging corporate entities to contribute to addressing the challenges associated with these conditions.

Bioprospecting

• Bioprospecting is the process of discovering and commercializing new products derived from biological resources. While indigenous knowledge has long been intuitively valuable, it has only recently been integrated into bioprospecting efforts to target the screening of bioactive compounds more effectively.
• On the other hand, biopiracy refers to the unauthorized use of biological resources by multinational companies and other organizations without proper consent from the countries and communities that have a stake in these resources, often without providing fair compensation.
• Biomining is a technique used to extract metals from ores and other solid materials. This process typically involves the use of prokaryotes or fungi, which secrete various organic compounds capable of chelating metals from the environment. These chelated metals are then transported back to the cells where they are commonly used to facilitate electron coordination.
• An example of biopiracy occurred when the USDA (United States Department of Agriculture) and an American multinational corporation, W.R. Grace, sought a patent (No. 0426257 B) from the European Patent Office (EPO) in the early 1990s for a "method for controlling pests on plants using hydrophobic extracted neem oil." This patenting of neem's fungicidal properties was considered an instance of biopiracy, as it involved the appropriation of indigenous knowledge without appropriate authorization.

Biomaterials

• Biomaterials are substances that have been specifically designed to interact with biological systems for medical purposes, serving either therapeutic (to treat, enhance, repair, or replace a bodily tissue function) or diagnostic roles.
• The Transplantation of Human Organs (Amendment) Act of 2011 covers the aspect of tissue donation and the establishment of tissue banks.

Various biomaterials can potentially be retrieved and stored for medical applications, including:

• Skin: Used as a biological dressing, particularly in cases of severe burns. It aids in infection prevention and requires less frequent replacement, providing patients with adequate recovery time.
• Bones: Limb bones can be stored and utilized to replace damaged or diseased bone segments. Bone grafts from tissue banks serve as supportive scaffolds, applicable in cases of bone loss due to trauma, sports injuries, or diseases like cancer, where parts of bones and joint cartilage have deteriorated.
• Ligaments and tendons: Useful in cases of sports injuries involving multiple ligaments, especially when using the patient's own tissues is challenging or insufficient. Commonly retrieved ligaments and tendons include the Achilles tendon (ankle), Peroneal tendon (leg to ankle), Patellar tendon (front of the knee), and Meniscus (shock absorber between the thigh and leg bones).
• Bone products: Bone powder is produced by grinding bones, typically those replaced during hip replacement surgeries. It finds applications in various procedures, such as dentistry and skeletal and joint reconstructions.
• Amniotic membrane: Derived from the amniotic sac wall, it serves as a biological dressing for conditions like burns, bedsores, diabetic ulcers, and skin reactions to radiation therapy.
• Heart valves: Harvested heart valves can be stored for use in valve replacement surgeries. They offer benefits such as not requiring blood thinners and being cost-effective compared to artificial valves. However, they have a lifespan of around 15 years and may necessitate subsequent procedures.
• Corneas: Corneal transplants are employed when the cornea becomes opaque due to factors like injuries, infections, birth defects, or rare complications following surgeries.

Escherichia Coli

• Escherichia coli, often abbreviated as E. coli, is a type of Gram-negative bacterium with a rod-shaped structure. It is considered a facultative anaerobe, which means it can thrive in both oxygen-rich and oxygen-deprived environments. E. coli is commonly found in the lower intestines of warm-blooded organisms, including humans.
• E. coli holds the distinction of having its genome fully sequenced, a milestone achieved in 1997. It is one of the most extensively studied bacteria globally and serves as a crucial model organism in various scientific fields, especially molecular biology, genetics, and biochemistry.
• One of the reasons for E. coli's prominence in research is its ease of cultivation in laboratory settings, and it is generally safe to handle. Moreover, E. coli exhibits rapid growth rates, enabling researchers to study numerous generations within a short timeframe. In optimal conditions, E. coli cells can double their population every 20 minutes.

Luciferase

• Luciferase is a term used to describe a group of oxidative enzymes that play a crucial role in producing bioluminescence, a natural phenomenon where living organisms emit light. It is typically differentiated from photoproteins, another class of light-producing molecules.
• Luciferases have a wide range of applications in biotechnology, microscopy, and as reporter genes, serving many of the same purposes as fluorescent proteins. However, unlike fluorescent proteins that require an external light source for fluorescence, luciferases need luciferin, a consumable substrate, to generate light.
• In laboratory settings, luciferases can be produced through genetic engineering for various purposes. This involves synthesizing luciferase genes and inserting them into organisms or introducing them into cells. This genetic modification has been successfully applied to a variety of organisms, including mice, silkworms, and potatoes, to enable them to produce luciferase.
• The luciferase reaction results in the emission of light when luciferase interacts with the appropriate luciferin substrate. The emitted photons can be detected using light-sensitive equipment like a luminometer or modified optical microscopes.

Photodynamic therapy

• Photodynamic therapy (PDT) is a medical treatment that utilizes a photosensitive drug to become active when exposed to light. This activated drug then converts molecular oxygen into reactive oxygen species, which are highly reactive molecules that can effectively kill cancer cells.
• PDT involves the use of special drugs known as photosensitizing agents, which are administered in one of two ways depending on the location of the cancerous cells. They can either be introduced into the bloodstream through a vein or applied directly to the skin. Once inside the body, the photosensitizing agent is absorbed by the cancer cells over a specified period. Subsequently, light is directed at the treatment area. When the photosensitizing agent interacts with oxygen upon exposure to light, it triggers the formation of a chemical compound that has the capacity to destroy the cancer cells.
• In addition to directly targeting cancer cells, PDT may also contribute to treatment by disrupting the blood vessels that supply nutrients to the cancer cells and by stimulating the immune system to mount an attack against the cancer. This multi-pronged approach enhances the therapy's effectiveness in fighting cancer.

Bacteriophage

• Bacteriophages, often referred to as phages, are a type of virus that exclusively infects bacterial cells. They achieve this by injecting their genetic material, typically DNA, into the host bacterium. Once inside the bacterial cell, the phage's genetic material takes control of the host's cellular machinery, effectively hijacking it to produce more phage particles.
• The injected phage DNA undergoes a selective process of replication and gene expression within the host bacterial cell. This results in the production of numerous new phage particles, which ultimately exit the host cell, typically through a process that bursts and kills the host (known as the lytic pathway). These newly formed phages can then proceed to infect neighboring bacterial cells, continuing the cycle.
• It's important to note that some phages only follow a lytic lifecycle, which means they exclusively utilize the burst-and-kill strategy to propagate. This ability to transfer genetic material from the phage genome to specific bacterial hosts during viral infection has led scientists to explore the potential of using specially designed phage-based vectors as valuable tools in gene cloning experiments. Two well-known phages, lambda (ë) and M13, have undergone extensive modifications to develop them into effective cloning vectors.

CHARGE syndrome

CHARGE syndrome is a rare congenital disorder that affects an estimated 1 in 20,000 individuals globally. This syndrome is characterized by a constellation of severe and often life-threatening issues that present in newborns. These issues can include:

• Facial Bone and Nerve Defects: Individuals with CHARGE syndrome may have abnormalities in the development of facial bones and nerves. This can lead to difficulties in breathing and swallowing.
• Sensory Impairments: CHARGE syndrome commonly causes sensory impairments, including deafness and blindness.
• Heart Defects: Many individuals with CHARGE syndrome are born with congenital heart defects.
• Genital Abnormalities: Genital problems can also be present in those with CHARGE syndrome.
• Growth Retardation: Individuals with this syndrome may experience growth retardation, leading to smaller stature.

It's important to note that while CHARGE syndrome can result in life-threatening complications, early intervention and corrective surgeries, particularly for heart and bone defects, can improve outcomes and allow affected individuals to lead relatively normal lives.
CHARGE syndrome is primarily caused by defective embryonic development, and in about two-thirds of cases, it is associated with sporadic mutations in a specific gene called CHD7. This gene plays a crucial role in normal development, and mutations in it are strongly linked to the development of CHARGE syndrome.

Bionics

• Bionics is indeed a fascinating field that involves the application of biological principles to engineering. It's not limited to a specialized science but rather encompasses a multidisciplinary approach to designing and developing innovative solutions, often inspired by nature, to address various challenges.
• In the context you mentioned, bionics can refer to the creation and engineering of artificial organs or body parts that can replace or augment diseased or non-functional ones within the human body. This can include devices like artificial limbs, cochlear implants, or even more complex systems like artificial organs.
• It's important to note that bionics is distinct from bioengineering or biotechnology, which typically involves using living organisms or biological processes to perform specific tasks or produce desired products, such as using microbes for waste treatment or genetic engineering for drug production.
• Bionics, on the other hand, often focuses on the integration of biological and engineering principles to create synthetic systems that mimic or enhance natural biological functions. This field has the potential to revolutionize healthcare and improve the quality of life for individuals with various medical conditions.

Autophagy

• Autophagy, a term derived from the Greek words "auto" meaning self and "phagy" meaning eating, is a vital physiological process in the body that involves the degradation of cells. It plays a crucial role in maintaining normal functioning and homeostasis by facilitating the breakdown of cellular proteins and recycling components of damaged or unnecessary cell organelles to generate new cellular material.
• Autophagy becomes particularly active during periods of cellular stress, which can result from factors like nutrient deprivation or a lack of essential growth factors. In response to stress, autophagy ramps up, providing the cell with an alternative source of intracellular resources and energy to support its survival.
• One interesting aspect of autophagy is its dual role. On one hand, it can help cells survive by providing the necessary building blocks and energy during stress. On the other hand, it can lead to a form of programmed cell death known as autophagic cell death, which is distinct from apoptosis, another form of programmed cell death.
• Autophagy plays a pivotal role in maintaining a balance between creating new cellular components and breaking down damaged or superfluous organelles and cellular constituents. This process has a wide range of functions, including:
  • Providing a rapid source of energy and materials for cellular renewal during times of starvation and stress.
  • Eliminating invading intracellular pathogens, such as bacteria and viruses, after infection.
  • Contributing to embryonic development and cell differentiation.
  • Removing damaged proteins and organelles, acting as a quality control mechanism against aging-related issues.
• Disruptions in autophagy have been associated with various diseases, including Parkinson's disease, type 2 diabetes, and cancer. Mutations in autophagy-related genes can lead to genetic disorders. Consequently, intense research is underway to develop drugs targeting autophagy to treat a wide range of diseases.
• Yoshinori Ohsumi's groundbreaking research in the 1990s significantly enhanced our understanding of autophagy and its crucial roles in physiology and medicine. For his pioneering work, he was awarded the Nobel Prize in Physiology or Medicine in 2016, bringing well-deserved recognition to this essential biological process.

Triple Drug Therapy

• The World Health Organization (WHO) has recommended the use of a three-drug treatment called IDA (Ivermectin, Diethylcarbamazine citrate, and Albendazole) to accelerate the global elimination of lymphatic filariasis, a neglected tropical disease.
• Lymphatic filariasis is caused by parasitic worms that reside in the lymphatic system of infected individuals. The larvae of these parasites, known as microfilariae, circulate in the blood and are transmitted from person to person through mosquito bites. Once infection occurs, the disease takes time to manifest, resulting in the alteration of the lymphatic system, abnormal enlargement of body parts, severe disability, and social stigmatization of those affected.
• The parasites responsible for lymphatic filariasis are transmitted by various mosquito species, including Culex, Mansonia, Anopheles, and Aedes.
• To combat this debilitating disease, the WHO recommends the annual use of the IDA triple drug therapy in areas where it is expected to have the greatest impact. This treatment approach aims to eliminate lymphatic filariasis and reduce the suffering caused by the disease.
• In India, the government has taken steps to scale up the use of Triple Drug Therapy (IDA) in a phased manner, starting from November 2019. This initiative is part of a global effort to eliminate lymphatic filariasis by 2021, with the goal of improving the health and well-being of affected populations worldwide.