Gene editing Flashcards
1
Q
basic principle of editing
A
addition or remove of a part of the genome so that it can serve a desired purpose
2
Q
why do we want to edit genes?
A
- model human genetic disease in animal models, study human pathways
- correct pathogenic mutations in cell lines for therapy and personalised medicine
- improve key organisms for biotech
3
Q
what are ds breaks associated with?
A
homologous dependent repair
4
Q
proceess of fixing a ds break
A
ds break > resection > strand invasion, D loop
5
Q
classical double stranded break repair
A
- ds break; damaging agents
- resection; nuclease degradation ss 3’ tails
- homology searching; RAD51
- D-loop; invading strand forms a loop, acts as a primer for DNA synthesis
- Holliday junction; intercrossing DNA during recombination.
6
Q
synthesis-dependent strand-annealing pathway
A
- no cross over events
- newly synthesised strand displaced from template and returns to processed end of non invading strand
7
Q
non-homologous end joining
A
- primary pathway for repair of a ds break though cell cycle
- relies on Ku protein to thread onto each broken DNA end
8
Q
meganucleases (MNs)
A
- endodeoxyrubonucleases.
- predicted to recognise idential sequence once for a genome 20x size of the human genome
- can tolerate 1 or 2 MMs
- can insert DNA by HR or mutations by NHEJ
- difficult to specifically target.
9
Q
zinc finger nucleases (ZFNs)
A
- ZFs are DNA binding domains
- each finger (30aa) recognises 3bps
- an array of zf domains; recognises unique genomic sequences
- ZF use on Fokl domain; Fokl needs to dimerise to cut, ZFN come as pairs
- complex, expensive, can have innacurate cleavage (domain interaction)
9
Q
what do we want from an endonuclease?
A
- specific recognition of long target sequences (ideally one per genome)
- adaptability for retargeting to other genomic loci
10
Q
TALENS
A
transcription activator-like effector nucleases
11
Q
what are TALE proteins?
A
xanthomonas
plant pathogenic bacteria
12
Q
what are DNA binding domais of TALEN?
A
series of tandem repeats ; 33-35aa
13
Q
TALEN vs ZFN
A
- much easier to design than ZFN, fewer targeting constraints
- much larger than ZFNs, harder to deliver
14
Q
variations of TALEN
A
aa 12+13 confers specificity nucleotides
