Flashcards in The Science and Ethics of Genome Editing Deck (17)
Briefly describe the required "equipment" for genome editing
- molecular scissors (nuclease enzyme to make double stranded cut in DNA)
- homing device to recognise specific DNA sequences (derived from DNA binding proteins)
List some methods of genome editing
- zinc finger nucleases
- TALENS (transcription activator like effector nucleases)
- CRISPR-Cas9 (clustered regulatory interspaced short palindromic repeats_ or (RNA-guided genome editing)
How does CRISPR-Cas9 work?
Cas9 is an enzyme acting as "molecular scissors"
Guide RNA aids the targeting of Cas9
After Cas9's guided cut, the cells recognises the DNA damage and tries to repair it- this is where DNA repair machinery introduces gene changes
Methods of CRISPR-Cas9
guide RNA + Cas9 + PAM (protospacer adjacent motif)
guide RNA + Cas9
guide RNA + dCAS9 cytosine deaminase
Advantages of CRISPR-Cas9
- simple to make
- simple to introduce into cells
- highly specific (DNA can be v. finely modified)
Uses of genome editing categories?
-research (basic + preclinical)
- clinical (somatic- nonheritable interventions and germline- heritable interventions)
Advantages of somatic gene therapy
- corrects defective gene, rather than introducing a construct that could mutate another gene
- appropriate regulation, instead of using promoter sequences in the vector
Uses of somatic gene therapy?
- edit cells ex vivo and reinsert them (e.g. blood cells for cancer treatment, sickle disease)
- edit cells in vivo w/ viral or particle delivery (e.g. liver cells for metabolic diseases, muscle cells for muscular dystrophy)
Challenges for somatic gene therapy?
- capacity of viral vectors
- tissue specificity
- immune response
- off target events
Methods of potentially heritable genome editing?
- edit cells that give rise to sperm OR via iPS cells and in vitro derived gametes to eggs or sperm ]- allows verification of edits
- edit fertilised egg (zygote)]- difficult to verify edit
What is homology directed repair (HDR)
a mechanism in cells to repair double strand DNA lesions-most common form is homologous recombination
[NB: can only be done if homologous DNA is present in nucleus]
What is non-homologous end joining (NHEJ)
Also repaird d-stranded DNA lesions, but here the break ends are directly ligated without needing a template
[NB: typically uses microhomologies to guide repair]
How to increase accuracy of homology-directed repair (HDR)?
HDR occurs in late S-G2 phase, whilst NHEJ occurs anytime:
- NHEJ inhibitors
- fuse Cas9 to geminin (DNA replication inhibitor)
What is improved gene editing via oviductal nucleic acid delivery (i-GONAD)?
Inject DNA component into oviduct -> electric current given -> it works
Advantage of i-GONAD
- easier to do
- simple equipment
(compared to zygote injection)
Concerns of i-GONAD
- Genetic changes may be inherited by next generation
- No consent for affected pts
- Interfering w/ nature
- could lead to less acceptance for the disabled