4.1 Principles of genetic engineering

Genetic engineering revolutionizes biotechnology by manipulating DNA to create organisms with desired traits. This field combines molecular biology, genetics, and biochemistry to modify genes and create new biological systems.

From recombinant DNA to CRISPR-Cas9, genetic engineering tools have advanced rapidly. These techniques enable scientists to develop transgenic organisms, gene therapies, and synthetic biological systems with wide-ranging applications in medicine, agriculture, and industry.

Genetic Modification Techniques

Recombinant DNA and Molecular Cloning

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• Recombinant DNA involves combining DNA fragments from different sources to create a new DNA molecule with desired characteristics
• Relies on the use of restriction enzymes to cut DNA at specific sites and DNA ligase to join the fragments together
• Molecular cloning is the process of inserting recombinant DNA into a vector (plasmid or virus) and introducing it into a host cell (bacteria or yeast) for replication
• Cloning allows for the production of large quantities of the recombinant DNA molecule for further study or application

Gene Manipulation and Synthetic Biology

• Gene manipulation involves altering the DNA sequence of a gene to change its function or expression
• Can be achieved through site-directed mutagenesis, which introduces specific mutations into a gene using synthetic DNA primers
• Synthetic biology takes gene manipulation a step further by designing and constructing entirely new biological systems or organisms
• Involves the creation of synthetic genes, gene circuits, and even entire genomes (minimal genome of Mycoplasma mycoides)
• Synthetic biology has the potential to create novel organisms with specific functions (biofuel production, drug synthesis)

Applications of Genetic Engineering

Transgenic Organisms

• Transgenic organisms contain genes from another species that have been introduced through genetic engineering
• Transgenic plants have been developed to improve crop yields, resistance to pests and diseases, and nutritional content (Bt corn, Golden Rice)
• Transgenic animals are used as disease models (Alzheimer's mice), for the production of pharmaceuticals (goats producing human antibodies), and for xenotransplantation (pigs with human-compatible organs)
• Ethical concerns surrounding the creation and use of transgenic organisms include potential ecological impacts, animal welfare, and the safety of genetically modified foods

Gene Therapy

• Gene therapy involves the introduction of functional genes into cells to replace or correct defective genes causing genetic disorders
• Can be performed ex vivo (cells modified outside the body and then transplanted) or in vivo (gene delivery directly into the patient)
• Viral vectors (retroviruses, adenoviruses) are commonly used to deliver the therapeutic gene into target cells
• Gene therapy has shown promise in treating inherited disorders (severe combined immunodeficiency, sickle cell anemia) and acquired diseases (cancer, HIV)
• Challenges include ensuring the safety and long-term efficacy of gene therapy, as well as addressing ethical concerns about germline modifications

Advanced Gene Editing Tools

CRISPR-Cas9 and Genome Editing

• CRISPR-Cas9 is a powerful gene editing tool derived from the bacterial adaptive immune system
• Consists of a guide RNA (gRNA) that directs the Cas9 endonuclease to a specific DNA sequence, where it creates a double-strand break
• The break can be repaired by non-homologous end joining (NHEJ), often resulting in gene knockouts, or by homology-directed repair (HDR) using a donor template to introduce precise modifications
• CRISPR-Cas9 has revolutionized genome editing due to its simplicity, efficiency, and versatility compared to earlier tools (zinc finger nucleases, TALENs)
• Applications include studying gene function, creating disease models, and developing gene therapies for genetic disorders (Duchenne muscular dystrophy, cystic fibrosis)
• Ethical concerns surrounding CRISPR-Cas9 include the potential for off-target effects, the creation of designer babies, and the need for responsible governance of the technology