Lecture 10: CRISPR & Genetic Manipulation I

Question 1

forward mutageneis:

Question 2

reverse mutagenesis:

Question 3

how easy is it to knock out a gene in yeast?

Answer 3

very easy

Question 4

how effective is gene knockout targeting in mammalian cells and why?

Answer 4

• very hard - if a billion cells are transferred, you will only get about 100 hits
• homologous arms need to be very long (>4kb)
• gene targeting frequencies very low (<0.1%)

Question 5

how do we overcome the difficulties of gene knockout targeting in mammalian cells?

Answer 5

we use RNA interference - this is a gene knock-down, not a knock-out = incomplete depletion of protein

Question 6

upsides and downsides to RNA interference in the gene knock-down of mamillian cells:

Answer 6

• stable proteins with long half life cannot really be infected by RNA interference
• transient (not long-lasting)
• not all cells are affected
• off target effects
• fast and easy

Question 7

how to carry out siRNA experiment:

Answer 7

• couple of controls: dont add siRNA, scrambled [same sequence as the one to knock out the gene but randomly scrambled so it doesn’t target any gene anymore] & actin [loading control] to ensure there is some protein here and that the gel is flowing correctly

-

Question 8

why RNA interference isn’t that good of a technique in mammalian knockouts:

Answer 8

• only lasts a short while and doesn’t target all genes [therefore incomplete depletion of protein]

Question 9

siRNA screening:

Answer 9

384 well plate - each well will target to knockout a different gene

• promoter added before a GFP gene

look up to finish

Question 10

what major advance has meant that we don’t have to rely on yeast for mammalian gene knockouts:

Answer 10

the major advance of CRISPR in the past decade

Question 11

where did CRISPR derive from?

Answer 11

CRISPR is a ribonuclear-protein called cas-9 that is found in in the bacterial immune system

Question 12

what is CRISPR?

Answer 12

• CRISPR is a ribonuclear-protein called cas-9 that is found in in the bacterial immune system
• it is efficient at engineering a double strand break anywhere in the genome

Question 13

how does CRISPR function?

Answer 13

• gRNA guides Cas9 to a specific target sequence
• Cas9 makes a double-strand break at target site

[Protospacer Adjacent Motif (PAM) site also needs to be present for Cas9 to cut e.g. NGG]

Question 14

CRISPR Cas-9 cannot cut unless:

Answer 14

PAM - protospacer adjacent motif sites are present

Question 15

how do you go about getting CRISPR to target a specific gene:

Answer 15

design a guide RNA to any gene you like

Question 16

with CRISPR, DNA double strand breaks can be repaired in 2 ways:

Answer 16

• homologous directed repair
• non-homologous end joining

Question 17

what can NHEJ do and what does it introduce?

Answer 17

• NHEJ can insert/delete a small no. of bp at the DSB
• this in turn introduces a frameshift in the gene

Question 18

what to aim for when designing your guide RNA:

Answer 18

• your knockouts must target an early exon [exon 1/ 2]
• choose a guideRNA sequence adjacent to a PAM in the target sequence
• minimise ‘off-target’ effects