Zinc Finger Nucleases
Zinc finger nucleases were
perhaps need to look further back to what other enzymes cut DNA sequences
specific DNA sequences and whether we could modify them or not so obviously
restriction enzymes exist in lots of prokaryotes and we have a large catalog
of these enzymes which will cut a specific sequence and the idea was to try and
modify those enzymes so by understanding them created crystal structures of
them doing lots of mutagenesis on them potentially we can take an enzyme that
cuts sequence X and persuade it to cut sequence Y specifically well actually
most restriction enzymes cannot be readily adapted to cleave new sequences.
Fok 1
in general, have got very short recognition
sequences which are not helpful that they'll cut many times in the genome and
are not cut to unique sequences and when you persuade them to cut another
sequence that cuts them very weakly so really that wasn't a route to develop a
genome editing however there's a class of enzymes the type 2's restriction
enzymes. which are quite interesting an example is the enzyme Fok 1
and these type 2's restriction enzymes have separate cleavage and DNA
recognition domains and importantly the cleavage domain has no specificity and
can work on its own it just needs to be recruited to the DNA so for example
here in the case of huaquan which functions as a dimer that's an important
feature of Fok 1 is a dimeric protein so two molecules of the same protein
in green and in blue here and this domain is the DNA recognition domain thought
bond binds this particular C once GG 80g and then the cleavage domain is here
and a key feature with this type to s restriction assumptions they tend to bind
in one place and cut a specific distance away and the sequence that they cut it
doesn't matter it can cut out all the sequences here so type 2’s reduce
restriction enzymes are really useful in synthetic biology for lots of
different techniques but in the case of genome editing we can take this
cleavage domain which will only function as a dimer and add it on to another
DNA binding domain so we've got the cleavage parts we need the DNA binding part
and the concept was to fuse this catalytic domain of Fok 1 on to a different
DNA recognition domain and when you look through the genomes of mammals.
Zinc finger domain
the most common DNA binding
domain out there is the Zinc finger domain so, for example, there are nearly 1500 human genes that have got zinc finger motifs and there's a
large family of genes that contain a certain kind of zinc finger called the sis
to hiss to us or C to H to Zinc finger domain and simple Beta
Beta Alpha fold and follow a very common amino acid motif so these were
discovered in 1985 and Zinc finger domains were heavily
studied in the 1990s and from this very substantial amount of work a DNA
recognition come up code emerged from comparing the protein amino acid sequence of Zinc
finger domains and DNA sequence
that they specifically bound to so if we look here I'm here's a crystal
structure of zinc finger proteins bound to DNA and so they bind to normal B DNA
and a Zinc finger domains interdigitate or into the major groove of
DNA.
Formation of zinc finger
nucleases
Zinc finger domains have an alpha
helix and two beta-sheets here and it's all held together with a molecule of
zinc hence the name zinc finger and then there are these specific residues on
the Alpha helix and on this beta tone here which are the ones which bind to DNA
and depending on which amino acid in which position in this triplet will determine which nucleotide is bound and
proteins would bind to a sequence of
interest and indeed this is possible can
then create zinc finger nucleases. so zinc finger nucleases are where you've
got an array of zinc finger proteins usually three or four each zinc finger
binds to three bases so in the case of four zinc fingers is by mister twelve
bases.
Types of
domains
·
Falkland domain
·
Fuckwad domain
fuckwad only functions as a dimer
so you need to make two of these proteins have to assemble two proteins so are
forcing fingers with a one cleavage domain at the c-terminus and Fok 1
needs six bases of space to bind to and cleave so your target sites are these
12 bases there's a gap and then these 12 bases and there are tools to help you
design these so key features of zinc finger nucleases is that the cleavage
domain has no sequence specificity so you can cut what whatever you want to you
do need to make two zfn proteins but this does increase sequence specificity so
you've now got twenty basis of specificity which is pretty good there are
mutants of the Falkland domain that were developed which are obligate heterodimers
so there's a left fuckwad mutant and there's a right well mutant and what this
means is that if one of these let's say this left ZF Xenophon protein bound at
an off-target site through its 12 bases it wouldn't be able to homodimer eyes
with itself and therefore cut that off-target site so by creating a look at
heterodimers will really restrict the possibility of
Xenophon's cutting off target.
Off-target activity
Due to some Off-target activity but
it's greatly reduced the four times in finger domains is about as good as it
gets longer race generally don't work so you're looking at 24 bases of
specificity at best assembling zinc finger arrays now is quite straightforward
because there are wonderful synthetic biology tools out there to join fragments
of DNA together quickly in cells, the real problem with zinc-finger nucleases is
the vast majority of Zinc finger domains do not function
very well when assembled together in these arrays but for reasons that are not
fully understood so the best approach is to take a sort of mass action approach.
Conclusion
it's a superb tool for genome
editing there's nothing fundamentally wrong with zinc-finger nucleases it's
just that they're difficult to make if you don't have one of these huge
assembly platforms available to you a key advantage of zinc finger nucleases over
all the methods that the fans are quite small so they're much easier to deliver
into cells and tissues than some of the more advanced tools other use later on
so they certainly do have their place in genome editing although you'll find in
the modern era, not many people use them so cell phones were first successfully
used for genome editing in 2003 they've been used in many species for a very
wide variety vadik editing applications and a lot of the genome editing
strategies and approaches that we know news today were developed using
zetas fans at first so some of the foundation papers that are out there all use
that offends so another reason for knowing about them there are over 20 zfn
based and genetic therapies that are going through different stages of clinical
trials so they have their place but they were slow to make there was a low
chance of them functioning well particularly when created in in in most
standard research laboratories and a big breakthrough came in 2009 when the DNA
recognition code of a different protein domain family was solved and that leads
us on to learning about talyn's in the next step.
