What are the components of a Zinc Finger Nuclease?
Endonuclease (DNA cutting) domain, DNA recognition domain
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Where is cleavage?
asymmetric sequences adjacent to recognition site
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What is cut as a result?
Non-specific DNA
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How do ZFNs recognise specific DNA?
Use 3 x Zinc finger motifs that recognise 3 bps each - link to nuclease
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What cuts do ZFN produce?
4bp 5' overhang
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Disadvantage of ZFN?
Difficult to make, difficult to access
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What does TALEN stand for?
TALE Nuclease
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What are TALEs?
Bacterial transcription factors secreted during plant infection
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What do TALEs contain?
TAL repeats
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What do TAL repeats recognise?
Each TAL recognises 1 bp
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How do you create a TALEN?
Link multiple TALEs to a nuclease
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How many TALs can you have?
Up to about 24 - 24bp run is probably unique in a genome
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Are TALENs good?
Work very well, but are difficult to make and use
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What did CRISPR-Cas evolve as?
A bacterial immune system against bacteriophage
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How does CRISPR-Cas work as an immune system?
Bacteriophage DNA taken up into genome as a spacer, seperated by repeats, which altogether form a CRISPR array. The array is transcribed to form pre-cRNA, and then CRISPR effector nucleases. CRISPR nucleases recognise and cleave foreign DNA
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What are Class 1 CRISPR systems?
Protein complex effector nuclease
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What are Class 2 CRISPR systems?
Single protein effector nuclease
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What is more desirable?
Class 2 - simplicity is easier to work with
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What is an example of a Class 2 Type II system?
Cas9
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What is an example of a Class 2 Type V system?
Cas12
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What is the PROTOSPACER?
Sequence on target DNA which is homologous to CRISPR spacer
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What is the PAM?
Protospacer Adjacent Motif - Short sequence adjacent to the protospacer that is unique to the particular Cas9
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What is Strep p PAM?
5' - NGG - 3'
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What are the components of the Cas9 system?
tracrRNA - constant, binds to repeat sequence of crRNA, there is also the spacer part of the crRNA
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What happens?
Cas9 recognises PAM, if protospacer and spacer are same, DNA is unwound, R-loop is formed when RNA bound into dsDNA, then Cas9 blunt end cuts the DNA. simple!
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What are the domains of the Cas9 protein?
RuvC, HNH, Helical lobe, CTD
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What are the nuclease domains?
RuvC and HNH
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What is a gRNA?
guide RNA - tracrRNA and crRNA sequences joined together
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