The Science and Ethics of Genome Editing

Question 1

Briefly describe the required “equipment” for genome editing

Answer 1

Requires:

• molecular scissors (nuclease enzyme to make double stranded cut in DNA)
• homing device to recognise specific DNA sequences (derived from DNA binding proteins)
• template

Question 2

List some methods of genome editing

Answer 2

• zinc finger nucleases
• TALENS (transcription activator like effector nucleases)
• CRISPR-Cas9 (clustered regulatory interspaced short palindromic repeats_ or (RNA-guided genome editing)

Question 3

How does CRISPR-Cas9 work?

Answer 3

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

Question 4

Methods of CRISPR-Cas9

Answer 4

guide RNA + Cas9 + PAM (protospacer adjacent motif)
OR
guide RNA + Cas9
OR
guide RNA + dCAS9 cytosine deaminase

Question 5

Advantages of CRISPR-Cas9

Answer 5

• simple to make
• simple to introduce into cells
• highly specific (DNA can be v. finely modified)
• efficient

Question 6

Uses of genome editing categories?

Answer 6

• research (basic + preclinical)

- clinical (somatic- nonheritable interventions and germline- heritable interventions)

Question 7

Advantages of somatic gene therapy

Answer 7

• corrects defective gene, rather than introducing a construct that could mutate another gene
• appropriate regulation, instead of using promoter sequences in the vector

Question 8

Uses of somatic gene therapy?

Answer 8

• 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)

Question 9

Challenges for somatic gene therapy?

Answer 9

• efficiency
• capacity of viral vectors
• tissue specificity
• toxicity
• immune response
• off target events

Question 10

Methods of potentially heritable genome editing?

Answer 10

• 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

Question 11

What is homology directed repair (HDR)

Answer 11

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]

Question 12

What is non-homologous end joining (NHEJ)

Answer 12

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]

Question 13

How to increase accuracy of homology-directed repair (HDR)?

Answer 13

HDR occurs in late S-G2 phase, whilst NHEJ occurs anytime:

• NHEJ inhibitors
• fuse Cas9 to geminin (DNA replication inhibitor)

Question 14

What is improved gene editing via oviductal nucleic acid delivery (i-GONAD)?

Answer 14

Inject DNA component into oviduct -> electric current given -> it works

Question 15

Advantage of i-GONAD

Answer 15

• easier to do
• simple equipment

(compared to zygote injection)

Question 16

Concerns of i-GONAD

Answer 16

• Genetic changes may be inherited by next generation
• No consent for affected pts
• Interfering w/ nature
• could lead to less acceptance for the disabled

Question 17

Things to consider in the future of genetic enhancement

Answer 17

• safety of methods
• social justice
• understanding views of pts and families
• “rogue clinics” (unsafe, untested methods)