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1. Molecular mechanism of CRISPR/Cas editing1.1.Scientists initially thought that the CRISPR/Cas9 system was similar
to the eukaryotic RNAi silencing mechanism. Explain the crucial
difference between the two.
The crucial difference is that RNAi can induce transient (tijdelijke) knockdown of
gene expression and CRIPR/Cas 9 nuclease can induce permanent damage of the
target DNA
CRISPR/Cas targets DNA and RNAi target RNA. They can both cleave. Both guides
at RNAi and CRISPR/cas are RNA molecules.
1.2.In the context of antisense oligonucleotides you learned about the
enzyme RNase H and its activity in LE 8-9. Explain why the CRISPR/Cas9
enzyme is sometimes referred to as DNase H.
Because RNase H is an enzyme that degrades the RNA strand of RNA-DNA
hybrids and the CRISPR/Cas 9 enzyme uses guide RNA to direct the Cas9
nuclease to a specific DNA sequence and this forms a RNA-DNA hybrid. The cas9
enzyme recognized this hybrid and it cleaves the DNA strand. So the function is
kind of the same as RNase H but RNase H cleaves the RNA strand and Cas 9
cleaves the DNA strand, this is why it is sometimes referred to as DNase H.
1.3.Explain the enormous success of CRISPR/Cas9 technology. Take into
account a comparison with zinc finger nucleases (ZNFs) and TALENs.
Give at least three arguments why CRISPR/Cas9, rather than ZFNs and
TALENs, has become so popular.
- You can use the same enzyme but change the RNA for specificity with
CRISPR/Cas which makes it more cheaper and easier to use.
- It is possible to modify several genomic sites simultaneously with CRISPR
- CRISPR has a higher efficiency and the design is easy for any genomic targets
- It can be used for other purposes (you can use it as a guide)
- easy to use
- Easy to adapt
- More than only a nuclease
- Quick
- Reliable
- Cheap
1.4.The first restriction enzyme was identified in the early 1950s.
Currently >3000 different restriction enzymes are known and >600 of
them are commercially available. Draw a parallel between the activity
, and the development of restriction enzymes and that of the
CRISPR/Cas9 technology.
Include differences and similarities with respect to:
a. recognition site
Restriction enzymes have short and fixed recognition sites, while CRISPR/Cas
9 can be edited to target almost any specific DNA sequence.
b. enzymatic activity
Restriction enzymes only cut at recognition sites and Cas9 can be coupled
with different guide RNAs to target and cut almost any DNA sequence.
c. use and application
Restriction enzymes are mainly used for DNA manipulation in laboratory settings,
whereas CRISPR/Cas9 enables direct manipulation of genomes in living
organisms, opening up new possibilities for gene therapy, disease modeling, and
synthetic biology.
1.5.Study a scheme that illustrates the CRISPR/Cas mechanism and find
out what the PAM sequence is. What is it made of: DNA, RNA or protein?
What does it bind to? What is its function?
PAM is a short DNA sequence containing the sequence NGG. It found 3-4
nucleotides downstream from the cut site. The PAM sequence is adjacent to the
target DNA (protospacer). And the ribonucleoprotein complex binds to the
protospacer so the Cas enzyme can make a precise cut in the DNA. The PAM
sequence plays a part in distinguishing self vs non self and ensures that the
CRISPR locus doesn’t get targeted by Cas enzymes and destroyed. The Cas
enzyme will not be able to bind or cut the target DNA if the PAM sequence isn’t
next to the target sequence.
Where does it bind to?
- The PAM is not hybridizing to anything in the guide. It binds to the enzyme
itself. If you change the enzyme, then it will bind to another PAM. But the
PAM binds to the enzyme itself. It has no covalent bond between the
enzyme and the PAM!! Because then it will be stuck to eachother. It has
electrostatic interaction probably. GGN and then the three basepairs later it
cuts! The PAM has NGG sequence. The PAM is not that specific but the
bacterie has the guide RNA and you need both for the enzyme to be able
to cleave
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