BIO421Lecture 11

Advanced GeneticsGenome Editing

  1. Why should we edit the genome?
  2. To induce mutations that are molecular nulls
  3. These mutations eliminate the production of the protein or severely alter it (stop or non-sense codon alterations, for instance)
  4. To perform reverse genetics by knocking out a gene whose sequence suggests it is involved in a pathway of interest to us.
  5. To knock in sequences such as reporters or make designer mutations
  6. What do we need to do to edit genomes?
  7. Induce a double strand break
  8. Repair the break
  9. Non-homologous end-joining (NHEJ)
  10. Homologous directed repair (HDR)
  11. New protocols that induce double strand breaks
  12. Zinc-finger nucleases
  13. Utilize zinc-finger motifs from common transcription factors
  14. These can be designed to recognize nearly any sequence.
  15. Each zinc-finger repeat recognizes 3 base pairs.
  16. Combined with the endonuclease domain from the Fok1 restriction enzyme
  17. Drawbacks:
  18. not guaranteed that engineered zinc-fingers will actually bind sequence desired
  19. large risk of off-site binding and DNA damage
  20. TALENs (Transcription Activator-like Effector Nucleases)
  21. Combines the TALE DNA binding domain with the Fok1 nuclease
  22. Easier to design the TALE DNA binding domains, and they are more likely to bind targeted site and less off-site damage
  23. CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats)
  24. Discovered as a bacterial immune system
  25. The palindromic repeats separate captured sequences from invading nucleic acids, like viral genomes and plasmids
  26. The CRISPR-Cas system destroys invading nucleic acids that match those in the captured sequences.
  27. The CRISPR-Cas system and been engineered to create double strand breaks (DSBs).
  28. The researcher needs to design a single guide RNA that targets a sequence with a PAM site (NGG).
  29. The researcher then transforms their organisms with the Cas9 sequence and the single guide RNA sequence, and a DSB will be made at the proper site near the PAM sequence.
  30. Base pairing between the sgRNA and the target sequence provides the specificity
  31. Much easier to work with than Zinc-finger nucleases or TALENs
  32. Examples
  33. spe-44 in C. elegans
  34. FAH in mice
  35. CCR5 gene in humans to create immunity to HIV
  36. CRE-lox
  37. This system is not new – it has proved useful for many years
  38. Uses loxP sites engineered into the genome.
  39. CRE recombinase induces recombination between loxP sites to alter the genome in multiple ways.
  40. Putting CRE recombinase under a tissue or cell-specific promoter can allow gene knockout only in those tissues or cells.