Genome Editing Flashcards
What are the goals of genome editing:
- Gene KO: ds breaks are repaired such that indels are created at break site (NHEJ)
- gene correction:homologous template is provided
- gene addition/knock-in: provide donor template with some homology + new sequence (always use homologous recomb)
genome editing tools are —
site specific DNA nucleases
examples of genome editing tools?
- transcription activator like effector nucleases (TALENs)
- homing-endonucleases or meganucleases
- sinc-finger nucleases
- CRISPR
these are “nucleases”
How are the 4 site-specific DNA endonucleases different?
- DNA recognition mechanisom
- size
- subunit composition
What is a meganuclease? Advantages and Disadvantages?
- large nuclease
- 6-8 nucleotide recognition sequence is normal, but these restriction endnucleases have 30-35
- such large sites are rare (advantage)
- very large = hard to put in cell (disadv)
What are zinc finger nuclease (ZFNs) proteins?
TWO PARTS
DNA Binding:
- have zinc-finger DNA binding domain
- recognize 3 bases in target site
- can design ZFNs to recognize any triplet or a group of triplets in increase specificity
DBS:
- nuclease Fok1 (nonspecific restriction enzyme) fused to ZFs
- dimer makes staggered cuts (can be designed specifically one for each strand)
What are TAL proteins?
- found in bacteria; pathogen of rice
- DNA binding protein
- made of repeated units of 32-34 AAs that only differ from each other by 2 residues called the repeat variable diresidue (RVD)
- each RVD contacts only one base
HD for C; NG for T; NI for A; NN for G
How is TAL better than ZFs?
more programmable -> one-to-one residue base-code instead of triplets
How is TAL similar to ZFs?
TAL is a DBD w/ no nuclease activity
- add to FokI head-to-head for genome cutting
What are TALENs?
- transcription activator like effector endonucleases
- TAL = DBD
- FokI nuclease domain
