Genome editing Flashcards
How are genomes edited?
- cut genome at specific sites.
- Cut repaired by cell. NHEJ or homology-directed repair.
How can an artificial chimera using two domains be done?
- DNA binding proteins w/ target sepcificity that can be programmed.
- Restriction enzyme FokI w/out its DNA binding domain.
What are zinc finger motifs?
Zinc finger (Znf) domains are relatively small protein motifs which contain multiple finger-like protrusions that make tandem contacts with their target molecule. Some of these domains bind zinc, but many do not; instead binding other metals such as iron, or no metal at all.
What do Znf domains make contact w/?
3x3=9 nucleotides in a sequence specific manner
How do TALENs bind DNA?
Transcription activator-like effectors nucleases.
DNA binding domain of TALE has 2 alpha helices but the DNA protein interaction is not via the helices but via 2 AA in btwn the helices.
2 AA to 1 nucleotides but 1 domain to 1 nucleotide -need for more domains.
What are the pros of Znf and TALEN?
Protein based method
Very specific
Cons of Znf and TALEN?
Expensive and time consuming.
Every target requires a novel protein being made.
Costs a lot
Takes a year to make an edit.
What does cas mean?
CRISPR associated genes
How does the bacterial CRISPR immunity work (type I eg e. coli)?
- bacteria encounter foreign DNA.
- Small fragments of foreign DNA integrate into the CRISPR locus - memory of previous infections.
- It is transcribed and crispr RNA processing by some of the cas enzymes occurs.
- They become Cas proteins.
- RNA mediated targeting of DNA during re-infection w/ same bacteriophage.
- Cas - crispr RNA-DNA complex induces nuclease acivity of Cas.
How does the bacterial CRISPR immunity work (type II eg streptococcus pyogenes)?
Two small RNAs generated (tracrRNA and crisprRNA).
Cas9 forms complex w/ RNAs and cuts target DNA.
What is the PAM site important for?
Important for nuclease activity of Cas9.
PAM is an essential targeting component which distinguishes bacterial self from non-self DNA, thereby preventing the CRISPR locus from being targeted and destroyed by the CRISPR-associated nuclease.
How does the CRISPR-Cas9 synthetic system do?
Add Cas9 protein and single guided RNA (sgRNA) to target which must contain the PAM sequence.
Pam required for cutting not for finding target.
What happens when you change the specificity of the Cas9 nuclease to cut DNA only on one strand - ‘nickase’?
Will generate overhangs after cleavage that cannot be targeted by NHEJ so no possibility of insertion/deletion (eliminates no desired mutations caused by NHEJ).
Only the desired edit will take place.
What happens when defective Cas9 that cannot cleave DNA?
Defective cas9 fused to transcriptional activators. Induces the expression of specific target genes.
Can also be fused to repressors to block expression of specific target genes.
Can be fused to GFP to monitor specific chromosomal locations.
What is a homing endonucleases (HEG)?
Typically found inserted btwn 2 specific sequences of DNA w/in genome.
HEG codes for the production of an enzyme that recognises theses two specific coding sequences when they are not interrupted by presence of HEG.
