CRISPR/Cas9

Question 1

What is tracrRNA and pre-crRNA translate into?

Answer 1

pre-crRNA and tracerRNA

Question 2

What is pre-cRNA and tracerRNA processed into?

Answer 2

which is processed to shorter forms by Cas9 and RNase III

Question 3

What happens after the Cas9, tracerRNA, crRNA complex is formed?

Answer 3

search for the DNA sequences that match the spacer sequence

-PAM is also required

Question 4

Where does Cas9 bind?

Answer 4

• a target site with a match between the crRNA and the target DNA
• 3 bases upstream of the PAM site

Question 5

What did Jinek discover?

Answer 5

a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing

Question 6

What are the two types of editing?

Answer 6

• nonhomologous end joining (NHEJ)

- homology directed repair (HDR)

Question 7

Which editing type is imprecise?

Answer 7

• NHEJ-mediated repair

- can produce insertion and or deletion mutations of variable length at the site of the DSB

Question 8

Which editing type is precise?

Answer 8

• HDR-mediated repair

- can introduce precise point mutations or insertions from a single-stranded or double-stranded DNA donor template

Question 9

How are the custom repair templates generated for HDR?

Answer 9

• digesting a dual-marker template vector with two convenient restriction enzymes
• PCR-amplifying appropriate homology arms
• perform a Gibson assembly
• transformation

Question 10

What are the steps of Gibson assembly?

Answer 10

1. exonuclease chews back 5’ end
2. DNA fragments anneal
3. DNA polymerase extends 3’ ends
4. DNA ligase seals nicks

Question 11

Why leave it up to the users to excise the cassettes?

Answer 11

1. many deletion knockouts are in essential genes and are therefore lethal. The GFP acts as a dominant selectable marker (balancer) so one doesn’t lose the strain
2. Conversely, many strains with single deleted genes are virtually wild type in appearance and behaviour. Again, the GFP acts as a selectable marker to follow the deletion in genetic crosses
3. The Cre/Lox excision step is quick and easy to do

Question 12

What are the Cas9 delivery methods and what are their advantages?

Answer 12

plasmid DNA: simple, low cost
mRNA: rapid gene expression, and eliminated the risk of insertional mutagenesis; can be efficiently delivered to cell types that are resistant to transfection with plasmid DNA
Protein: the most rapid pulse of genome editing activity and reduce the possibility of off target cleavage events; can be efficiently delivered to cell types that are resistant to transfection with plasmid DNA