CRIPSR

Question 1

Summarise 2007 experiment

Answer 1

tested bacteria against bacteria who did or did not have homology to the bacteriophage
When the spacer region was knocked out, sensitivity increased
Moving the repeat region also increased sensitivity

Question 2

Stage 1: Adaptation

Answer 2

Foreign DNA enters cell
Recognised by Cas complex
Which cleaves a segment and is integrated into spacers

Question 3

Stage 2: Biogenesis

Answer 3

Transcribed as a single RNA strand
tracrRNA is transcribed, and binds to crRNA
○ Has homology to the repeats - allowing it to bind to the strand
- RNAase III cuts the strand into smaller fragments
Cas9 binds to make a complex

Question 4

Stage 3 Interference (immunity)

Answer 4

Cas9 looks for the spacer, endonuclease is activated and causes a double stranded break
Additional requirement
○ What is stopping the cas9 binding back to its own chromosome?
Targets DNA if PAM site presence

Question 5

PAM sequence

Answer 5

mechanism so that Cas-9 doesn’t target their own genes
present on invading viral/plasmid sequence and Cas-9

Question 6

Non homologous end joining

Answer 6

Cas 9 induces double stranded break
spontaneous reforming of DNA that is next to each other
Doesn’t check the sequence, just sticking it together
Can form insertions or deletions

Question 7

Homologous directed repair

Answer 7

supply a donor sequence which will be integrated into ds breaks

Question 8

Arrayed screen

Answer 8

need to manually synthesize reagents and constructs in individual wells

Question 9

Pooled screen

Answer 9

reagents are synthesized as a pool, then the DNA leftover is read by next gen sequencing
- compare abundance of different transgenes in each sample

Question 10

anti-CRISPR mechanisms

Answer 10

mutation leading to mismatches and inefficient cleavage (e.g. PAM sequence)
recombination in polylysogenic genomes
anti-CRIPSR proteins: Acr blocks PAM binding
Phage carrying CRISPR cas elements
- turns CRISPR against its own host