ML 2

Molecular Tools in Genetics

  • Introduction to Molecular Tools
    • Molecular tools are essential for diverse applications in genetics and public health.
    • The Polymerase Chain Reaction (PCR) is a widely known molecular tool, particularly used during the COVID-19 pandemic for testing.
    • The focus of this discussion is on CRISPR as a significant tool for genome or genetic engineering.

Central Dogma of Molecular Biology

  • Understanding the central dogma is critical since the processes involved can be harnessed to develop various molecular tools.
  • Examples of applications derived from the central dogma include:
    • Genetic Engineering
    • Gene Expression Control
    • Turning genes on/off
    • Analyzing gene expression effects in diseases (e.g., cancer)
    • Diagnosis, Prognosis, & Treatment Outcomes
    • Sequencing Genomes
    • The evolution and engineering of molecules allow for accurate high-throughput genome sequencing.
  • mRNA Vaccines during COVID
    • mRNA vaccines were produced using RNA polymerases and purified DNA to generate worldwide supplies of mRNA for vaccination.
    • This represents an impactful application of molecular tools in manufacturing.

Genome Engineering Focus

  • Definition of Genome Engineering
    • The goal is to make precise changes to the genetic material of an organism to correct mutations causing diseases.
    • Example Case: A mutation in human DNA leads to a faulty messenger RNA and then to a dysfunctional protein, which may cause disease.
    • The modification aims to correct the original mutation so that the resultant protein functions normally.
  • CRISPR-Cas9 Tool Development
    • Introduced as a tool effective for genetic modifications within the last decade.

Background to CRISPR-Cas9

  • CRISPR research originally stemmed from microbiologists and genome sequencing scientists.
  • Initial Discoveries:
    • Observations were made on bacteria's defenses against bacteriophages (viral infections of bacteria) resulting in interest from yogurt makers.
  • Genome sequencing efforts revealed repetitive sequences (CRISPR) associated with Cas genes found in bacterial chromosomes.
    • These assist bacteria in protecting against infections by storing sequences from previous phage attacks.

Mechanism of CRISPR-Cas System

  • Bacterial Defense Mechanism
    • The CRISPR locus transcribes RNA molecules that form a structure capable of recognizing and cleaving the DNA of invading plasmids (bacteriophages).
    • The guide RNAs (gRNAs) play a critical role in this recognition by pairing with the DNA.
  • Understood Functions
    • When introducing CRISPR-Cas9 into genetic modification applications, the potential of directing cuts at any DNA target was realized, expanding beyond bacteriophages.

Engineering CRISPR-Cas9 for Biotechnology

  • Application to Gene Editing
    • The Cas9 protein can use guide RNAs to create double-strand breaks in desired DNA sequences.
    • The cell's repair systems engage, repairing the break through two main pathways:
    1. Non-Homologous End Joining (NHEJ)
      • Can introduce insertions or deletions, creating gene knockouts.
    2. Homologous Recombination (HR)
      • Allows for precise editing by supplying a donor sequence that is integrated at the cut site.
  • Nobel Prize Achievement
    • Emmanuel Charpentier and Jennifer Doudna won the Nobel Prize in Chemistry in 2020 for their discoveries leading to CRISPR-Cas9's applicability in genetic engineering.

Structural Studies of CRISPR-Cas9

  • CRISPR-Cas9 structures have been resolved to understand the protein's mechanics, including guiding RNAs and target DNA interactions.
  • Importance of Motility:
    • Motions of Cas9 are critical for its functionality, ensuring precise cuts at targeted DNA sequences.
  • Fidelity Studies:
    • Investigations into how mutations impact the accuracy of Cas9 action provide insights into making CRISPR technology more precise.

Current Applications and Future Directions

  • Uses of CRISPR Technology
    • Engineering animal models for human diseases (e.g., mice with cancer models).
    • Generating crops resistant to stressors like drought and pests.
    • Targeting genetic diseases for treatment, with sickle cell disease as a notable example.
  • Sickle Cell Disease and Genetic Engineering
    • The disease results from a single mutation in hemoglobin.
    • Utilizing CRISPR to correct this mutation in red blood cell precursor cells targets an effective therapeutic pathway.
    • A successful case involved a patient named Victoria Gray, who underwent CRISPR treatment for sickle cell anemia, marking historic advancements in gene editing therapies.

Challenges and Ethical Considerations

  • Cost and Duration of Drug Development
    • Drug development processes can take ten to thirty years, costing around a billion dollars on average.
  • Comparison of Therapeutic Markets
    • Successful drug markets (e.g., diabetes medications) contrast with under-invested areas such as new antibiotic discovery, highlighting the funding dilemma in medical research.

Molecular Basis of Cancer

  • Cancer Development and Process
    • DNA damage from resources such as radiation, chemicals, and viruses leads to mutations in genes responsible for cell growth and repair.
    • Tumorigenesis involves oncogenes and tumor suppressor genes, with mutations causing unchecked cell division and failure of cell death mechanisms.
  • Example of Oncogenes and Tumor Suppressors
    • Oncogenes: Mutations in genes like RAS lead to excessive cell growth.
    • Tumor Suppressors: For instance, p53 is a key tumor suppressor whose dysfunction is seen in many cancers, hindering the cell’s ability to regulate itself.
  • Summary of Cancer Statistics
    • Data indicates that cancer remains a leading cause of death globally and emphasizes the need for targeted research in therapies.
  • Emphasis on Research in Cancer Treatment
    • Understanding molecular mechanisms allows for the design of innovative therapies targeting critical pathways in tumor development.