Lec 9 (gene editing) done Flashcards
1
Q
Define gene edited/cisgenic organisms
A
This is when the editing of host genes or the introduction of genes could be achieved by breeding.
-haven’t introduced foreign material, instead the process is sped up naturally using CRISPR/Cas9
2
Q
Genetically modified is aka ?
A
transgenic
3
Q
Define transgenic organisms
A
aka genetically modified
occurs when genes are introduced to a host organism from another species.
4
Q
What main methods can be used to introduce Cas9 into cells
A
4 main methods
- Electroporation
- Plasmid DNA
- Viral methods-also in vivo
- Cell penetrating peptides
5
Q
__________ injected himself with CRISPR in a bid to grow bigger muscles
A
Josiah Zayner injected himself with CRISPR in a bid to grow bigger muscles
6
Q
Josiah Zayner injected himself with _____ to____
A
Josiah Zayner injected himself with CRISPR in a bid to grow bigger muscles
7
Q
Genome engineering toolbox consists of __ main types. What are they
A
4
- Zinc finger protein
- Meganuclease
- TALE
- CRISPR/Cas9
8
Q
T/F there are a lot of commercial applications for the main genome engineering tools
A
T
9
Q
Zinc finger proteins are hard/easy to engineer
A
hard
10
Q
Meganucleases are easy/hard to engineer and have extreme/low specificity
A
hard
extreme
11
Q
TALE tools emerged __ years ago
A
15
around 2005
12
Q
___ tool improved on Zinc finger proteins by
A
TALE
recognised as a single base rather than three
13
Q
CRISPR/Cas9 came around __ years ago. However, the first hints were discovered in __ by___
A
6 2015
first discovered in 1993 by Francisco Mojica (spain) researching extremophiles
looked at bactera in salt marches
14
Q
What exactly did Francisco Mojica find?
And how did other scientists improve on his findings?
A
Found strange repeated regions in the genomeof bacteria.
Several gens of scientists found that these regions are involved in immune defense in bacteria
15
Q
What did Jennifer Doudna do
A
published in vitro data in the journal science about CRISPR/Cas9
also filed patent
16
Q
What did Feng Zhang find out?
A
he was the first to demonstrate genome editing in eukaryotic cells
also filed patent
17
Q
The CRISPR patent war occured between ___ and __
A
Zhang and Doudna
18
Q
What does the ruling of the CRISPR patent war state?
A
there is no interference between the patents, but which should companies license
19
Q
CRISPR/Cas9 stands for
A
Clustered Regularly InterSpaced Palindromic Repeats =sequences of DNA
Cas9 = CRISPR associated protein 9 = that acts as a nuclease to cut the DNA
20
Q
CRISPR/Cas9 protein complex is made up of __ components. What are they?
A
3
- Cas9 = CRISPR associated protein
- crRNA (CRISPR RNA)
- tracrRNA (trans activating CRISPR RNA)
21
Q
The ___ component of the CRISPR/Cas9 protein complex are what guides the Cas9 towards its _______
A
The 2 RNA components of the CRISPR/Cas9 protein complex are what guides the Cas9 towards its genome target
(crRNA and tracrRNA)
22
Q
_________this is the part of the RNA that has complimentary binding to the target genome.
A
crRNA
this is the part of the RNA that has complimentary binding to the target genome.
This associates with a tracrRNA
23
Q
tracrRNA does not bind to the genome it is targetting, instead it_________
A
tracrRNA does not bind to the genome it is targetting, instead it forms a complex with crRNA to stabilise and help it load into the Cas9 protein
24
Q
PAM stands for? and what does it do
A
protospacer adjacent motif
=in the target genome determines if cleavage occurs-Cas9 recognition
25
_____repeats can be read from either direction
the short palindromic repeats can be read from either direction
26
Francisco Mojita found short palindromic repeats, what are these actually?
crRNA = CRISPR RNA sequences that target phage genomes
therefore cas9 can be thought of as a very primitive form of immune system for bacteria
27
What is the CRISPR array?
CRISPR array is where the sequences are loaded
28
What happens when the CRISPR array is expressed?
CRISPR array is expressed and gets loaded into the Cas9 protein
29
In terms of bacteria, what can Cas9 be thought of as?
cas9 can be thought of as a very primitive form of immune system for bacteria
30
What are the functions of other Cas proteins
other cas proteins are involved in creating the crRNA and tracrRNA processing so that they fit into the Cas9 complex.
31
What happens if a virus, that the bacteria has already been exposed to, comes along?
If a phage/virus comes along and infects the bacteria, and there is an element of the phage/virus sequenced in the CRISPR array then CRISPR can target and cut the phage genome so that the virus won't infect the bacteria
32
Is CRISPR an adaptive system? Why/why not?
CRISPR is an adaptive system because
bacteria can keep picking up these crRNA pieces and integrate them into the CRISPR array. Therefore, the array gets longer and longer
33
What happens if the bacteria is exposed to a new virus?
crRNA will take a small piece of the viral genome, put it in the CRISPR array and then will be able to target and cut the virus genome if it invades in the future.
34
___ is a requirement in the targeted genome for cutting to occur
PAM
35
What stops CRISPR/Cas9 from cutting the host genome (suicidal)
PAM sequence
protein/bacteria has evolved to recognize a PAM sequence
36
PAM sequences are not present in the genome of the __ but will be present in __
host but will be present in the genome of the target
37
What are PAM
Protospacer adjacent motif -
short stretch of nucleic acids that define whether or not cutting should occur.
38
If a protospacer adjacent motif is present in the target genome determine what?
if cleavage occurs
i.e whether or not cutting occurs.
39
Why have scientists engineered a single guide RNA that is a combination of tracrRNA and crRNA
So that it is easier to introduce the RNA sequences as 1 sequence.
of a known concentration, ratio etc..
40
What is one of the big breakthroughs/modifications of CRISPR/Cas9 for genome engineering?
the engineering of the single guide RNA by combing the crRNA and tracrRNA
41
What are guide sites?
elements that the crRNA sequences recognize
42
Do all guides work the same? why or why not
not all guides work equally well due to many reasons such as chromatin accessibility etc..
DNA can be compacted- therefore the accessibility can be limited
43
why do we edit/change guides?
so that we can predict which guides may/may not work
44
why do we check guides experimentally
to figure out which guide works best for the required sequence
45
How can we use online applications to choose guide sites?
insert the GOI and the application will show crRNA sequences
each of the crRNA sequence will have a PAM sequence at the 3' end of the guide.
guides are scored out of 100. 100=better and more likely to have on-target activity (more likely to be efficient)
46
in terms of X, briefly describe on-target effects
how well X works at the targeting what you want
47
What can you do after cleavage has occured?
1st and simplest = NHEJ
48
Put the following in order:
1. mediate a cut of the DNA
some mechanisms are shared between bacteria and higher order cells
2. design Cas9
3. introduced guide site into cells
4. got a perfect guide site
1. design Cas9
2. got a perfect guide site
3. introduced guide site into cells
4. mediate a cut of the DNA
some mechanisms are shared between bacteria and higher order cells
49
What can you do after cleavage has occured?
1st and simplest = NHEJ (repair DNA) using random bases)
2. HDR more precise (sequence integrated seamlessly into genome)
3. MMEJ
50
Why can DNA repair occur after cleavage
DNA repair is an evolutionary method of ensuringDNA is intact. if genes are chopped in half then you lose gene function- which would eventually be lethal to the cell
51
disadvantage of NHEJ
a non-template method of DNA repair
puts random bases in the middle of the double-stranded break (gene) which could cause a lot of problems as framshifts, delections ans insertions could occur) and disrupt gene function.
52
Why are NHEJ useful for knocking out a gene
NHEJ add random bases to repair breaks in genes, therefore this may result in frameshift, deletion and/or insertion mutations which would disrupt the gene function and would be easier to knock out from the genome.
53
HDR stands for
homology-directed repair
of DNA
repair construct undergoes HDR in which the new sequence can be integrated seamlessly into the genome (ds-break)
54
What is known as the 'power' of CRISPR/Cas9 ?
HDR
the ability to direct targeted repair to the genome at efficiencies that is useful
55
after cleavage, how can we correct a mutation that was part of the sequence that is now removed due to the ds-break?
If there was a mutation in the genome that is now removed due to the ds-break. We can correct this mutation by inserting a gene that does not contain the mutation. therefore, when this new sequence is integrated into the ds-break, the genome will function as if there is no mutation
56
MMEJ stands for
microhomology-mediated end joining
57
Why is NHEj best for frameshift knockouts?
insertion of a single base => farmeshift of aas => alters entire protein
(non-frameshift) insertion of a single codon=> changes aas => diff aa => diff protein function
58
Why is NHEJ best for frameshift knockouts?
insertion of a single base => farmeshift of aas => alters entire protein
(non-frameshift) insertion of a single codon=> changes aas => diff aa => diff protein function
59
HDR uses ___ to mediate an exchange/insertion of new DNA
strand invasion
60
Briefly describe HDR
homology-directed repair
- high fidelity(precision) repair mechanism (scarless)
- can use a sister chromosome
- Best for Gene insertion
61
What post-cleavage method is used for
1. Gene insertion
2. Frameshift knockout
1. HDR
| 2. NHEJ
62
in HDR, what occurs with a donor template that has a a point mutation?
If we would like to introduce a point mutation into the genome. we have strand invasion by the host genome DNA into the repair construct.
HDR results in a site-specific gene correction or single base-pair change
63
In HDR, what occurs with a donor template that has an insertion
HDR results in targeted gene insertion
64
When desgining the repair template for HDR, there are a few considerations. What are they?
2x homology arms
| gene
65
What is the homology arm, in terms of designing a repair template for HDR?
parts of the DNA that match the target site that we are going to introduce to the insert gene
the arms need to be of a certain length
larger the arms = harder it is to deliver into the cells
66
Very briefly descibe the design variables when designing a repair template
4
1. homology arm length
2. ssODN vs dsODN
3. source and purity of DNA (CpG mods)
4. Selection of edited cells (e.g fluorescent marker)
67
ssODN stands for
single stranded Oligodeoxynucleotide (DNA template)
68
dsODN stands for
double-stranded Oligodeoxynucleotide
69
oligodeoxynucleotide aka __
DNA template
70
HDR/NHEJ is more efficient than HDR/NHEJ and why is this a problem?
NHEJ is more efficient than HDR
Creating a ds-break makes HDR1000x more efficient than without a break
If you have rampant NHEJ you may mediate a scarless insert, however, the majority of the cells will be screwed up .
And may target oncogenes if not careful
because NHEJ= random bases= more uncertain
HDR= template= more certain and less scarless
71
Creating a ____ makes HDR1000x more efficient than without ____
ds-break
a break
72
In terms of efficincies, HDR rates are often ___ , whereas, NHEJ rates are ___.
How can we change this ratio
```
HDR = 1-10 %
NHEJ= 50-90 %
```
Inhibitors and repair template design can influence this ratio
73
There is intense medical interest in improving HDR/NHEJ?
HDR
74
gene edited organisms are ones that could've been bred, but are created using _____ to ____the breeding process.
by
CRISPR/Cas9 could make the ____ that will result in the desired product that would occur naturally over ________
gene edited organisms are ones that could've been bred, but are created using CRISPR/Cas9 to speed up the breeding process.
by
CRISPR/Cas9 could make the point mutations that will result in the desired product that would occur naturally over several gens or several breeding
75
How do the worldwide rules differ on GMOs?
EU-strict ruling, (all mutagenesis techniques make GMOs)
Aus- genes edits =non-GMO
US- allows gene edited crops with a lighter regulatory process than for GMOs
76
In terms of GMOS, what does NZ determine?
NZ first determined gene editing did not always make GMOs but this has been overturned in 2003
77
Briefly describe the pitfalls (dangers) of CRISPR/Cas9?
1. off-target cleavage of the genome
2. PAM NGG limitation
3. How do you get Cas9 into cells?
78
Why are off-target cleavage of the genome, a disadvantage?
if there are mismatches (point mutations that differ from the guide RNA) and these can still be found in the genome
CRISPR/Cas9 can still bind and cut DNA in regions that you don't want it to cut.
79
Why could the PAM requirement be a disadvantage?
PAM requirement
need a GG motif - which occurs once every 16 basepairs.
there are regions in the genome that are AT rich and do not have the PAM motif, therefore, cutting would be hard.
80
How do we detect off-target activity?
2 main methods
1. Low throughput (inverse PCR)
2. High-throughput (DISCOVER -Seq)
81
Describe inverse PCR
use a restriction enzyme to randomly digest the target genome (that was targetted by CRISPR/Cas9)
Then self-ligate the fragments that have been digested out of the genome,
This can be used as a 'landing pad' with primers to sequence this unknown DNA- this tells us where in the genome it is gone.
82
Describe DISCORVER-Seq
use a gene e.g
binding some of the proteins responsible for NHEJ repair in order to pull down DNA sequences and sequence them.
We can sequence DNA exactly where the ds-break occurred and find where all the breaks were made
83
How many sites can be found by DISCOVER-Seq?
a lot
gene VEGFA 417
off-target cleavage sequence
RAPD7 = 191
about 50% of the cleavage detected was in a completely random off-target gene = do not want
84
How do we engineer Cas9 to enhance its specificity?
DNA is extremely negatively charged
The binding site of Cas9 has a lot of Lysine/Arginine residues (+charge). Therefore, the DNA will bind in binding site well.
However, if the DNA binds too well then the specificity requirement is lost.
If there is too much positive charge then any DNA will bind to it and its the frequency that will cut.
85
What is one way
Made changes to the aaseq of the protein
e.g changed a positive residue into an alanine(uncharged)= this reduces the amount of positive interactions with DNA= this increases specificity.
86
protein engineering gives nearly no off-target activity with comparable activity. Explain how this is done
VEGFA gene
Creates a series of guide RNA targets with mismatches and looking at how well each of the cas9s is able to target VEGFA and how much off-target there is cleaving this mismatch
lose a little bit in overall efficiency but you've affectly eliminated all of that off-target activity.
87
Describe Cpf1 and how it is different from Cas9.
An alternate version of Cas9.
The PAM stream is far away from the cut-site. In cas9, the PAM sequence is close to its cut site.
88
Cas 13
RNA guided RNAse
instead of binding DNA, this molecule targets RNA seqs
Therefore, it does not make inherited changes to a genome as the underlying DNA is not changed, however, you could use this to treat a genetic condition e.g
like fixing RNA that is transcribed from the genome without having to create a GMO.
89
alternate version of Cas9 include
Cpf1, Cas13
90
Why is it an advantage to have a cut-site away from the PAM sequence? Give an example of where this is found.
Found in Cpf1
second-chance cutting
You might not disrupt the PAM sequence when you cut, this enables the cpf1 molecule to keep cutting if it doesn't work the first time.
With NHEJ and you don't make too many mutations so the guide can't bind, you get second-chance cutting. Which is one way this is more efficient.
If the PAM seq hasn't been destroyed, cutting can occur again and again
91
Argonaute
A DNA guided DNA editor
retracted cause no one could replicate it
92
Cas9 ___ activity can be removed to make ___
nuclease
'dead' Cas9
93
What is an example of Cas9 fusions
Cas9 fused to a restriction enzyme to mediate cleavage, however, 2 copies of the restriction ezy are required in order for it to cut
Therefore, there is an increased specificity requirement. Thus, you'll only get a cleavage event if both the guide RNAs bind in the required places.
If 1 guide RNA is off-target and only the other binds then the molecule will not cut.
94
What does VP64 do, in terms of Cas9 fusion
it recruits other proteins to activate gene transcription
| upregulate gene expression without creating mutations
95
Give an example of base-editing, in terms of CRISPR/Cas9
Fusing CRISPR/Cas9 with cytidine deaminases
96
CRISPR/Cas9 can be thought of as a blunt tool, it generates these ds-breaks with the competing NHEJ and HDR mechanisms attempting to fix the break.
Scientists have fused CRISPR/Cas9 to these enzymes to make single base pair changes.
cytidine deaminase removes the amine group from cytidine. This mediates a change that converts C to a T.
Therefore, if you have a codon of 3 aas, changing the nucleotides=> change what the codons code for => change protein sequence= > allows a single point mutation to occur.
97
Why is it particularly hard to get Cas9 into mammalian cells compared to other eukaryotic cells?
Because Cas9 has to be transported into the nucleus of the mammalian cells. as well as across the lipid membrane.
98
How does electroporation work and what can this be used for, in terms of CRISPR/Cas9?
Electroporation can be used to introduce Cas9 into cells.
99
How does electroporation work and what can this be used for, in terms of CRISPR/Cas9?
Electroporation can be used to introduce Cas9 into cells.
Electroporators deliver short, sharp pulses of electricity that open transient pores in the membrane and by diffusion the cas9 can move into the cell.
100
How can we use modified viral particles to introduce Cas9 into cells?
e.g Lentivirus, adenovirus
strip awy all the infective and enable the virus to replicate.
insert CRISPR/Cas9 components encoded within the viral particle
-enter cells
101
what is a physical approach to introduce Cas9 into cells?
cell penetrating peptides (normally very charged) migrate across the cell membrane
102
Gene editing plasmid
GFP to fluroresce when Cs9 has been made.
103
Surveyor nuclease (T7) detects on-target effects
detects mismatches between bases.
Eg if a mutation was created and some of popl wasn't edited.
If both are heated, strands are pulled apart, reannealed.
Some of the mutated pieces of DNA that have annealed to the unmutated DNA seq. the Ezy will recognise and cleave = we can observe products on a gel
104
On-target vs off-target effects
on-target = target of interest.
e.g on-target effect of Ritalin = increased dopamine
off-target = unrelated to target of interest
e.g off-target effect of ritalin = increased anxiety
105
Describe the first step of gene editing
1. preparation
- design CRISPR targets
- order primers (next day)
- cell seeding
106
gRNA stands for
guide RNA
107
Describe what occurs in the 2nd day of gene editing
2. make guide RNA (3-4 hours)
- assemble gRNA template
- IVT reaction
- purify guide gRNA
3. Deliver guide RNA and Cas9 to cells (1 hour)
- complex (guideRNA + Cas9 protein)
- Lipid or electroporation
- Incubate 24-48 hours
108
What is the last step in gene editing
4. Analyse editing effciency
| e. g using gel
