Genome Editing and Therapy Flashcards
what are the basic principles of genome editing?
cut and paste mechanism, cutting chunks out and replacing with corrected version of mutations. Or putting mutations in. Or causing a double stranded break
why do we want to use this technology?
model human gene*c disease in animal models, study human
pathways
correct pathogenic mutations in cell lines for therapy and personalised
medicine
improve key organisms for biotechnology
e.g plants, livestock, yeast and bacterial strains
where can Double stranded breaks (DSBs) occur?
occur naturally in the cell
can occur due to reactive oxidising agents or ionising radiation or UV light. These cause DSBs at random postions in the genome.
what is the repair mechanism?
the DSB is nucleated causing resection of the broken strand leaving a 3 prime overhang.
Then have strand invasion where specific proteins bind to the DNA and then allow the DNA to search for homologous region on other chromosome and then this acts as primer for synthesis for broken strand.
Then either have:
classic uses this strand to synth the corrected sequence and you have crossover events called holiday junctions. these are resolved using enzyme called resolvase and nicked together using ligase and you have repaired strand.
or
Synthesis dependent best strand annealing: get invasion of the broken strand whichthen synthesise against homologous chromosome and then is returned to orignial broken part of genome and acts as template for other part which is missing
Describe classic double stranded break repair (DSBR)
DSB: DNA damaging agents
Resection: nuclease degradtation leaving single stranded 3 prime tails overhand
Homology searching strand: RAD51 (recombination protein) when it finds an area of homology, it displaces the part of the DNA and causes the structure known as D-loop.
D-Loop: invading strand forms a loop and acts as a primer for DNA synthesis.
cross over events during recombination (holliday junction) which are the resolved using resolvase and ligated into place and you have a corrected strand
Describe synthesis-dependant stand annealing pathway
you have the resection, the homolgy searching event, the D-loop. But the synthesised strand return to the OG parent strand and acts a template to fill in the rest. No cross over events
what happens when no homologous dna can be found?
Non homologous end joining (NHEJ)
what is NHEJ?
A much quicker repair mechanism than other two but is open to more errors - imperfect repair mechanism
Get break
Complex known as DNA PK complex which attracts certain ligases and bring DSB together.
Often had addition of nucleotides as imperfect repair. USED to advantage when trying to creat mutant animal model - usually get 3-6 more nucleotides usually causing out of frame mutation or stop codon.
How many genome editing techniques are there?
4 ``
what is a meganuclease (MNs)?
Genome editing tool
Endodeoxyribonucleases, like restriction enzymes that target large sequences.
characterized by a large recognition site (double-stranded DNA sequences of 12 to 40 base pairs); as a result this site generally occurs only once in any given genome. For example, the 18-base pair sequence recognized by the I-SceI meganuclease would on average require a genome twenty times the size of the human genome to be found once by chance (although sequences with a single mismatch occur about three times per human-sized genome).
examples of MNs?
I-SceI (baker’s yeast), I-CreI(green algae), I-DmoI(Archaebacteria)
I-Scel, 18 bp Recognition. Site.
what do we want from an endonuclease?
Specific recognition of long target sequences (ideally one per genome)
Adaptability for retargeting to other genomic loci
what are zinc finger nucleases (ZFNs)
ZFs are DNA binding domains, these work by having a ZF array which recognises a certain strand of the DNA, this is fused to a endonuclease known as FokI.
FokI only cuts when its a heterodimer so you have to have it forming a complex together to make it cut.
To develop these zfn need two sets of zfn’s which target either side of the region of interest. In such that the targetting region will allow the two portions of the endonucelase to interact and cut at a specific region
explain zinc fingers
each fingerhas 30aa and each zf will recognise 3 bps
each zf can be designed to be specific to a triplet code and can build array of ZF to target certain areas.
con of ZFN?
making them is expensive, complex and can have inaccurate cleavage (domain interaction).
what are transcription activator-like effector nucleases (TALENs)?
originate from a plant pathogenic bacteria - xanthomonas
these act in these bacteria by targeting promoter region in the plant which activate certain genes which enables them to be infected by the bacteria.
TALENs are an array of aa which will target single base pairs. Diff from ZFN which target triplets. This makes its easier to target certain gene of interest bc can design in way where you can pick nucelotides rather than aa which identify certain triplets.
Each TALEN consist of 35aa
use FokI domain, fused to the TALEN array. Use it as a pair in order for it to cut. FokI heterodimerises and then causes cleavage in DNA.
Variaons at
aa
12 + 13 confers specificity to nucleodes.
Con of TALEN
Much larger than ZFNs (3kbVs1Kb), harder to deliver
off-targe concern
what is CRISPR/Cas9 System
Clustered regularly interspaced short palindromic repeats.
Works in collaboration with another endonuclease known as Cas9.
Originiate as a bacterial adaptive immune system, used to chop up viral infection. Intergrates portion of viral dna into the CRISPR setup and the bacteria can then target and chop viral DNA up.
Has now been adapted to cause DSBs.
Starts with guide RNA. Guide RNA consist of proto-spacer (specific to region of interest, however region you pick has to have PAM sequence next to it within the genome)
PAM (protospacer adjacent motif) seq consists of a nucleotide (any) and GG.
PAM seq occur every 8 bp’s.
Guide RNA has targeted seq, which is complimentary to the genomic are you are interested in but also has architectural DNA for binding of Cas9.
Then you intoduce guide rna and cas9 endonuclease. Guide rna binds to its target region, then cas9 binds to the protospace portion and then also bings to targetted region and sacffolding part of guide rna - causing a DSB 3 bps upstream from the PAM seq.
what is duchenne muscular dystrophy?
Most common sever form of childhood muscular dystrophy.
Mutated gene: dystrophin
Dystrophin is req for muscle integrity. Links the cytoskleton up to the sarcolemma in the muscle fiber. When not present, muscle integrity is lost.
Can effect skeletal and cardiac muscle.
By age 10-12 children unable to walk, die in 20s due to heart failure
Current treatments of DMD?
Corticosteroids (side effects),
morpholino based exon skipping - target gene specific translation or splice site (preventing slicing or causing exon skipping)
By skipping specific exon, get shorter protein, allows in-frame splicing of exon allowing for functional dystrophin protein.
Gene therapy diffuclt bc dystrophin is large protein. Difficult for viral delivery.
problem w morpholino?
Have to keep giving which increases chance of off-targetness, also has toxicity
explain mouse model for treatment of DMD?
Used a strain of mouse hwihc has a gene which has a ubiqitous promoter driving a red fluorexcent protein which has a stop casette at the begining (doesnt allow rfp to be produced), then use guide rna against stop cassette.
what experiments were conducted?
Tabebordbar et al 2016 :
Transfection of constructs into dystrophic satellite cells (in vitro)
AAV-CRISPR excises E23 + restores Dystrophin
expression (in vivo)
what are the ehtical concerns?
Balance of risks Vs
benefits
Ecological disequilibrium
Regula*on for consumers
Applica*on of CRISPR/Cas9 technique to human
germline
(inheritable changes)
Whose liable if things go wrong?
Genome edi*ng for enhancement: who gets this, rich? Social divide?
Formaon of animal chimeras for transplantaon
why is genome editing important?
can manipulate gene to correct genetic abnormatlities
shows us the mechanism of action for disorders in animal models
what natural processes repair DSB?
NEHJ
HDR
ETC
why is CRISPR/Cas9 favoured
less off target effects in animal models but still unclear
easy to create - dont have to create large array of aa
efficient at targeting gene of interest
cheap
what is the major conncern about genome editing techniques?
off-target effects
whether it is used in germ line cells - may cause off target effects which may not be the same within in generateions and may introduce cancers etc.
ethical issues - misuse
