Genome Editing and Therapy

Question 1

what are the basic principles of genome editing?

Answer 1

cut and paste mechanism, cutting chunks out and replacing with corrected version of mutations. Or putting mutations in. Or causing a double stranded break

Question 2

why do we want to use this technology?

Answer 2

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

Question 3

where can Double stranded breaks (DSBs) occur?

Answer 3

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.

Question 4

what is the repair mechanism?

Answer 4

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

Question 5

Describe classic double stranded break repair (DSBR)

Answer 5

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

Question 6

Describe synthesis-dependant stand annealing pathway

Answer 6

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

Question 7

what happens when no homologous dna can be found?

Answer 7

Non homologous end joining (NHEJ)

Question 8

what is NHEJ?

Answer 8

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.

Question 9

How many genome editing techniques are there?

Answer 9

4 ``

Question 10

what is a meganuclease (MNs)?

Answer 10

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).

Question 11

examples of MNs?

Answer 11

I-SceI (baker’s yeast), I-CreI(green algae), I-DmoI(Archaebacteria)

I-Scel, 18 bp Recognition. Site.

Question 12

what do we want from an endonuclease?

Answer 12

Specific recognition of long target sequences (ideally one per genome)

Adaptability for retargeting to other genomic loci

Question 13

what are zinc finger nucleases (ZFNs)

Answer 13

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

Question 14

explain zinc fingers

Answer 14

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.

Question 15

con of ZFN?

Answer 15

making them is expensive, complex and can have inaccurate cleavage (domain interaction).

Question 16

what are transcription activator-like effector nucleases (TALENs)?

Answer 16

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.

Question 17

Con of TALEN

Answer 17

Much larger than ZFNs (3kbVs1Kb), harder to deliver

off-targe concern

Question 18

what is CRISPR/Cas9 System

Answer 18

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.

Question 19

what is duchenne muscular dystrophy?

Answer 19

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

Question 20

Current treatments of DMD?

Answer 20

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.

Question 21

problem w morpholino?

Answer 21

Have to keep giving which increases chance of off-targetness, also has toxicity

Question 22

explain mouse model for treatment of DMD?

Answer 22

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.

Question 23

what experiments were conducted?

Answer 23

Tabebordbar et al 2016 :
Transfection of constructs into dystrophic satellite cells (in vitro)

AAV-CRISPR excises E23 + restores Dystrophin
expression (in vivo)

Question 24

what are the ehtical concerns?

Answer 24

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

Question 25

why is genome editing important?

Answer 25

can manipulate gene to correct genetic abnormatlities

shows us the mechanism of action for disorders in animal models

Question 26

what natural processes repair DSB?

Answer 26

NEHJ
HDR
ETC

Question 27

why is CRISPR/Cas9 favoured

Answer 27

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

Question 28

what is the major conncern about genome editing techniques?

Answer 28

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