Genome editing

Genome editing,
or genome editing with
engineered
nucleases (GEEN) is a
type of genetic
engineering in
which DNA is inserted,
replaced, or removed from
a genome using artificially
engineered nucleases, or
"molecular scissors."
Mechanism
The nucleases create specific double-
stranded break (DSBs) at desired locations in
the genome, and harness the cells
endogenous mechanisms to repair the
induced break by natural processes of :
homologous recombination (HR)
nonhomologous end-joining (NHEJ).
So based on these principles if one is able
to create a DSB at a specific location
within the genome, then the cells own
repair systems will help in creating the
desired mutations.
Site-specific double stranded
breaks

if genomic DNA is treated with a particular
restriction endonuclease many DSBs will be
created.

To overcome this challenge and create site-
specific DSB, three distinct classes of nucleases
have been discovered and bioengineered to
date:

Zinc finger nucleases (ZFNs),
Transcription-Activator like Effector Nucleases
(TALENs)
Meganucleases
Meganucleases
Meganucleases, found commonly in microbial
species, have the unique property of having very
long recognition sequences (>14bp) thus making
them naturally very specific.

But the challenge is that: not enough
meganucleases are known, or may ever be
known, to cover all possible target sequences.

Mutagenesis and high throughput screening
methods have been used to create
meganuclease variants that recognize unique
sequences.

Meganuclease
advantages and disadvantages.
very specific

the construction of sequence specific
enzymes for all possible sequences is
costly and time consuming.
As opposed to meganucleases, the concept
behind ZFNs and TALENs is more based on a
non-specific DNA cutting enzyme which would
then be linked to specific DNA sequence
recognizing peptides such as:

zinc fingers and
transcription activator-like effectors (TALEs)
The key to this was to find an endonuclease
whose DNA recognition site and cleaving
site were separate from each other, a
situation that is not common among
restriction enzymes.

A restriction enzyme with such properties
is FokI.
Zinc-Finger Nucleases
What is a Zinc Finger ?

In eukaryotes, often complex sets of
regulatory
elements control the initiation of
transcription
of genes. Upstream of the RNA
polymerase II initiation site there are
different
combinations of specific DNA sequences,
each of
which is recognized by a corresponding
site-specific DNA-binding protein. These
protein
are called transcription factor.

Transcription factors have two functionally
different domains, one that binds to specific
DNA
sequences and another that activates
transcription. And now NMR methods recently
have
been used to determine the 3D structure of
these
motifs zinc fingers, leucine zippers, and
helix-turn-helix motifs.

The zinc finger motif was first
described in 1985 in the laboratory of Aaron
Klug
at the MRC laboratory of Molecular Biology in
Cambridge, where it was inferred from an
analysis
of the amino acid sequence of the transcription
factor TFIIIA from Xenopus laevis.

Zinc finger domains
Large superfamily of protein domains
Characterised by 2 anti parallel b sheets and 1 a helix
Structure stabilised by binding of Zinc ion
Zinc binding mediated by specific cysteine (b sheets) and histidine
(a helix) residues

N terminal
C terminal
Zn
Role of zinc finger domains
Zinc finger domains make multiple contacts on target
molecule

Can bind to DNA, RNA, protein and/or lipids

Binding properties depend on specific type of zinc finger,
no of fingers and sequences therein

Versatility in binding results in specialised functions
including gene transcription, translation, mRNA
trafficking, cytoskeletal organisation and chromatin
remodelling.

Over 70 different classes of zinc finger domains
recognised

The structure of the zinc finger motif is formed by
coordination of the zinc(II) ion.


Zinc-Finger Nucleases have two domains
1. DNA-binding Domain
2. DNA-cleavage Domain

Chandrasegaran proved that cutting could be redirected by
substituting alternative recognition domain.

Three fingers of a ZF bound to DNA showed that each finger
contacts 3 bp of DNA in a remarkable fashion.

This suggest many different sequences can be attacked by
making novel assemblies of ZFs.
FokI enzyme
The enzyme FokI, naturally found
in Flavobacterium okeanokoites, is a bacterial type
IISrestriction endonuclease consisting of:

FokI restriction endonuclease recognizes the non-palindromic
penta-deoxyribonucleotide 5-GGATG-3:5-CATCC-3 in
duplex DNA and cleaves 9/13 nucleotides downstream of the
recognition site.

Synthetic derived from Fok I a Type II Restriction enzyme
which has physically separable binding and cleavage
activities.

an N-terminal DNA-binding domain and
a non-specific DNA cleavage domain at the C-terminal.
FokI dimerization
FokI has the advantage of requiring
dimerization to have nuclease activity and
this means the specificity increases
dramatically as each nuclease partner would
recognize a unique DNA sequence.
Mre11/Rad50/Nbs1 complex
Strand invasive protein Rad 51
DNA Polymerase
DNA Ligase

Chromosomal breaks are detected in cells as
potentially lethal damage, and one natural
pathway of DSB repair is copying from a
homologous template.

From this perspective, DSB-stimulated gene
targeting simply provides an exogenous template
for a natural repair process.

An alternative repair pathway for DSBs,
nonhomologous end joining, often joins the
broken ends inaccurately, creating deletions,
insertions, and substitutions at the break site.