L 15-16

Question 1

What are the 3 methods of genome editing?

Answer 1

ZF nuclease, TALENs, and Crispr

Question 2

Compare and contrast ZF nuclease, TALENs, and Crispr

Answer 2

W/Crisper Cas9, Cas9 has ability to cut DNA. Restriction enzymes can also cut DNA, they recognize certain sequences in DNA and when bound to them or near them, they cut at them.

2. ZFN
   - uses nuclease Fok1 to make ds breaks in DNA, linking it to ZF proteins
3. TALENs
   - uses nuclease Fok1 to make ds breaks in DNA, linking it to talen proteins

Question 3

Compare and contrast NHEJ and HDR

Answer 3

1. NHEJ
   - pathway is the predominant repair pathway in mammalian cells
   - Is error prone and simply re-ligates the 2 ends
   - Can result in processing which removes or inserts nucleotides
   - Active during G1
2. HDR
   - Based on HR and utilizes homologous donor sequence as a repair template
   - Mostly active during S and G2
   - Is the basis of both conventional gene targeting and ZFN-induced targeting
   - Cell can be directed to use this response if high quantities of donor are provided

Question 4

Describe the crispr components

Answer 4

1. Cas 9 enzyme (nuclease that cuts DNA) but needs to be directed to site where its gonna cut. Does that by incorporating w/in complex a guide RNA. Guide RNA has structure that allows it to sit inside cas 9 enzyme complex but also bind to DNA homologous to that RNA. This enzyme also needs a sequence called PAM sequence (a few CG nucleotides needed for Cas9 to cut DNA really close to that, not right at nucleotide but a way nucleotides away. With this technique, scientists have been able to design guide RNAS that will bind to any part of genome wherever they want. Only restriction is just need a PAM sequence w/in that region so that when ca9 sitting on sequence it’ll cut. But cause PAM sequence is so vague, ubiquitous, it is straightforward to engineer cutting anywhere. How do you I get Cas9 and guide RNA into my cell, then you can cleave DNA at specific site. Lots of cell lines they’ve generated which has cas9 integrated in them so can just induce its expression, can encode guide RNA on plasmid, inject it in, transfect it in etc.

Question 5

Slide 6

Answer 5

NA

Question 6

Describe crispr inhibition

Answer 6

1. A broken/dead Cas9 enzyeme will block binding of other proteins such as RNA pol, needed to express gene (turns off gene without cutting DNA)

Question 7

Describe crisper activation

Answer 7

An activating protein can be attached to a to broken cas9 protein to stimulate expression of a specific gene

Question 8

Describe crispr epigenetics

Answer 8

A broken cas9 can be coupled to epigenetic modifiers, such as those that add methyl groups to DNA or acetyl groups to histone proteins. Allow researchers to study how precisely placed modifications affect gene expression and DNA dynamics

Question 9

Describe inducible crispr

Answer 9

Cas9, either dead or alive, can be coupled to switches so that it can be controlled by certain chemicals or by light

Question 10

Define gene therapy

Answer 10

Any procedure to treat or alleviate disease which involves genetically modifying the cells of the patient

Question 11

What diseases can gene therapy treat?

Answer 11

inherited disorders
infectious diseases
cancers
immune system disorders

Question 12

What are the 4 scientific hurdles in gene therapy?

Answer 12

1. concept of vehicles called vectors (gene carriers) to deliver therapeutic genes to the patients’ cells
2. Once the gene is in the cell, it needs to operate correctly

3 Patients’ bodies may reject treatments

4. need to regulate gene expression.

Question 13

Compare the 2 gene therapy approaches

Answer 13

1. Classical
   Deliver genes to appropriate target cells with aim of obtaining optimal expression
   Once inside target cells, genes may:
   Produce a product the patient lacks (loss of function mutations)
   Kill diseased cells directly (cancer cells)
   Activate immune system to kill cells
2. Non-classical
   Inhibit expression of genes associated with pathologies (gain-of-function mutations, for example)
   Correct a genetic defect and restore normal gene expression (targeted gene replacement or correction)

Question 14

Compare somatic vs germline therapy

Answer 14

Somatic
Genetic defect is only corrected in somatic cells of affected individual
Any genetic changes are restricted to treated person
Few ethical concerns
Germline
Genetic modifications made to the gamete, fertilized egg or embryo
Any stable changes will be passed on to all future generations
Used to create transgenic mice
Research into such techniques is currently prohibited in most countries

Question 15

Somatic therapies: ex vivo vs. in vivo

Answer 15

Ex vivo

• suitable when affected tissues can be removed, genetically modified, reintroduced
• Good for blood/skin cells
• Used to treat ADA deficiency

In vivo

• Cloned gene is transferred directly in tissues
• Used when cells can’t be cultured in vitro (brain and lung)
• Used to treat CF

Question 16

Gene addition vs. replacement

Answer 16

Gene replacement

• ideally we’d like to replace the defective gene w/a normal copy
• would not be useful 4 in vivo therapy
• germline therapy would require precise gene replacement (major technological hurdle)
• new techniques developed using recombination/gene correction

Gene addition (most common technique in somatic therapy)

• transgene works alongside mutated gene
• cannot correct dominant mutation

Question 17

What are the factors to consider for vehicles/vectors for gene transfer

Answer 17

1. Efficiency of delivery to target cell
Specificity of delivery
Expression of introduced gene in target cell
Dividing versus non-dividing
Size of DNA that can be carried
Stability of gene in target cell
Induction of host immune response?