gene editing Flashcards
basic principle
cause cuts in dna and inset certain dna
why do we went to edit genomes
to study disease
aka model human genetic disease in animal
correct pathogenic mutations in cell lines and personalised medicine
improve organisms for biotech
DSB associated with HDR
break causes resection where 5’ end is nibbled back to leave 3’ over hand
rpa is attracted and turns it into homology searching arm. dloop formed
and nicked dna is ligated
2nd one is dependant strand annealing it uses sister chromatin to act as temploate
NHEJ
erorr prone
but quick
hetero dimer recruited to dsb
which recruits dna pk which attracts ligase to join double stranded ends
Meganucleases
MNs
restriction enzymes
recognise seq once for a genome 20x size of human
can tolerate 1 or 2 mis matches
can insert DNA by HR or mutations by NHEJ
difficult to specifically target
what do we need from endo nuclease
specific recognition of long target sequences
adaptability for retargiting to other genomic loci
ZINC finger nucleases
ZFs are DNA binding domains
each finger is 30aa and recognises 3 bps
an array of zf domains recognises unique sequences
zf are fused to fokl
fokl needs to dimerise to cut; ZFN come as pairs for specificity
complex, expensive and can have inaccurate cleavage
Transcription activator-like effector nucleases
recognise certain genes which help bacteria invade
each tail domains recognise single nucleotide
easier to design and larger than ZFN, hard to deliver
off target a concern
Crispr/cas 9
guide RNA binds to DNA and also to cas9 which finds sequence and causes cut
each signance needs a PAM sequence next to it
Target sequence recognises and cut made upstream of PAM
duchenne muscular dystrophy
DMD most comon form of dytrophy
skeletal _ cardiac
unable to walk - early desath
current treatments ; corticosteroids
gene therapy difficult,
startgy to fix DMD
NONsense mutation in Exon 23 and cut it it out and NHEJ
Very efective but
repeat needed
poor tissue uptake and toxicity
