Gene Editing Flashcards
1
Q
What natural repair mechanisms does gene editing take advantage of?
A
Double stranded break repair
- homology directed repair
- NHEJ
2
Q
Zinc finger nucleases
A
- Engineered proteins (DNA cutting endonuclease fused to Zn finger domain engineered to bind a specific DNA sequence) that bind and cut specific DNA sequences
- Indels at cut sites in living cells
- 10% success
3
Q
Transcription activator-like effector nucleases
A
- TAL effectors are secreted DNA-binding proteins from bacteria that are engineered to recognize specific sequences
- Indels at cut sites in living cells
- Increased specificity over ZFNs
4
Q
Naturally occurring bacterial CRISPR
A
Used as a bacterial immune system:
- After viral attack, bacteria collect pieces of viral DNA and incorporate them into their own genome at the CRISPR locus (cas genes + repeats and spacers)
- These fragments are transcribed, and next time the virus attacks, the crRNAs guide cas proteins to the virus to cleave DNA
5
Q
Cas9
A
Enzyme that makes a double stranded break
6
Q
gRNA
A
20 nt sequence complementary to the target attached to a longer RNA scaffold
7
Q
PAM sequence
A
- Protospacer adjacent motif
- 3 nt sequence needed for Cas activity (different for each Cas)
- Must occur in the genome next to the target region and increases specificity
8
Q
What is the success rate of CRISPR with homologous recombination?
A
< 10%
9
Q
What are the sources of template DNA for homologous recombination in CRISPR?
A
plasmids and ssODNs
10
Q
ssODNs
A
- single-stranded oligodeoxyribonucleotides
- used for single nucleotide gene correction
- 200 nt fragments with short homology arms
11
Q
plasmids (for use in CRISPR)
A
- 800-1400bp regions of homology flanking site of interest
12
Q
Germline therapies
A
change DNA in reproductive cells; changes will be passed on
13
Q
Somatic therapies
A
changes will only affect that individual
