Lecture 2 - RNAi ...

Question 1

RNAi

Answer 1

RNA interference

Question 2

siRNA

Answer 2

small interfering RNA

Question 3

shRNA

Answer 3

small hairpin RNA

Question 4

CRISPR

Answer 4

clustered regularly interspaced short palindromic repeats

Question 5

RNAi is a cell defense mechanism to …

Answer 5

degrade foreign dsRNA molecules (ie viruses)

Question 6

dsRNA triggers Rnai by recruiting ____. _____ cleaves the dsRNA into ____bp fragments called ____

Answer 6

Dicer,Dicer 23 SiRNAs- small interfering RNA

Question 7

siRNAs are loaded into the ___ complex, unwound and the _____ hybridizes with ____ traget

Answer 7

RISC, ssRNA, mRNA
Anything that originally had the same seq or very similar to the dsRNA will be removed

Question 8

The rapid degradation of mRNA in RNAi is done by

Answer 8

Argnoaute

Question 9

What is the shRNA principle derived from?

Answer 9

natural occurring regulatory microRNAs

Question 10

Pros and cons of siRNA

Answer 10

Pros
Easily titrated, fast, simple

Cons
non-renewable
transient effect
Diluted as cells divide
Limited to transfectable cells
Hard in vivo

Question 11

Pros and cons of shRNA

Answer 11

Pros
Long term knockdown
Delivery to any cell line (viral-based transduction)
Renewable
in vivo use

Cons
Cloning required
Challenging technically
Plasmid-based version is transfected at a lower eff than siRNA
-some countries require gov’t approval

Question 12

N-TER nanoparticle siRNA transfection

Answer 12

when cell penetrating bipartite peptides are complexed with siRNA they form nanoparticles that rapidly cross eukaryotic cell membranes

Question 13

How is CRISPR characterized?

Answer 13

by short direct repeats interrupted at regular intervals by unique spacer seqs

Question 14

What are the spacer seqs derived from?

Answer 14

foreign DNA = viral seqs that confer resistance to bacterial viruses with matching DNA steps

Question 15

CRISPR locus is transcribed as a ___

Answer 15

long RNA molecule

Question 16

sgRNA

Answer 16

single guide RNA

Question 17

What does sgRNA locate?

Answer 17

locates targets and Cas9 cleaves DNA with seq homology to the guide RNA

Question 18

Cas9 works with ____ to identify target seqs adjacent to a ____

Answer 18

sgRNA, PAM protospacer adjacent motif

Question 19

Cas9

Answer 19

DNA endonuclease that makes ds breaks (DSBs) in target seqs that base pair with the guide RNA

Question 20

What is the function of PAM?

Answer 20

The PAM (NGG) is required for a Cas nuclease to cut and is generally found 3-4 nucleotides downstream from the cut site.