RNAi and CRISPR

Question 1

Define RNAi.

Answer 1

Gene silencing by double-stranded RNA.

Question 2

What type of molecule does the cleaving of dsRNA into fragments produce?

Answer 2

Small interfering RNAs (siRNAs).

Question 3

What enzyme makes siRNA single-stranded?

Answer 3

Ago2.

Question 4

The dsRNA associates with a RISC. True or false?

Answer 4

False.

Question 5

What does RISC stand for?

Answer 5

RNA-induced silencing complex.

Question 6

What molecule activates the RISC?

Answer 6

Single-stranded siRNA.

Question 7

When does RNAi lead to full mRNA degradation?

Answer 7

When there is perfect complementarity between siRNA and target mRNA.

Question 8

What happens if the siRNA is not a perfect complement to the target mRNA?

Answer 8

Translation inhibition - RISC stays bound to mRNA.

Question 9

What are the three functions of Ago2 in RNAi?

Answer 9

1. Unwind double-stranded siRNA.
2. Transport ss-siRNA to RISC.
3. Assist RISC in mRNA degradation.

Question 10

What is the name of the RNase that produces the siRNAs?

Answer 10

Dicer.

Question 11

What are two applications of RNAi?

Answer 11

1. Knockdown studies.
2. Inhibition of protein synthesis in cancer cells.

Question 12

The bacterial system where short RNA transcripts hydrolyse complementary DNA is called…

Answer 12

CRISPR-Cas.

Question 13

What does CRISPR stand for?

Answer 13

Clustered Regularly Interspaced Short Palindromic Repeats.

Question 14

Define CRISPR-Cas9.

Answer 14

A genome-editing system using Cas9 protein.

Question 15

What does gRNA stand for?

Answer 15

Guide RNA.

Question 16

Cas9 is complementary to the target gene. True or false?

Answer 16

False.

Question 17

What part of gRNA recognises the target gene?

Answer 17

5’ guide sequence.

Question 18

How long is the gRNA guide sequence?

Answer 18

20 bp.

Question 19

What part of the gRNA binds to Cas9?

Answer 19

3’ gRNA scaffold.

Question 20

Cas9 is a DNA-guided endonuclease. True or false?

Answer 20

False.

Question 21

Cas9 cuts both strands, called a…

Answer 21

Double-stranded break (DSB).

Question 22

When Cas9 complexes with gRNA, the molecule is referred to as a…

Answer 22

Ribonucleoprotein.

Question 23

What does PAM sequence stand for?

Answer 23

Protospacer Adjacent Motif sequence.

Question 24

What is the motif of PAM?

Answer 24

5’-NGG-3’

Question 25

Cas9 unwinds the genomic DNA. True or false?

Answer 25

True.

Question 26

What is the DSB repair method that lets the cell repair itself naturally?

Answer 26

Non-homologous end joining (NHEJ).

Question 27

What is the DSB repair method that uses a DNA repair template?

Answer 27

Homology-directed repair (HDR).

Question 28

Which DSB repair method is best for knockout experiments?

Answer 28

NHEJ - prone to loss-of-function mutations.

Question 29

Homology-directed repair allows the editing of the genome. True or false?

Answer 29

True.

Question 30

RNAi exists naturally in bacteria and archaea. True or false?

Answer 30

False.

Question 31

What sequence must be immediately upstream of the gRNA’s target sequence?

Answer 31

PAM sequence.

Question 32

CRISPR exists naturally in some bacteria and archaea. True or false?

Answer 32

True.

Question 33

Ago2 helps to locate the mRNA in RNAi. True or false?

Answer 33

False.