Hot topic - Crispr/CAS9

Question 1

Where did the CRIPSR/Cas9 system originate?

Answer 1

It is a prokaryotic immune defence response against invading bacteriaphage

Question 2

How does it work?

Answer 2

The CRISPR system is modified for genome editing in humans.
Cas9 is bound to a RNA targeting sequence, used to direct the nuclease activity of the Cas9 to the site of interest.

Cas9 and RNA are incorporated into a plasmid vector and delivered to the site of action.

Cas9 nicks the DNA (depending on the Cas9 used, this may be dsDNA or ssDNA nick) ready for a gene to be removed or added.

Subsequent repair of the site is carried out by NHEJ or HRR.

dead Caspases, without nuclease activity, can also be used to target sequence or epigenetic modification; promoters can be targeted to turn genes on/off.

Question 3

Why is it controversial?

Answer 3

Any changes may be introduced into the germline and become heritable.

There is a current moratorium preventing the use of this system for germline editing.

Question 4

Give examples of how this system is showing promise for treatment of rare disease.

Answer 4

In cell lines it has been demonstrated to turn methylated genes on. This may be useful for FrAX treatment or Angelman syndrome to increase expression of UBE3A from the paternal allele.
It has been shown to correct a specific DMD mutation and restore function
It has also been shown to raise FXN levels in Friedriech’s Ataxia patients.

Question 5

What is the main issue with CRISPR?

Answer 5

Delivery of gene editing components to target sites is still difficult, and studies have demonstrated off-target effects.