Lecture 3 - gene editing

definition of gene editing

restriction enzymes for gene editing

So issue it can only use for sites with specific seuqnce

Also sites are ubiquotous - small number of base pairs and repeated often

So possible to get a lot of off target effects

zinc finger nucleases

both restriction enzymes and zinc finger nucleases can be used to

DNA NHEJ

Don’t need to know al this detail, just what they each are and when you would use them etc

homologous recombination

problems with previous tools

what is CRISPR/Cas9

CRISPR discovery

Cas9

causes a double stranded break

programmable - uses guide RNA to find the correct segment of DNA to edit

Cas is resposible for integrating foreign DNA into the genome and cleaving target DNA

PAM acts as a target for the guide RNA - specific sequence of DNA just prior to the matching sequence - allows ediitng every 8 base pairs

drawbacks of CRISPR-cas9

cas12

sticky ended breaks allow us to make complementary overhangs on our DNA insert sequences that are homologous to the sticky end

this tends to favour donor DNA insertion to the cell DNA during repair, leading to more efficient (more likely) knock in

Opsogenetics - opsons are proteins which are ion channels = a way that a cell can respond to light - not expressed in most mammalian cells

So can use crispr to make mammalian neurons to express opsin which usually wouldn’t do this - means can selectively turn neurons on or off just by shining light at them

Means can find stuff out about brains without doing heaps of damage to brains

This is just one example of how this technology can be used

Cas 13

directed against RNA and not DNA

So can change proteins (or more accuratley the instructions for proteins) without permenanrlt changing the host DNA

Cre-Lox conditional knockouts

huntingtons disease - therapy

normal role of huntintin in cells

exising HD treatments

single dose gene replacement therapy for spinal muscular atrophy

SMA is a monogenic ddisorder affecting motor neurons, typical fatal withint 2 years of birth