basic principle of editing
addition or remove of a part of the genome so that it can serve a desired purpose
why do we want to edit genes?
- model human genetic disease in animal models, study human pathways
- correct pathogenic mutations in cell lines for therapy and personalised medicine
- improve key organisms for biotech
what are ds breaks associated with?
homologous dependent repair
proceess of fixing a ds break
ds break > resection > strand invasion, D loop
classical double stranded break repair
- ds break; damaging agents
- resection; nuclease degradation ss 3’ tails
- homology searching; RAD51
- D-loop; invading strand forms a loop, acts as a primer for DNA synthesis
- Holliday junction; intercrossing DNA during recombination.
synthesis-dependent strand-annealing pathway
- no cross over events
- newly synthesised strand displaced from template and returns to processed end of non invading strand
non-homologous end joining
- primary pathway for repair of a ds break though cell cycle
- relies on Ku protein to thread onto each broken DNA end
meganucleases (MNs)
- endodeoxyrubonucleases.
- predicted to recognise idential sequence once for a genome 20x size of the human genome
- can tolerate 1 or 2 MMs
- can insert DNA by HR or mutations by NHEJ
- difficult to specifically target.
zinc finger nucleases (ZFNs)
- ZFs are DNA binding domains
- each finger (30aa) recognises 3bps
- an array of zf domains; recognises unique genomic sequences
- ZF use on Fokl domain; Fokl needs to dimerise to cut, ZFN come as pairs
- complex, expensive, can have innacurate cleavage (domain interaction)
what do we want from an endonuclease?
- specific recognition of long target sequences (ideally one per genome)
- adaptability for retargeting to other genomic loci
TALENS
transcription activator-like effector nucleases
what are TALE proteins?
xanthomonas
plant pathogenic bacteria
what are DNA binding domais of TALEN?
series of tandem repeats ; 33-35aa
TALEN vs ZFN
- much easier to design than ZFN, fewer targeting constraints
- much larger than ZFNs, harder to deliver
variations of TALEN
aa 12+13 confers specificity nucleotides
CRISPR/Cas9 system
- clustered regularly interspaced short palindromic repeats
- protospacer; target sequence of guide RNA
- guide RNA binds to strand of genomic DNA, Cas9 endonuclease binds to non protospacer portion of gRNA + PAM of DNA, DSB 3bp upstream of PAM
what is duchenne muscular dystrophy (DMD)
most common severe form of childhood muscular dystrophy
mutate gene; dystrophin
what happens in DMD?
-affects skeletal + cardiac
- unable to walk by 10-12 ys, death by early to mid 20s (heart failure)
current treatments of DMD
corticosteroids (side effects)
morpholino based exon skipping (prolonged ambulation)
- gene therapy is difficult; dystrophin 11kb (AAV)
targeting specific genes with CRISPR
shown effective in antisense oligonucleotides
but; repeat administration, poor tissue uptake, toxicity
in vivo techniques of treating DMD
AAV+ CRISPR; excises E23
= restoration of dystrophin expression
gene edited babies
C-C chemokines receptor type 5 (CCR5) ; chemokine receptor involved in immune response
what is CCR5 linked to?
- main HIV coreceptor, involved in viral entry and cell-to-cell spread
- CCR5 genetic polymorphoisms associated with HIV-resistance
things to consider with gene editing
- risks vs benefits
- ecological disequilibirium
- regulation for consumers
- application of CRISPR/Cas9 technique to human germline (inheritable changes)
- libaility if things go wrong on who?
- genome editing for enhancements goes to who?
- formation of animal chimeras for transplantation
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lecture 1: genes & genomes35
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Studying gene expression II11
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regulation of transcription factors I33
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regulation of transcription factor II23
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RNA interference and its applications40
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Gene editing24
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Mechanism & regulation of translation I40
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Mechanism and regulation of translation II54
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DNA repair I28
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DNA repair II14
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V(D)J recombination11
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DNA transposition10
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relevance of transcription11
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basal transcriptional machinery20
