AMB18 Flashcards
(20 cards)
What are the problems with gene knockout and gene knock down
gene knock out = hard and expensive
gene knockdown = cheap but not always efficient.
what is gene knockout
A gene knockout (abbreviation: KO) is a genetic technique in which one of an organism’s genes is made inoperative (“knocked out” of the organism).
What are the two pathways used to repair double stranded breaks in DNA
homology directed repair - uses homologous donor DNA as template to repair breaks
Non homolgous end joining - does not require donor dna often leads to small insertions or deletions.
How can regions of the genome be deleted or inverted
make two double stranded breaks in cis on a single chromosome, by programmable nuceases, the flanking region cna be ddeleted or inverted
how can translocations be induced
when two double stranded breaks are generated on two different chromosomes
what are zinc finger nucleases
protein domains that form finger like structures which bind specific DNA sequences,
what can zinc fingers be engineered to do?
bind any three bp sequence
how can zinc fingers be used to create a protein that can binds specific DNA sequences
by combiniding multiple fingers
How can you use zinc fingers to cut a target gene sequence
fuse a Fok1 endonuclease to the zinc fingers
what two functional domains make up a zinc finger nuclease
- dna binding domain - two finger modules each recognisesing unique hexamer sequence of DNA - stitched together
- dna cleaving domain comprised of the nuclease domain Fok1, whent he DNA binding and DNA cleaving domains are fused together, a highly specific pair of genomic scissors are created.
what is the specificity of each zinc finger module
around 24bp.b
what is a TALEN
transcription activator like effector nucleases
proteins from plant pathogenic bacteria - containing modular repeats that recognise individual base pairs.
fused to fok1 nuclease and trasnfect target cells/tissues
what can TALEN offer compared to zinc finger nucleasesa nd meganuclease
more versatility and flexibility in targetting a specific DAN sequence, at any location in any given gene
briefly describe the CRISPR/Cas system
bacteria able to recognise and destroy nucleic acids in the cytoplams of viruses plasmids etc
transcripts of clustered interspersed repeat sequences homolgous to viral or plasmid dna are pcoressed into short crRNA sequences that complex with an endonuclease - Cas9
what is crispr
acquired prokaryotic immune system conferring resistance to exogenous seqeunces such as plasmids and phages.
what are RGENs
RNA guided endonucleases
- programmable engineering tools that are adapted from CRISPR/Cas system
Cas9 forms a sequence specific endonuclease when complexed with two RNAs one of which guides target selection (crRNA) and the other is a constant component (tracrRNA)
RGENs recognise a target specific seqeunce that is 23bp in length endng with two guanines. GG
this minimal requirement for target site selection means that RGENs can be designed to cleave any region of the genome (every 8p in theory)
how can new small Cas9 be introduced into the cell
viral mediated transduction as small
what can nuclease dead Cas9 still do
still bind in an RNA dep fashion dCas9
what is sgRNA
single guide RNA - stops the need for two RNA guides
what does fusing an activation domain to cas9 allow
turn it into an RNA guided gene activator
