L4 - Crispr Technology Flashcards
Where was crispr first identified / how
Bacterial defence against bacteriophage dna upon reinfection
What are the 3 components
Cas9 nuclease
Crispr rna - has comp sequence/ protospacers and binds tracr rna
Tracr rna- binds crrna and is a hairpin
Both form the grna
What are the steps during first infection with Phage dna
Dna is cut by cas1 and 2 into small fragments called protospacers which are then stored in the crispr locus amongst identical repeat arrays (to form crrna)
What happens during reinfection
Crispr locus txn so the complementary protospacers will be transcribed and tracr/crrna with the spacers will bind cas9 and direct it to the comp sites of phage invading dna
Ds dna breaks occur preventing infection
What is the cas operon in crispr locus
Operon transcribing diff cas proteins eg cas1,2 and 9
What’s the diff between cas1/2 and 9
Cas1/2 are multi protein complexes whereas cas9 is a single protein but with multidomains
Explain what happens to form mature cr rna
Protospacers sequences amongst repeat arrays will be joined
Protospacers will be the point which the rna will be complementary to invading dna whereas the repeat array part will bind tracrrna hairpin
Which domains in cas9 are required to recognise and guide cas9 to the grna/dna duplex
Rec1 and 2
Which lobe is the hnh nuclease domain which cleaves comp dna on
The nuc lobe (not rec lobe)
There is another domain which cleaves non comp dna sequence for it to be a double strand break. What’s it called
RuvC
Which domain recognises and interacts with the Pam sequence downstream of cleavage site
PI domain
What is the Pam sequence and why important
It’s a small bp sequence about 3-4 bp adjacent to the cut site usually downstream
Required to be present on the invading dna for cas9 to cause cleavage
This is because this distinguishes bacterial self dna from invading dna as upon first infection the Pam sequence is removed from crispr locus after protospacers insertion by cas1/2
So Pam is not present in bacterial crispr locus and prevents cleavage of this by own cas9
Which sequence commonly used in research as a Pam site
NGG strep pyogenes
What have they done to grna for actual genome editing in research which makes it easier
Linked crrna with tracrrna by a linker loop to get a single grna
What are the 2 ways you can deliver cas9/grna into cells for research
Either through transfecrion with plasmids 1 with cas9 and one with the grna of interest which will then be translated
Or can make a grna/cas9 complex in test tube and deliver it into cell that way