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Lec 10 (gene editing) done Flashcards

1
Q

Give an example as to why gene editing is considered to be controversial

A

Josiah Zayner injected his own left forearm with the gene-editing tool CRISPR in a bid to grow bigger muscles

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2
Q

What is CRISPR?

A

a gene-editing tool

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3
Q

Give examples of what can be found in the genome engineering toolbox

A
  1. Zinc finger protein
  2. Meganuclease
  3. TALE
  4. CRISPR/Cas9
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4
Q

What genome engineering toolbox is difficult to engineer but has extreme specificity?

A

meganucleases are huge proteins that are difficult to engineer but have very extreme specificity

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5
Q

______ improved on Zinc finger nucleases and recognised as a single base rather than 3

A

TALE repeat domains improved on Zinc finger nucleases and recognised as a single base rather than 3

(difficult to engineer)

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6
Q

CRISPR/Cas 9 came out around __ years ago

A

CRISPR/CAS 9 came out around 6 years ago

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7
Q

First hints of CRISPR/Cas9 were discovered in _____ by _____ when ____

A

1993 by Francisco Mojica (Spain)

when researching extremophiles in salt marches

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8
Q

what did Francisco Mojica discover?

A

Hints of CRISPR/Ca9
he was studying bacterial salt marches and found repeat regions in the genome of the bacteria.

Several gens of scientists studies these regions

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9
Q

CRISPR/Cas9 can be used in _____cells

A

CRISPR/Cas9 can be used in high-order eukaryotic cells

Jennifer Doudna et al

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10
Q

What significant event(s) occured following the discovery of CRISPR/Cas9 ?

A

The CRISPR patent war

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11
Q

What does the ruling of the CRISPR/Cas9 patent war state?

A

“no interference” between the patents, but which should companies license?

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12
Q

patent aka __

A

legal ownership

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13
Q

What does CRISPR/Cas9 stand for?

A

Clustered Regularly InterSpaced Palindromic Repeats sequences of DNA

Cas9 is CRISPR associated protein 9 that acts as a nuclease to cut the DNA

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14
Q

Define palindrome?

A

word that is the same backwards and forwards

e.g tacocat

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15
Q

What is Cas9?

A

it is the CRISPR associated protein 9 that does the cutting of DNA (acts as a nuclease)

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16
Q

What components make up the CRISPR/Cas9 complex?

A

3 components

  1. Cas9 (CRISPR associated protein 9)
  2. crRNA = CRISPR RNA
  3. tracrRNA = Trans Activating CRISPR RNA
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17
Q

What does the tracrRNA stand for?

A

Trans Activating CRISPR RNA

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18
Q

What components of the CRISPR/Cas 9 complex drives the cas9 towards the genome target

A

The 2 RNA components of the CRISPR/Cas9 complex drive the Cas9 towards the genome target

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19
Q

What does crRNA do in the complex?

A

crRNA is the part of the RNA that has complimentary binding to the target genome

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20
Q

What does the tracrRNA bind to

A

crRNA associates with a tracrRNA

tracrRNA does not bind to the genome it is targeting but instead it forms complex with crRNA to stabilize and help it load into the Cas9 protein

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21
Q

Adaptive immunity that targets phage genome is associated with which component of the CRISPR/Cs9 complex?

A

crRNA

CRISPR RNA

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22
Q

___ are short palindromic repeats

A

crRNA are short palindromic repeats that target the phage genome

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23
Q

What does CRISPR/Cas9 protect bacteria from

A

Viruses

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24
Q

what does PAM stand for and what is it

A

PAM = protospacer adjacent motif

PAM is a requirement in the targeted genome for cutting to occur.

PAM determines if cleavage occurs-cas9 recognition

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25
Q

What are PAM?

A

PAM= protospacer adjacent motif

short stretch of nucleotides that define whether or not cutting of DNA should occur

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26
Q

PAM sequences are not present in the genome of the host but will be present in the genome of the target

A

CRISPR/Cas9 can bind the invading genome and if a PAM sequence is present the DNA can be cut

However, if we find the same DNA sequence in our own genome we will not cut it -as a PAM sequence would not be present there

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27
Q

How is a guide RNA engineered?

A

By combining crRNA and tracrRNA

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28
Q

engineered single guide RNA

A

efficiency

(allows both crRNA and tracrRNA to be infused into the complex together)

instead of having to fuse them in individually

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29
Q

What is a modification of CRISPR/Cas9 for genome engineering

A

the engineering of the single guide RNA

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30
Q

Not all guides work equally well because

A

DNA is tangled around histones, compacted etc

accessibility can be an issue therefore, tools have been made to predict which guides may or may not work

The guides are to be checked experimentally

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31
Q

what happens after cleavage?

some mechanisms are shared between bacteria and higher-order cells

A
  1. once the ds-break is produced- where cas9 cleaves both strands in the target DNA
  2. repair DNA - evolutionary benefit (fixes DNA) =
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32
Q

what main processes can occur after cleavage?

A

DNA repair methods

3

  1. NHEJ
  2. MMEJ
  3. HDR
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33
Q

Describe NHEJ

A

= is a non-templated process = means it doesn’t use another piece of DNA to mediate the repair

=similar to recombination during meiosis

= is used in the nature to introduce diversity e.g antibodies

= best for frameshifting sequences

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34
Q

How does NHEJ work on frameshift mutations

A

Insertion mutation (frameshift) = insertion of single basepair => frameshift the amino acid sequence which alters the entire sequence (protein)

insertion mutation (non-frameshift) = insertion of an entire codon = results in a change in 1 amino acid => could change overall protein

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35
Q

briefly Describe HDR

A

=uses strand invasion in order to mediate an exchange/insertion of new DNA

=high fidelity repair mechanism (scarless)

=can use a sister chromosome

=best for gene insertion

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36
Q

HDR stands for

A

Homology-directed repair

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37
Q

crRNA is a __ recognition sequence that determines ___ where the Cas9 can go in the genome

A

20bp

where the Cas9 can go in the genome

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38
Q

What are the components of CRISPR/Cas9 that mediate the cut

A

RuvC and HNH domains (nuclease domains)

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39
Q

how have scientists made CRISPR/Cas9 more accessible?

A

engineered a single guide RNA

which is composed of tracrRNA and crRNA

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40
Q

What is guide RNA composed of

A

2 parts

  1. target specific crRNA
  2. tracrRNA (helps with stability and load into the Cas9 complex)
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41
Q

sgRNA stands for

A

single guide RNA

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42
Q

What is the function of crRNA

A

aka Spacer

it directs Cas9 to the genome

43
Q

Why do PAM sequences exist?

A

Because in the original bacterial systems, we don’t want the Cas9 to target the host genome.

Because crRNA seq is present in the host genome

44
Q

Why is it a disadvantage to have a PAM sequence

A

it narrows the specificity/no. of regions that can be targeted with CRISPR/Cas9

however, there are many variants of CRISPR/Cas9 from diff species that have different PAM restrictions. therefore, slightly diff nucleotides that they recognize.

45
Q

For ____ Cas9 the spacer is __bp

A

S.pyrogenes

20

46
Q

No cleavage is mediated unless ___ in the genome is recognised by __

A

PAM seq

Cas9 protein

47
Q

Cleavage occurs ~__bp before/after the PAM

A

3 bp before the PAM

48
Q

Describe MMEJ

A

microhomology directed end joining

Similar to NHEJ, in that frameshifts, deletions and insertions can occur.

However, instead of having a smooth ds-break like in NHEJ, there are v small regions (microhomology regions) that help to 2 strands anneal. The result is pretty much the same.

49
Q

Which DNA repair mechanism can we used to insert a whole gene after cleavage?

A

HDR

seamlessly insert a gene in place of the double-stranded break

can also insert a single point mutation

50
Q

What main applications can CRISPR/Cas9 be used for?

A
  1. in non-mammalian/ in vitro applications
  2. mammalian systems
  3. in humans
51
Q

What applications of CRISPR/Cas99 can be used for non-mammalian applicants?

A

+ in vitro

  • sensors
  • information storage
  • gene drivers
  • editing of crops
52
Q

What applications of CRISPR/Cas99 can be used mammalian systems?

A
  • protein engineering

- animal models

53
Q

What applications of CRISPR/Cas99 can be used in humans?

A
  • gene therapy

- cell therapy (CAR T cells)

54
Q

Describe how CRISPR/Cas9 biosensors are used

A

a diagnostic chip that is designed to detect Duchene Muscular Distrophy.

Characterised by point mutations in a couple of exons in the gene.

if you design a gRNA, load it into a Cas9 that we have knocked out the nuclease activty. When it binds it won’t cut.

You have a way of detecting any 20bp sequence you want.

Immobilised CRISPR/Cas9 on the surface of a graphene chip. Applied a voltage across the chip. When the DNA gets close to the chip’s surface, (highly negatively charged) influences the graphene to cause a change in current.

If CRISPR/Cas 9 with its guide, detects DNA that has this mutations (e.g for musclular distrophy), you will get a signal.

No binding = no signal

55
Q

What happens if there are new variants of Covid19.

A

look for regions of the genome that are currently easily mutated- may be essential for function

OR

switch to gRNA, which allows you to detect each variant specifically.

56
Q

How is CRISPR/Cas9 used to encode a digital movie

A

CRISPR/Cas9 is used in bacteria to defend against viruses. It is able to process sequences of viral DNA and put them into the CRISPR array. (array grows = remembers = if it is exposed again- kills it)

Instead of supplying viral RNA, pixtechs were supplied.

These were incorporated into the bacterial genome. EAch triplet in the random DNA seq encodes a diff colour in the image. And the end seq determines there will be 144 different pixtechs to encode the entire image.

If you generate 144 of these DNA seq, supply them to the bacteria, bacteria takes them up and puts them into a CRISPR array in the genome.

The image has been loaded into the genome.

To get the image out of the genome - you can se to recover the DNA pieces and rebuild the image.

57
Q

Why was CRISPR/Cas9 used to store the image? What was the underlying message

A

That CRISPR/Cas9 can be used to store large quantities of information for a really long time.

58
Q

What are gene drives

A

Gene drives using CRISPR/Cas9 are a way to ensure that a mutation/gene is passed through a population even if its not beneficial to the individuals

59
Q

Describe gene drive in mosquitos

A

They have targeted a gene responsible for female reproduction.

By inserting a construct into 1 of the 2 chromosomes in the mosquito genome. This insert will disrupt the gene so that when the protein goes to get translated it it can’t.

There is a cas9 in the middle of the construct. This cas9 contains a guide RNA that targets the same gene on the 2nd chromosome. Therefore, Cas9 expression drives it to cut the sister chromosome and HDR can create 2 copies of the gene within the organism. Therefore, it forces the transfer of the gene to a sister chromosome. So when the mosquitos breed, instead of having a heterozygous pop, there will be homozygous pop with the disrupted gene. This results in any offspring from this parents to have the gene.

60
Q

____ + ____ = 100% chance of passing it on

A

altered gene + gene drive = 100% chance of passing it on

61
Q

What are the limitations of gene drives

A

3 (mosquitos)

  1. point mutations to the CRISPR guide target induce resistance
  2. Once released there isn’t an easy way to recall them
  3. How to target select populations?
62
Q

How can you use gene editing in plants?

A

Rice plant

Used CRISPR/Cas9 to create an albino dwarf rice plant. Has a very easy phenotype so can be seen easily if the experiments worked or not.

63
Q

what is known as speedy domestication, in terms of editing plants

A

speeding up the domestication process

tomatoes can have either 1) large quantities of fruit or 2) more robust tomato plant

if they were crossed= they don’t work well

You can use CRISPR/Cas9 to create a combined genotype to get more robust fruits.

Therefore, CRISPR/Cas9 was able to provide a short cut towards making new varieties.

64
Q

Some crop mutations can’t be combined by breeding due to the other accumulated mutations elsewhere in the genome. What can we use to combine crop mutations instead?

A

Gene editing e.g CRISPR/Cas9

65
Q

___ can be used as a protein engineering tool

A

CRISPR/Cas9

66
Q

BRAC1 gene is involved in __

A

breast cancer

= tumour suppressor gene TSG

67
Q

a mutation in the BRAC1 gene will __

A

increase the chances of getting cancer

68
Q

Explain protein engineering using BRAC1 gene

A

a CRISPR library was created with all the combinations of hexamers. And placed in the promoter of the gene. To study how the diversity in the promoter influences expression of the gene.

Since there are a lot of variation in the population, are there specific mutations that make people more at risk of cancer.

HDR was used afterwards to mediate the changes in the promoter region. They let the cells grow and over time observed relative expression of the gene and find specific mutations in the promoter region that would signal an increased risk of cancer.

This is a way of speeding up what normally occurs in the natural population.

69
Q

Give an example as to how you can fuse Cas9 to speed up evolutionary processes

A

The fusion of an error-prone DNA polymerase to Cas9 generates random mutations

When the gene replicates, point mutations are introduced randomly throughout the gene. This is able to be screened in a large variant for a function that you want.

e.g if you wanted an antibody that had better binding to a target. you may be able to use the CRISPR/Cas9 polymerase to quickly mutate that gene and generate lots of diversity.

70
Q

Genome wide screening

A

multi-guide

Libraries have been crated to target every single gene in the human genome.

~70-100 k CRISPR Cas guides. Each
one pf those 20bp seq target a human gene.

If these guide RNA + CRISPR/Cas9 are put into a viral system, we can transduce/infect a pop of cells and screen them to see what might drop out

71
Q

Genome wide screening

A

multi-guide

Libraries have been crated to target every single gene in the human genome.

~70-100 k CRISPR Cas guides. Each
one pf those 20bp seq target a human gene.

72
Q

give an example of genome wide screening (Long answer)

A

PLX (vemurafenib) = a common chemotherapy drug is used to treat melanoma.

However, people naturally develop resistance.

If you treat human cells with this entire knockout library of guide RNAs. And then supply the chemotherapy drug. You can observe which guide RNA remain present in the cell pop over time.

If the cells are killed by the dug = that guide RNA (that was inside the cell) gets lost from the pop.

If the gRNA makes a knockout/mutation = it is enhancing the resistance for the drug. then these cells will propagate. Overtime we can see which gRNA become more abundant.

73
Q

In terms of genome wide screening, if the guide RNA + CRISPR/Cas9 from the library are put into a viral system, what can we do?

A

If these guide RNA + CRISPR/Cas9 are put into a viral system, we can transduce/infect a pop of cells and screen them to see what might drop out

74
Q

How can transgenic animal models be created using CRISPR/Cas9

A

directly inject Cas9 into the embryos and then transfer them into a surrogate mother.

75
Q

What can we use transgenic animal models for?

A

can make human disease models
e.g

  • cancer oncogenes
  • genetic disorders
  • autoimmunity
76
Q

What is generally used for intranuclear injections?

A

Cas9 mRNA

77
Q

Inserting genes into the genome by HDR is inefficient. state whether it is efficient or inefficient to use a ds-break vs without a ds-break

A

Without a ds-break =
very inefficient

With a ds-break =
efficiency increases

78
Q

What occurred in 2015 in terms of editing human embryos

A

Work was performed on a double-fertilized (non-viable) human embryo.

they were able to correct embryos 25% of the time

79
Q

What occurred after scientist edited a non-viable embryo?

A

In 2018, 2 twin girls were born with a gene knockout in CCR5

(genetically engineered humans)

He Jianjui, suggested that the girls would be HIV resistant in the future. There are other HIV entrance receptors and he only knocked out 1 of them.

80
Q

Why was the Berlin patient significant?

A

Berlin patient had HIV and lymphoma Cancer and got cured because he had a bone marrow transplant with cells that had a truncated receptor that HIV uses to get into cells and the donor had a mutation which meant that the HIV could no longer enter the cells and the immune system was able to clear it.

81
Q

How to deliver Cas9 therapeutically?

A

therapeutic systemically = to the entire body

adenovirus or nanoparticle technologies can be used where they are injected and they will circulate throughout the body.

OR use knockouts of latent viruses in humans

many viral infections lie latent in the genome and are difficult to treat. Therefore, if the viruses are dormant, we may be able to target it using CRISPR/Cas9. generate random mutations by NHEJ and might be curative for some diseases

82
Q

Give examples of knockouts of latent viruses in humans

A
  1. Herpes Simplex Virus
  2. Epstein Barr Virus
  3. Human papilomavirus
83
Q

____basically a repurposed HIV

A

Adenovirus is basically a repurposed HIV

84
Q

What occur in the early 1990s with gene therapies

A

A couple of people died because of immune side effects of some of the viruses that were used.

Therefore, people have become very cautious.

85
Q

exons in red =

A

exons in red = problem with gene
frameshift, truncation

Therefore, the entire gene is truncated

86
Q

Describe the traditionally gene target for therapy by knockout

A

Duchenne Muscular Dystrophy

People have discovered that using double CRISPR/Cas9 cuts either side of the red exons allow you to remove the exon red exon

the protein, despite missing a whole exon, can still function as it still has some of the domains downstream that still work well.

This gene therapy has been fast-tracked by the FDA

87
Q

Immunotherapy involves adaptive immunity. Immune system is able to respond to new threats by 1 of __ ways by____

A

2 ways

1) making T-cells
2) making B-cells

88
Q

T-cells kill infected cells by ___

A

=directly kill infected cells

recognizing markers on the surface of the cell that were infected

89
Q

How do B-cells work?

A

B-cell make antibodies that travel around the body and

a) kill viruses or
b) bind to pathogen and mediate cellular destruction

90
Q

Immunotherapy involves the transfer of cells. Describe the transfer methods.

A

allogenic transplant
=transfer of cells from 1 patient to another with some mods
(similar to a stem-cell transplant)

Autologous transplant =take cells out of a person, do mods, reinvest them

91
Q

What are CAR T cells?

A

Genetically engineered T cells

Carry the antibody that tracks and kills any cell that the antibody targets
e.g CD19

Can be redirected to target cancer cells instead.

92
Q

What is CD19

A

found on the surface of B-cells and lymphoma (cancer)

93
Q

What are B-cells

A

a type of WBC that produced antibodies.

part of the adaptive immune system

94
Q

What does CAR T stand for

A

Chimeric antigen receptor T-cells

95
Q

Where does the antigen binding site bind to

A

antigen that is on a tumour cell

96
Q

The constant region joins _____

A

The constant region joins the two arms together

97
Q

How have CAR T cells been engineered?

A

By taking the normally soluble antibody sequences and integrated them into the membrane of T-cells to redirect the T-cell response

Therefore, they are using the targeting specificity of an antibody and combining it with the really potent cell function of T-cells

98
Q

How can you use CAR T cells to target lymphomas.

CD19 protein that is found on the surface of B-cells

A

By having an antibody that targets CD19 and having it on the surface of the T-cell will allow you to target the lymphomas that are circulating in the body

99
Q

How many types/generations are there of CAR T cells?

A

4

1st gen = blue and bind domain makes the antigen binding region

4th gen = TRUCKs

100
Q

What have they included with successive generations of CAR T cells?

A

1st gen
= removed all the constant domains

included in the cytoplasmic domain of the CAR domains to help stimulate
(hyperactivate) the T-cell.

TRUCKs=
a small regulatory element is released to induce expression of cytokines to get a local immune response around the tumour cell.

101
Q

How can we use CAR T cells to treat cancer? (long answer)

A

Take a blood sample from people, isolate WBCs , stimulate T-cells to make them expand so there are billions of them.
insert CAR into the cells (transduction) and then reinfuse the killer T-cells back into the patient. (autologous transplant)

= no immune response as it is your own cell that has been modified and reintroduced.

102
Q

What can lead to a cytokine storm?

A

CAR T cells treatment

If CAR T cells are killing a tumour there will be a lot of inflammation. This can kill patients.

CAR T cells struggle to treat solid tumors. Other cell types are now being explored (Natural Killer Cells)

103
Q

What is the future of CRISPR

A
  • better coverage of the genome
  • greatly reduced off-target mutations -safety
  • better development regulations both in NZ and abroad

can you access all the parts of the genome you need to target

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