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 cell’s 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 cell’s 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.

Meganucleaseadvantages 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 domains1. DNA-binding Domain2. 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.