CRISPR-Cas9

• Gene transfer between species

  1. Using bacterial plasmids

  2. Viruses

  3. Microinjection
• CRISPR use

  Edit and/or transfer genes
• Gene Delivery Systems In Bacteria and Plant Cells

  • Bacterial Plasmids
  • Viral Vector
• Gene Delivery Systems in Animal cells

  • Bacterial Plasmids
  • Viral Vector
  • Microinjection
• Bacterial plasmid as a vector

  • An extra-chromosomal, circular piece of DNA in bacteria and some yeast
  • Capable of self-replication
• Inserting isolated gene of interest DNA into the plasmid

  Recombinant DNA is taken up by the bacteria (transformation)
• Viral vectors

  1. Transfer the gene of interest to the viral DNA

  2. A virus containing recombinant DNA binds to the cell membrane and enters the cell to infect it
• Microinjection

  1. Requires a glass micropipette/fine glass needle

  2. Sharp tip punctures the cell membrane

  3. DNA of the gene of interest is injected through the cell membrane into the nucleus
• Microinjection use in transgenic animals

  1. Inserting recombinant DNA into the embryo 12-14 hours after fertilization

  2. Results in every cell in the growing embryo containing the new DNA
• CRISPR

  • Clustered Regularly Interspaced Short Palindromic Repeats
  • Consists of Cas9 enzyme and guide RNA to introduce changes into the DNA
• Genetic Engineering Recombinant DNA Tech

  1. Identify the gene of interest using labeled complementary RNA/DNA probes

  2. Isolate gene from donor genome using restriction enzymes

  3. Insert recombinant DNA using vectors such as plasmid, virus, CRISPR-Cas9
• CRISPR-Cas9

  1. identify gene of interest using complementary guide RNA (gRNA)
  2. isolate the gene using Cas9
  3. insert the CRISPR-Cas9 system (bacteria way of inactivating viruses that infect)