Gene Editing

Question 1

What does a CRISPR/Cas9 plasmid contain?

Answer 1

Promotor, guide RNA, CRISPR RNA, another promotor, CAS9 gene, fluorescence gene.

Question 2

How is the bacteria CAS9 modified to engineering use?

Answer 2

Added nuclear signalling sequence.

Question 3

What must follow the DNA sequence targeted by CRISPR/Cas9?

Answer 3

Protospacer adjacent motif (PAM).

Question 4

What is the protospacer adjacent motif?

Answer 4

Any nucleotide followed by GG.

Question 5

What is the size of the DNA sequence recognised by a pair of Z-fingers, TALENs and CRISPR/Cas9?

Answer 5

18-36, 28-40, 22, respectively.

Question 6

What are the trageting Z-fingers, TALENs and CRISPR/Cas9?

Answer 6

Z-fingers: Difficult to target non-G-rich sequences
TALENs: 5’ targeted base must be T
CRISPR: PAM site

Question 7

List Z-fingers, TALENs and CRISPR/Cas9 in decreasing order of difficulty to engineer.

Answer 7

Z-fingers, TALENs and CRISPR/Cas9.

Question 8

How can Z-fingers, TALENs and CRISPR/Cas9 be delivered ex-vivo?

Answer 8

Electroporation and viral transduction.

Question 9

List three disease that can be treated with Z-fingers.

Answer 9

Haemophilia B, HIV, SCID

Question 10

List three diseases that can be treated with TALENs

Answer 10

DMD, Hep B, B-cell accute lymphoblastic leukaemia.

Question 11

List seven disease that can potentially be treated with CRISPR/Cas9.

Answer 11

HIV, DMD, cataracts, cystic fibrosis, hereditary tyrosinemia.