Case Unit 4: Disease Models

Question 1

What does CRISPR stand for?

Answer 1

Clustured Regularly Interspaced Palindromic Repeats

Question 2

What is Cas9?

Answer 2

Bacterial endonuclease that cleaves DNA at a target sequence

Question 3

What is Sp, in relation to CRISPR?

Answer 3

Streptococcus pyogenes - the bacterial species that CRISPR technology has been adapted from

Question 4

What is gRNA?

Answer 4

‘Guide’ RNA - a RNA sequence of 20 bases that is identical to the target DNA sequence to be removed by CRISPR

Question 5

What is PAM?

Answer 5

Protospacer Adjacent Motif. A short sequence that must be adjacent to the target sequence to enable CRISPR to work

Question 6

What is dCas9?

Answer 6

Nuclease deficient Cas9

Question 7

What is NLS?

Answer 7

Nuclear localisation signal - directs proteins to the nucleus

Question 8

What is AmpR?

Answer 8

Ampicillin resistance gene

Question 9

How is CRISPR delivered into cells?

Answer 9

Genes for the guide RNA and Cas9 protein are put into a plasmid
Plasmid inserted into cells

Question 10

What are the features of a good target site for CRISPR?

Answer 10

Few other regions in the genome with a similar sequence
Target site present in an exon rather than an intron
Target early within an exon so the rest of the gene gets frameshifted - no partially functional protein produced

Question 11

How does CRISPR work?

Answer 11

Nuclease causes a double stranded break at a target region
Cell repairs the break using NHEJ
Results in random deletions and frameshift mutations

Question 12

Why might you include an EGFP protein in the CRISPR plasmid?

Answer 12

To make the Cas9 visible so you can make sure it has entered the nucleus

Question 13

Why might you include an AmpR gene in the CRISPR plasmid?

Answer 13

Treat the bacteria with ampicillin, only those that have incorporated the CRISPR plasmid will survive. Purifies the useful cells from the non useful cells

Question 14

Example of how a disease model can be made chemically for Type 1 diabetes

Answer 14

Type 1 diabetes is autoimmune destruction of the insulin producing beta pancreatic cells, leads to hyperglycaemia
Hyperglycaemia can be induced in animals by chemically destroying their beta cells (e.g NOD mouse)
Can then be used to test therapies for lowering blood sugar

Question 15

Example of how a disease model can be made chemically for Parkinson’s

Answer 15

Parkinson’s is caused by a loss of dopamine-releasing neurons that control movement
Marmosets can be given parkinsons by chemically destroying their dopamine neurons
Test therapies such as deep brain stimulation

Question 16

Example of how a disease model can be made genetically

Answer 16

In vitro cells can be genetically modified to mimic CML cells by introducing the BCR-ABL fusion gene
Can then test drugs like Imatinib to see if they result in cell death of not

Question 17

Pros/Cons of in vitro disease models

Answer 17

More ethical - no animal is harmed
May not reflect exact in vivo conditions e.g. in vitro conditions are normoxic whereas tumours are usually hypoxic
Cannot replicate exact complexity of conditions/signals found in body
Less cell-to-cell contact

Question 18

Pros/Cons of mice as animal models

Answer 18

Similar cortical structure to humans
Mammalian
Fast breeding - quick to see offspring effects
Tight regulations on living conditions - expensive to keep
Highly inbred
Don’t always have same pathway leading to cause of disease as in humans

Question 19

What is a SCID-NOD mouse?

Answer 19

Immunodeficient mouse (No T, B or NK cells)
Tissue can be grafted onto it and won’t be rejected
Developed by inbreeding

Question 20

What is a Lep mouse?

Answer 20

Leptin-deficient obese mouse

Has a predisposition to developing Type 2 diabetes

Question 21

What are mutant mice?

Answer 21

Mice that have been genetically engineered to have a gene ‘knocked in’ or ‘knocked out’

Question 22

How is a knockout mouse made use embryonic stem cells?

Answer 22

Genetically engineer an embryonic stem cell with the desired gene knocked in/out
Inject stem cell into mouse blastocyst
Inject blastocyst into pseudopregnant mouse
Mouse gives birth to chimeric offspring (some are affected with gene, some aren’t)
Screen the offspring to check for transmission of the mutation

Question 23

What are tissue specific knock-outs?

Answer 23

The target gene is only knocked out in certain areas of tissue, using Cre recombinase

Question 24

Why is a tissue specific knock out useful?

Answer 24

Useful for when the gene is essential to life, and the organism would not survive if every cell was affected

Question 25

Disadvantages of using mice as disease models and example

Answer 25

The model doesn’t always accurately mimic humans with the disease
e.g. in humans with CF, upregulation of proton pumps fills the lung lumen with H+ ions, which suppresses the immune system so they are more susceptible to infections, but this doesn’t happen in mice

Question 26

Two methods for making a knockout mouse

Answer 26

CRISPR and Embryonic Stem Cells

Question 27

Benefit of using CRISPR over embryonic stem cells to create knockout mice?

Answer 27

CRISPR can be injected directly into an oocyte - don’t need to create blastocysts

Question 28

What are Avatars?

Answer 28

Disease models made from flies
Induce ‘personalised’ mutation pattern of someone’s cancer in a fly
Fly mimics cancer
Test drugs on fly

Question 29

What is the dCas9 activation system used for?

Answer 29

Instead of causing a double stranded break in the DNA, it employs transcriptional activators to increase expression of certain genes