Lecture 21 - Gene Manipulation Strategies Flashcards
What does crispr do?
Defence mechanism from bacteriophage
What does crispr involve?
1) Cas9 endonuclease and a guide RNA (gRNA) cleave both DNA strands.
2) The gRNA should contain specific 20nt protospacer (target sequence) and designed upstream of a protospacer adjacent motif
What does crispr 9 recognise and what does this do?
Recognises PAM site and makes double stranded break 3 nucleotides upstream of PAM
What are the 3 ways crispr can introduce genomic modifications?
1) repaired by NHEJ - gene knock outs
2) repaired by HDR: use short Oligocene or DNA vectors with short homologous arms that flank the cleavage site
- introduce specific mutations, reporter genes (GFP etc) or loxP sites etc.
3) two gRNA target sites can be chosen - delete large genomic regions
Is crispr 9 officiant?
Yes all cells which undergo this will express the mutants
How do you generate mice with several mutations using crispr?
Inject cas9 and gRNA’s into pronucleu. Repair in Vigo using HDR (allows rapid generation of transgenic animals carrying specific modifications_
What do you do to stop off target effects as cas9 and gRNA is not absolutely specific?
Choose target sequences within genes with minimal homologous to other genes or regulatory regions
Wha reduces off target effects?
Mutant cas9 (nickase).
How does mutant cas 9 work?
Cleaves only one DNA strand leaving an overhand when used with 2 gRNA’s specific for opposite DNA strands.
NHEJ or HDR methods are used to knockout or introduce reporters
would 2 close gRNA target sites be unlikely to be present elsewhere in the genome and therefore increase the chance of 2 nicks being near one another?
Yes the are unlikely to be elsewhere in the genome but no they would reduce the chance of nicks being near one another
Did they efficiently generate genetically modified mice using crispr 9?
Yes - they put GFP in the reporter for EGF to show where it was in the cell.
What is conditional modification of the gene?
Conditional deletion of te gene
Why are conditional mutations useful?
- avoid early lethality of animal to analyse role of gene
- to activate mutations
Conditional mutations - what is cell specific activation useful?
analyse role of gene in a defined cell (cell specific inactivation)
Conditional mutations - why is inducible inactivation done?
analyse the role of gene at certain development stage (inducible inactivate)
Conditional modification - why is cell fare experiments done?
- permanent take cels with reporter gene to determine fate (cell fate experiments)
How do you conditionally translate a mutations?
DNA recombination can be driven in-vivo by expressing Cre-recombinase from a transgene
loxP sites are previously inserted by gene targeting to flank a critical sequence and create a loop
Results in loss of sequence from genome
What can you use instead of cre recombinase and loxP?
Flp recombinase and frt recognition sites
How do you make an inducable deletor mouse?
- Cre-ERT2 constructs to generate deletor mice: Cre fused to a mutant estrogen ligand-binding receptor domain ERT2, which responds to synthetic analogue oestrogen called tamoxifen.
ERT2 keeps Cre in cytoplasm but upon addition of tamoxifen can move to the nucleus and cause recombination
Once cre is in the nucleus what happens?
It binds to LoxP ad causes deletion/recombinant
Epithelial self-renewal is what?
An example of inducible deletions
How did they work out how the epithelial self renews?
They made knock out mice and fluorescents the Lgr5 expression at the bottom of the crypt
