lecture 4 genome editing

• Modern biotechnology


  The application of in vitro nucleic acid techniques, including recombinant DNA technology or direct injection of nucleic acid into cells or organelles, and fusion of cells beyond the taxonomic family that overcome natural physiological reproductive or recombination barriers and that are not techniques used in traditional breeding and selection
• Waves of biotechnology


  • First wave
  • Second wave
  • Third wave
  • Fourth wave
  • Fifth wave
• Gene editing tools


  • Zinc finger nucleases (ZFN)
  • TALENs (transcription activator-like effector nucleases)
  • CRISPR-CAS
• Gene editing tools


  • Site of action is specific
  • Can result in removal of single base or several bases or mutation to knock out gene or repair gene
  • Can also insert a new gene for a new or improved trait
  • Other parts of vector may be removed or not incorporated in genome
• 2020 Nobel Prize in Chemistry was awarded to Emmanuelle Charpentier and Jennifer Doudna for discovering the CRISPR/Cas9 "genetic scissors" used to cut DNA
• CRISPR


  A very old defense mechanism in bacteria where they use clusters of DNA repeats with unique sequences in between to recognize and defend against invading viruses
• Cas


  A set of enzymes that can precisely remove the DNA out of the invading viruses
• Gene editing


  1. Guide RNA binds to target sequence

  2. Cas9 enzyme binds to guide RNA

  3. Cas9 enzyme cuts both strands of DNA

  4. This cut is repaired introducing mutation
• Delivery systems for gene editing

  • Agrobacterium tumefaciens delivery
  • Particle bombardment
  • Lipid transfection
  • Electroporation
  • Microinjection
• Lipid or Liposome-mediated gene delivery


  Use of cationic lipids like DOTMA which are better than neutral lipids because they are more efficient and easy to use
• Electroporation


  A physical transfection method that uses electrical pulse to create temporary pores in cell membranes through which substances like nucleic acids can pass
• Direct DNA transfer - naked DNA


  Physical injection of DNA directly into the nucleus of cultured cells, which is technically difficult and laborious but can result in integration of foreign DNA and expression for several generations
• Applications in health


  • Using CRISPR to edit T cells to remove the PD-1 gene and re-introduce them to treat lung cancer
  • Using CRISPR to correct the defective promoter in Hunter's syndrome
• Applications in agriculture


  • Anti-browning white button mushrooms by knocking out a PPO gene
  • Waxy corn with altered starch composition by deleting the waxy gene
  • High oleic acid, low linoleic acid soybean by deleting the FAD2 genes
• CRISPR-edited agricultural products have been determined as not regulated by USDA APHIS