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Lab 3 > Gene therapy & editing > Flashcards

Gene therapy & editing Flashcards

1
Q

cause of flu virus?

A

influenza A

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2
Q

name 2 stomach flu - viral gastroenteritis vomiting bug causing virus names

A

norovirus
retrovirus

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3
Q

what virus causes chickenpox?

A

VZV
or dormant VZV for shingles

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4
Q

what virus causes cold sore?

A

herpes simplex

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5
Q

what viruses may cause hand foot mouth disease?

A

coxsackievirus A16
enterovirus

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6
Q

What is gene therapy?

A

process of introducing foreign genomic materials (i.e. nucleic acids) into host cells to elicit a therapeutic benefit

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7
Q

What are the 2 nucleic acids that are delivered to treat disease in gene therapy?

A

DNA or siRNA
(using genetic material as drug, pack into DDS and deliver)

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8
Q

gene therapy = new tool to cure human disease and involves transfer of what?

A

genetic material into cells/tissue to prevent/ cure a disease

transfection

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9
Q

What are 3 gene alterations gene therapy is used to make?

A
  • introduce a new gene
  • replace defected/mutated gene
  • silence gene not working properly (STOP protein expression)
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10
Q

What genetic material is introduced as a new gene or replacing a defected/mutated gene?

A

plasmid DNA

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11
Q

What genetic material is used to silence a gene not working properly?

A
  • small interfering RNA (siRNA)
  • short-hairpin (shRNA)
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12
Q

why do you need a delivery vector to deliver plasmid DNA?

A

else DNA will degrade

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13
Q

What are the 2 types of genetic diseases that gene therapy is used to treat?

A
  • single gene defect disease
  • polygenetic or non-hereditary
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14
Q

Give an example of a single gene defect disease.

A

haemophilia - a hereditary (genetic) disease affecting blood’s ability to clot

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15
Q

Give examples of polygenetic or non-hereditary diseases?

A
  • cancer
  • CVD
  • hepatitis
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16
Q

polygenetic/nonhereditary conditions are harder to treat than single gene defect, why?

A

more than 1 gene involved, along w environmental factors

origin can be complex

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17
Q

gene therapy is used to treat but also prevent, as in the case of SARS-CoV-2 vaccines.

outline how these work

A

prevent infection by causing body to produce antibodies against SARS-CoV-2 spike proteins
elicit immune response

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18
Q

problem with influenza and sars-cov-2 vaccines?

A

the virus may change by nest year, body wont recognise -> infection

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19
Q

What are the 2 main classes of delivery vectors used in gene therapy? to induce transfection of cells

A

viral
non-viral

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20
Q

give examples of non-viral delivery vectors

A

(1st term)
liposomes
polymers
CNTs

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21
Q

What are 2 categories of transfection methods in gene therapy?

A
  • in-vivo
  • ex-vivo
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22
Q

What is meant by in-vivo gene therapy?

A

within living:

gene transferred to cells inside patient directly using vector

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23
Q

Which is more common: in-vivo or ex-vivo?

A

ex-vivo

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24
Q

Which is more risky: in-vivo or ex-vivo and why?

A

in-vivo
immune reaction possible from vectors used
could be rejected straight away

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25
Q

What are examples of in-vivo vectors? 3

A

viruses
bacterial plasmids
NPs

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26
Q

Methods used in in-vivo gene therapy 3

A

tissue injection- recombinant virus

systemic infusion- DNA liposome

(biolistic gene gun- plasmid DNA)

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27
Q

Outline the steps of how in vivo gene therapy works.

A

DNA (gene) encapsulated in vector
gene therapy- inject in
gene released into cell
gene expresses proteins
- receptor
- secreted

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28
Q

What is meant by ex-vivo gene therapy?

A

cells modified outside body then transplanted back in again

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29
Q

what does ex-vivo gene therapy allow?

A

targeting of specific cells

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30
Q

Explain how ex-vivo gene therapy works.

A
  • cells from px blood/bone marrow removed and grown in lab
  • cells exposed to gene therapy vector carrying desired gene (i.e. virus enters cells then inserts desired gene into cells DNA)
  • cells grow in lab then returned by IV injection
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31
Q

What are the 3 limitations to ex-vivo gene therapy?

A
  • complex as cells require maintenance in vitro
  • indirect introduction: immune response
  • modified/transfected cells may not fulfil function
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32
Q

In what ways can ex-vivo gene therapy transfected cells not fulfil function?

A

may:

  • not function as desired due to patient rejection
  • malfunction, triggering an immune response
  • not work at all once transplanted
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33
Q

Diagram of ex-vivo gene therapy (retrovirus)
Summarise ex-vivo gene therapy….
slide 17

A
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34
Q

Summary of differences between in and ex-vivo gene therapy
slide 18

A
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35
Q

With viral drug delivery (virus as vectors), what is the basic essence of what we do?
how do they work?

A
  • remove original disease-causing genes
  • replace w genes needed to stop disease
  • then insert altered viruses into px diseased cells in-vivo or ex-vivo
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36
Q

What are the 2 viral cycles?

A

lytic and lysogenic

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37
Q

What is lytic or virulent viral cycle?

A

virus enters host, replicates, lyses (burst open) host cell causing its death

(cell opens and releases stuff)

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38
Q

What is the lysogenic or latent viral cycle?

A

viruses become dormant in cell and integrate its own DNA into host cell DNA, later activating in response to external signal

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39
Q

2 examples of lysogenic/ latent viruses?

A

HIV and Herpes viruses

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40
Q

do you get integration of viral DNA into host chromosomes/DNA with lytic or lysogenic viral cycles?

A

lysogenic

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41
Q

What are examples of viral vectors?

A
  • retroviruses
  • adenoviruses
  • adeno-associated viruses (AAV)
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42
Q

Structure of a retrovirus

A

viral DNA, enz, reverse transcriptase in core and around: envelope of proteins + lipids

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43
Q

What is the size of retroviruses?
nm diameter

A

100nm in diameter - considered nanomedicine

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44
Q

What genetic material is found within retroviruses?

A

RNA

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45
Q

How do retroviruses work?

A

uses reverse transcriptase to make DNA from RNA which can be inserted into the genome

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46
Q

What do retroviruses do when they infect host cells?

A

inject RNA and reverse transcriptase enzyme into cytoplasm of that cell

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47
Q

What is an example of a retrovirus?

A

HIV

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48
Q

what enzyme do retroviruses require to work? why?

A

reverse transcriptase

as want RNA later to be converted back to DNA

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49
Q

Are retroviruses used in in or ex-vivo gene therapy?

A

ex-vivo

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50
Q

What cell type do retroviral vectors affect?

A

divided

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51
Q

What is the therapeutic gene size that retroviruses can deliver?

A

8Kb

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52
Q

What genetic material is found within adenoviruses?

A

double-stranded DNA

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53
Q

What types of infections do adenoviruses cause in humans? 3

A
  • respiratory
  • intestinal
  • eye
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54
Q

What is an example of an adenovirus?

A

common cold

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55
Q

whats the size range in nm of adenovirus?

A

90-100nm

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56
Q

What cell type do adenoviral vectors affect?

A

dividing and quiescent

eg cancer and dormant cells

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57
Q

What is the therapeutic gene size that adenoviral vectors can deliver?

A

37Kb
huge

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58
Q

what type of virus is AAV?

A

adeno-associated.
small DNA viruses

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59
Q

How do AAVs work?

A

non-pathogenic, small DNA viruses that integrate into chromosome 19 in humans w the helper virus (cannot replicate alone)

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60
Q

what helper virus may be used with AAV?

A

adeno!! or herpes virus

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61
Q

are AAV pathogenic or non?

A

non-pathogenic
not known to cause disease

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62
Q

What type of genetic material is found within AAVs?

A

linear single-stranded DNA

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63
Q

What cell type do AAVs infect?

A

dividing and non-dividing

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64
Q

therapeutic gene size of AAVs?

A

4.5kb
tiny

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65
Q

difference between viral vectors and CNTs (non-viral) in terms of genes you can incorporate.

A

CNT: can put multiple on surface but here limited

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66
Q

whats the easier choice out of the 3 viral vectors studied?

A

adenovirus. nothing req like reverse transcriptase (retro) or helper virus (AAV)

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67
Q

What is found within the AZ/Oxford vaccine (ChAdOx1)?

A

chimpanzee adenovirus vector containing spike protein of COVID-19

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68
Q

AZ/oxford vaccine can generate strong immune resoibse from 1 dose.
its been genetically changed so its impossible for it to do what?

A

grow in humans

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69
Q

How do non-viral delivery vectors change in change when complexed w therapeutic gene?

A
  • lipoplex
  • polyplex
  • carboplex
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70
Q

What charge must non-viral delivery vectors have?

A

positive (cationic)

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71
Q

Why must non-viral delivery vectors be cationic?

A

DNA/RNA is negatively charged so it can increase its cellular uptake

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72
Q

with cationic DDS of non-viral vectors, hopefully no immune response so ->?

A

readministration can be acheived

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73
Q

What is the advantage of non-viral delivery vectors over viral vectors?

A

no limitation on size of therapeutic gene delivered

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74
Q

What is the advantage of viral delivery vectors over non-viral delivery vectors?

A

viral are more efficient at transfecting genetic material

in vivo efficiency higher than non-viral vectors

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75
Q

Explain how liposomes traffic genetic material.
trafficking: lipoplexes

A

1) complex liposome w gene to form lipoplex

2) lipoplex endocytosed into cell

3)a) if endosome used to transport in matures, DNA fragments as endosome forms lyosome/ degradation :(

b) if endosome mixes w lipid, gene escapes and reaches nucleus via nuclear pores

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76
Q

what do we want the non-viral vectors to escape in the cell?

A

endosome compartment
think low pH…

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77
Q

Which of the COVID-19 vaccines out of the Moderna, Pfizer and AZ/Oxford use mRNA?
non viral DDS

A

Moderna and Pfizer

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78
Q

What are the advantages of using mRNA as the therapeutic gene delivered?

A
  • safer than whole virus/DNA delivery. not infectious
  • processed directly IN cytosol (no need to reach nucleus like DNA)
  • has a short half-life so can be regulated by molecular design
  • not immunogenic
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79
Q

How is mRNA safer than whole virus or DNA delivery?

A
  • not infectious
  • cannot integrate into host genome
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80
Q

What does mRNA need to be used therapeutically?

A

a drug delivery system

81
Q

What are the requirements for mRNA to be transported safely and efficiently in vivo?

A
  • without degradation in circulation
  • crossing plasma membrane: mRNA needs help
  • reaching cytosol
82
Q

What DDS has been used to deliver mRNA in the Moderna and Pfizer vaccine?

A

positively charged lipid nanoparticles

83
Q

What are the advantages of the mRNA due to its DDS in Moderna and Pfizer vaccines?

A
  • more stable
  • resistant to RNase-mediated degradation
  • nanoparticles promote endosomal escape into cytosol
  • forms self-assembled virus-sized particles allowing various routes of administration
84
Q

How does mRNA in the Pfizer/Moderna vaccines work against COVID-19?

A

mRNA translated into antigenic proteins causing immune system to produce neutralising antibodies

85
Q

What is the disadvantage w mRNA used in Pfizer/Moderna vaccines?

A

long-term storage requires low temperatures leading to distribution logistic issues

need freezers… hot countried :/

86
Q

oxford/ pfizer/ moderna vaccines use:

  • viral DDS
  • adenovirus
  • double stranded DNA
A

oxford

87
Q

oxford/ pfizer/ moderna vaccines use:

  • mRNA
  • have lipid based NPs in them +ve
A

pfizer and moderna

88
Q

What are the main obstacles to gene therapy?

A
  • immune response depletes after repeated doses
  • viral vector issues
  • difficult to integrate therapeutic DNA into genome
  • polygenic disorders
  • insertional mutagenesis
  • expensive treatment
89
Q

What are issues w viral vectors that form an obstacle to gene therapy?

A

risk of toxicity, inflammatory responses

90
Q

What are problems w integrating therapeutic DNA that form an obstacle to gene therapy?

A

rapidly dividing cells means effect is short-term

need more doses

91
Q

What is meant by insertional mutagenesis?

A
  • integration of DNA in a sensitive place in the genome
  • e.g. in place of tumour suppressor gene potentially leading to cancer :(((
92
Q

What is an example of gene therapy used for cancer?

A

retrovirus encoding shRNA targeted ATP-binding domain to silence gene

93
Q

Non-Viral (Nanomedicine) Gene Delivery……

amino based therapeutics involve proteins and peptides (<50 aa). name 2 peptides

A

insulin
cyclosporin

94
Q

amino based therapeutics involve proteins and peptides (<50 aa). name examples of proteins used?

A

epo
vaccines
antibodies - targeting

95
Q

3 examples of nucleic acid based therapeutics

A

siRNA
oligonucleotides
plasmid DNA

96
Q

example of polysaccharide therapeutic

A

heparin

97
Q

viral gene carriers have the natural ability to target what?

A

nucleus
but risky

98
Q

2 types of non-viral gene carriers?

A

organic
inorganic - cationic

99
Q

what interactions are used to form diff types of complexes + deliver genetic material to cell (non-viral carriers)

A

strong electrostatic attraction

100
Q

how can you restore/ replace a defective gene?

A

increase protein expression
pDNA or mRNA

101
Q

how can you silence a defective gene?

A

decrease protein expression

  • antisence oligonucleotides
  • miRNA
  • RNA aptamers
  • siRNA
  • shRNA
102
Q

RNA aptamers used for active targeting how?

A

act like antibodies recognise specific protein/ peptide sequence

to dec protein expression and silence defective gene

103
Q

some formulation challenges?

A

degradation from enzymes
size
charge
hydrophilic
water soluble
- wont cross membrane on own+ too big to travel in between cells

104
Q

What are examples of routes that non-viral vectors can be administered via?

A
  • Parenteral
  • Intrathecal (spine)
  • Intravitreal (eye)
  • Intradermal
  • Intratumoural (tumour)
105
Q

why is immunogenicity not wanted with this therapy?

A

some of these gene materials produced using bacteria: can have lot of endotoxins left - problem if inject to px

106
Q

why is mucosal delivery for gene therapy not wanted?

A

mucosal sites have high enzymatic activity + RNA esp: suceptible to enzymatic degradation

strong risk of not getting enough therapeutic macromols at site of action

107
Q

usually what prevents gene therapy crossing mucosal barrier?

A

too big: cant cross sbetween cells
too hydrophobic: cant cross memb either

108
Q

What are the 3 routes of mucosal delivery possible?

A
  • transcellular
  • paracellular
  • receptor/carrier-mediated
109
Q

What types of molecules go through the transcellular route?

A

small hydrophobic molecules (lipid bilayer forms membrane)

110
Q

What type of molecules go through the paracellular route?

A

small water-soluble (limited by tight junctions)

111
Q

what is paracellular route?
and whats it therefore limited by?

A

in between cells

limited by tight junctions

112
Q

what is the transcellular route?

A

through membrane
apical + basolateral side

113
Q

What is the main obstacle to mucosal drug delivery?

A

enzymatic degradation activity

114
Q

What are techniques for improving transport across mucosal membranes?

A
  • increase hydrophobicity
  • add permeation enhancers
  • overcome enzymatic degradation (chemical modification)
  • co-administration inhibitors
  • encapsulation via nanomedicines
115
Q

How can we increase hydrophobicity to improve transport across a mucosal membrane?

A

lipidisation of peptides and oligonucleotides

116
Q

What are examples of permeation enhancers we can add to improve transport across a mucosal membrane and how do they work?

A
  • surfactants
  • cationic polymers
  • calcium chelators (e.g. EDTA)
  • opens tight junctions of cell
117
Q

What enzymes are responsible for the enzymatic activity within the mucosal membranes?

A

endo and exonucleases

118
Q

How do endonucleases work?

A

cut INSIDE DNA/RNA

119
Q

How do exonucleases work?

A

cut ENDS of DNA/RNA

120
Q

restriction enzymes recognise specific sequences and are useful for what?

A

molecular cloning, restriction fragment length polymorphism testing

120
Q

restriction enzymes recognise specific sequences and are useful for what?

A

molecular cloning, restriction fragment length polymorphism testing

121
Q

How does the enzymatic degradation activity change across the mucosal departments?

oral
buccal
nasal
rectal
transdermal
pulmonary

A

Oral > rectal > buccal = nasal > transdermal > pulmonary

122
Q

For a drug highly susceptible to enzymatic degradation, which mucosal compartment will it be degraded in the most?

A

oral

123
Q

For a drug highly susceptible to enzymatic degradation, which mucosal compartment will it be degraded in the least?

A

pulmonary

124
Q

which has highest -> lowest surface for absorption from…

oral
buccal
nasal
rectal
transdermal
pulmonary

A

rectal
oral
nasal
buccal
pulmonary
transdermal

125
Q

What are examples of chemical modifications that can be made to overcome enzymatic degradation?

A
  • replace O w S in oligonucleotides so enzyme can’t recognise where to cut (phosphorothioate)
  • PEGylation
126
Q

what does replacing O w S (phosphorothiodating oligonucleotides) do the molecule?

A

makes it harder for enz to recognise where to cut

inc stability of ON

127
Q

What is the danger of co-administration inhibitors?

A

can’t degrade RNA/DNA long-term causing toxicity
(body cant fight infections)

128
Q

why effect does encapsulating gene material in nanameds have?

A

enz wont see it so wont be degraded

129
Q

if using plasmid RNA/ shRNA.. getting to nucleus vs being in cytoplasm has impact bc if DNA in nucleus, what happens when cell divides?

A

DNA will be in cells produced, diff generations will still have that DNA

130
Q

will activity be longer or shorter ig gene therapy agent in nucelus and why?

A

bc will still exist there when cell divides

longevity

131
Q

What are examples of nanomedicines used as non-viral vectors?

A

polymers

liposomes

carbon nanotubes

metal nanoparticles

dendrimers

132
Q

with a polyplex etc what is it formed of?

A

= complex between +ve DDS and drug (gene material)

133
Q

once you administer non-viral gene therapy, what so you hope about the complex?

A

that its strong enough to prevent premature release of the gene therapy agent

134
Q

what happens if gene therpay agent is prematurely released afteer admin?

A

gene therapy agent: DNA/ RNA, would become susceptible to attack by nucleases + would be degraded quickly in systemic circ

135
Q

formation of the complexes relies on electrostatic interaction. So anything that could change the XXX could mask or hide or interact with any of the components of the complexes -> potential to change system PKs.

A

+ve charge on carrier

136
Q

anything that can change +ve charge on carrier could do what 3 things thus having potential to change system PKs?

A

mask
hide
interact w any complex components

137
Q

why do proteins like albumin have possibility of interacting w gene carrier and displacing gene therapy agent -> premature release + quick elim by enzymatic degradation?

A

Albumin is also -ve charged so therapy may interact with albumin and not genetic material.

cationic +ve gene carrier

138
Q

(SoM1 colloids): salt concentration (ionic strength) could be a problem w gene therapy why?

A

salt could mask charge on cationic gene carrier, making interaction w gene therapy agent weaker.. premature release, quick elim

139
Q

systems in lab, usually made in simple solutions where ionic strength not too high, no proteins.
So the 1st time 2 systems will be exposed to proteins and higher ionic strength - might be in body, could ->?

A

destabilisation of system.

140
Q

what can protein adsorption do to gene therapy agent in body?

A

potentially displacing gene therapy agent

could -> increase in size, increased opsonisation, = complexes removed faster from systemic circulation :(

141
Q

why do we need to ensure at pH in systemic circulation the charge will be +ve?

A

if not, interaction between carrier and therapy agent will be a lot weaker -> premature release of gene therapy agent.

142
Q

as using +ve gene delivery systems, able to interact with negatively charged membranes e.g. of RBC = those gene delivery systems could cause some X

A

haemolysis! = 1 of toxicities that we are worried about when we are working with +ve charged delivery systems.

143
Q

polyplexes have diff ways to use endocytosis to get into cell, and depends a lot on what?

A

size

144
Q

once inside cell, whatll happen depends on what?

A

gene therapy agent used

145
Q

polymers used often contain amino +ve function.
but what is done so we can escape the endosome eventually?

A

prefer mix of primary, sec, tert amines

146
Q

What is the proton sponge effect?

A

theory of what happens when polyplex inside endosome.

Protonation causes an osmotic gradient.
Swelling and an increase in osmotic pressure causes a rupture.

147
Q

the endosome inside goes through a process of acidification which is what?

A

conc of H+ inside increases

148
Q

if have polyplex, can accept H+ and act as buffer… get influx of protons into endosome
will the pH inside change or not?

A

no
as these wont be free protons, but attached to polymer form polyplex

149
Q

why does endosome eventually erupt in protonge sponge effect?

A

too many ions inside
water enters to try and dilute + maintain OP on either side of memb. swells then explode, releasing complex in process

150
Q

whats an alternative theory to proton sponge that better describes liposomes, lipids, lipoplexes after they enter endosome. 3 steps

A

protonation + polymer memb interaction
local hole formation

151
Q

protons can be used for coating what?

A

gold NPs (-ve) will complex w +ve polymers and conform hybrid gold NP polyplex for delivery

152
Q

Describe the endosomal release of lipoplexes?

A
  • lipoplex made w cationic lipid that can interact w negative membrane
  • phospholipids rearrange so +ve goes w negative lipids
  • Formation of neutral-phospholipid pairs.
  • DNA displaced and released into cell

Lipofectamine 3000 main one used

153
Q

What are the 2 pathways a lipoplex can undergo once inside an endosome?

A
  • endosome matures to lysosome leading to DNA fragmentation
  • liposome escapes from endosome
154
Q

the theory of endosomal release depends on type of lipids used, if using purely permanently +ve lipids, what theory is likely?

A

fusion + arrangement

(liposome membrane fusion
cel penetrating peptides)

155
Q

How can we exploit the way which DNA is released from a lipoplex?

A
  • use pH sensitive liposomes that become charged in acidic endosome -> memb disruption + endosomal degradation
  • helper lipids (DOPE) that enter charge conformation when endosome is acidic enough to trigger endosomal escape process
156
Q

give an example drug lipoplex that is PEGylated and using interaction with ApoE to -> hepatocytes in liver :)

A

Onpattro

157
Q

Why is PEG used with lipoplexes?

A

Used to hide negative charge

else complex way too toxic and would cause lot of haemolysis after admin

158
Q

What is the limitation of using charged lipids for lipoplexes?

A

can be too toxic causing embolisms in blood or activating immune system - need PEG

159
Q

why do we use corticosteroids before amdinistering Taxoter?

A

because of CARPA
interaction w complement = causing severe pseudoallergic reaction and we can admin corticosteroids/ antihistamines before to dec risk of interactn

160
Q

with PEG still not 100% sure as has some issues. Can use it but what must be carefully selected?

A

type of PEG

161
Q

What are the pros of non-viral gene delivery?

A
  • complexation
  • internalisation
  • protein interaction
162
Q

What are the cons of non-viral gene delivery?

A
  • toxicity
  • protein interaction
  • biocompatibility issues
163
Q

why is toxicity an issue w non-viral gene delivery?

A

using +ve systems. can have intercactions w membrane -> issues w immune system…

164
Q

whats the use of SCR (scrambled sequence) in research?

A

to check for non-specific effects
genotoxicity of carrier

  • same conposition as therapeutic macromols, diff order
165
Q

technique used for gene expression

A

PCR
important in diagnostics too inc COVID19 before LFTs

166
Q

technique used for protein expression - assessing efficacy by looking at protein levels

A

western blot

167
Q
    • -therapeutic macromols mini lectures on word—–
A
168
Q

Gene editing techniques….

what is the word for: genetic material that defines an organism?

A

genome

169
Q

a complete set of nucleic acids sequences encoded as DNA within what?

A

23 chromosomes

170
Q

How many genes from the 25,000 have been linked to diseases from the human genome?

A

3,000

171
Q

name 2 examples of well established diseases w genetic link

A

SC anaemia
haemophilia

172
Q

what has limited the efficacy of drugs esp those in cancer treatment eg kinase resistance and combination therapy

A

genetic mutation

173
Q

T/F: if we fix faulty gene at top, will fix 100% of problem

A

False

174
Q

What is gene editing?

A

process in which engineered nucleases are inserted, replaced, or removed DNA from a genome.

175
Q

How do the types of gene editing technologies differ?

A

by 3 different types of nucleases

176
Q

What are the 3 main gene editing technologies developed?

A
  • zinc finger nucleases (ZFNs)
  • transcription activator like effector nucleases (TALENs)
  • Clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas-based RNA-guided DNA endonucleases (CRISPR/Cas-9).
177
Q

What is the general process of gene editing?

A

1) double strand break at nuclease target site

2) either homologous directed repair or non-homologous end-joining

178
Q

What are the 2 gene alterations that are made w homologous directed repair?

A

gene insertion and correction

179
Q

whats the difficulty w homologous directed repair?

A

want template to figure out WHERE double strand break happened

180
Q

what is the first step of gene editing?

A

double strand break at nuclease target site

181
Q

What are the gene alterations that are made w non-homologous end-joining?

A

gene disruption

182
Q

Describe the basic structure of a ZFN.

A
  • DNA cleaving domain (Fok1)
  • DNA binding domain
183
Q

How do zinc finger nucleases create their DNA double strand break?

A

recognise 3 base-pair sequences and cleave DNA here

184
Q

why do we engineer ZFN before put in?

A

so knows exact sequence to recognise
(directed in proper place, not random)

185
Q

what type of gene editing:

targeted editing of genome by creating double strand breaks in DNA at user specified locations

A

ZFN

186
Q

Describe the basic structure of a TALEN.

A
  • DNA cleaving domain (Fok1)
  • DNA binding domains
187
Q

How do TALENS create their DNA double strand break?

A

recognise single bases

188
Q

How do TALEN and ZFN compare?

A

TALEN more sensitive however more difficult to generate as v precise

189
Q

What is CRISPR-Cas 9 based on?

A

bacteria DNA defence mechanism against virus DNA: RNA-guided DNA cleavage

190
Q

What types of genetic errors can CRISPR be used to target?

A

double strand break at nuclease target site

…cells translate + process sequence to give crRNA
these anneal to transactivating crRNAs (tracRNAs) - recruit + direct Cas proteins -> direct seq-specific cleavage (+ silence pathogenic RNA)

191
Q

How is CRISPR used in drug discovery?

A

Target identification and validation.

192
Q

Why is CRISPR better for screening than traditional methods?

A

CRISPR can screen for multiple cells at once.

193
Q

genes are involved in drug sensitivity in CRISPR/Cas9 positive or negative screening?

A

positive

194
Q

describie CRISPR Cas9 positive screening

A

put cells w crispr
use drug then study which cells alive after drug tretament

has potential to target and kill specific cells
spot several genes/ targets at same time :)

195
Q

describe CRISPR Cas9 negative screening

A

got 2 pools of cells
treat 1 w drug, keep 1 untreated
at end compare the 2 (which cells are now dead)

196
Q

T/F with crispr cas9 -ve screening, you get same results as +ve screening

A

true

197
Q

give example of crispr/cas9 screening

A

cells w/out p53
still alive after pt as cant kill
will survive Pt drugs = link between presence of p53 and killing w Pt

198
Q

main difference between crispr cas 9 +ve and -ve screening?

A

+ve: 1 pool of cell
-ve: 2 pools of cells

Lab 3 (21 decks)

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