Lecture 29: Genome editing

Question 1

How are genomes edited?

Answer 1

1. Cut genome at specific sites
2. Cut is repaired by cell via:
   a. Non-homologous end joining (NHEJ)
   repair = DISRUPT GENE
   b. Homology-directed repair (HDR) =
   CHANGE GENE

Question 2

What is needed to create a DSB?

Answer 2

1. Recognition sequence
2. Artificial chimera with two domains:
   a. DNA binding proteins with target
   specificity that can be “programmed”
   b. Restriction enzyme “Fok1”, without DBA
   binding domain

Question 3

What park of a zinc finer interacts with DNA

Answer 3

1. Alpha helix
   a. positions; -1, 3, and 6 make contact
   with 3 consecutive nucleotides
   b. 1 zinc finger to 3 nucleotides

Question 4

How do zinc finger nucleotides work

Answer 4

1. Chain of zinc-finger modules combined
   with Fok1 nuclease domain (1 or 2)
2. One chain +N1 binds to one side of DNA
   whilst the other +N2 binds to the other
3. N1 and N2 form obligate heterodimer -
   only active when dimerized

Question 5

How can ZFN work as a library?

Answer 5

1. Have a array of preselected finger
   modules
2. HOWEVER, interactions between
   modules can affect their specificity