Nuclease mediated genome engineering tools

Zinc Finger Nucleases (ZFNs), Transcription Like Effector Nucleases (TALENs), Clustered Regularly Interspaced Short Palidromic Repeats (CRISPR/CRISPR- Cas) are the useful tools of genome editing. They function via utilizing target- specific nucleases.

Two main components of these tools are the targetable nucleases in order to create Double strand Breaks (DSBs) and a DSB repair mechanism to reconnect separated DNA parts, with the addition exogeneous DNA fragment.

Two options for DNA repairing are Non-homologous End Joining (NHEJ) and Homology Directed Repair (HDR). Exogeneous genes connected by means of NHEJ repair generally gain deletion or/and insertion mutations. NHEJ is known to be error-prone as HDR is not, however HDR needs a repair template. This problem of HDR is overcome by co-introduction of exogeneous repair donors with target specific nucleases. NHEJ repair system functions regarless of phases of cell cycle in opposition to HDR which majorly works at S/G2 phase.

Because DSB repair machinaries can be encouraged in most of the organisms, target specific nuclease- mediated genome engineering is able to be applied in animals and plants.

ZFNs
ZFNs are artificial structures that are the combination of a restriction enzyme FokI domain and a C2H2 zinc-finger (ZF) DNA-binding domain.

In past, scientist observed the ability of ZF domain to interract and bind to a variety of nucleotide sequence so they engineered this domain for tagetting user defined site in the genome.

Main characteristics of ZF domain originate from a zinc ion coordinated by two cystein residue, located on two antiparallel β-sheets, and two histidine residues on the α-helix. In nature, the ZF domain is primarily responsible for connection with DNA, each finger is capable of detecting a 3 nucleotide unit, called target triplet, by means of its α- helix, thus named also recognition helix. Linkage to DNA only occurs at single strand. A 3 finger ZF can detect 9 base, at total. Furthermore, ZFNs basically works as dimers so recognition capacity at total is 18 base. In human genome, 18 base is a satisfactory length to specifically target a single site on the genome.

It is possible to observe unintended interractions between some parts of ZFNs and DNA. Another problem is that each finger strongly prefer to connect to GNN triplet. These issues harden design and limits target site repertoire for linkage of ZFN.

FokI nuclease cuts target DNA away from the recognition location. FokI work as a dimer, it is an obligation for functioning of the domain. Firstly one of the FokI binds and then second FokI freely travelling in the solution connects to just directly opposite direction of the first one, thus FokI become dimerized. After that the dimer cut DNA.

Nuclease of ZFN solely efficiently dimerize and cleave DNA when two of ZFNs link to target nucleotides that are separated via the spacer sequence. Therefore ZFNs obtain high specifity based on this property. However, it is probable to get off target effects, like cleavage of palidromic sequence. In order to escape from this type of unintended effects, obligate heterodimeric mutants of FokI restriction enzyme can be used. By the prevention of homodimerization of mutant nucleases, decline in cytotoxicity is reached. Wild type/nuclease-dead mutant FokI heterodimer cuts only single strand of DNA. As NHEJ repair is not able to recover this type of break, HDR takes over the repair work , though efficiency is enourmously low. Another advantage of HDR single strand cleavage repair is to block most of off targettings.

Nucleases are not solo molecules that are capable of fusing with ZFs. Site-specific activator repressor, methyltransferase and recombinase domains are the instances of structures which can be linked to ZFs.

ZFs and nuclease are connected via inter- domain linkers. These elements also play role in specifity of ZFNs. Emergence of TALENs, which is simpler to construct, restricted the use of ZFNs on a large scale. However, there are some ZFNs which are highly target specific.

TALENs
Precursor of TALE proteins gained from a plant pathogen bacteria genus Xanthomonas and related genera. TALE proteins include three domain can be counted as N-terminal domain, DNA- binding domain and C-terminal domain, which hosts an activator domain. Transportation of these proteins into target cell achieved via type III secretion system in the bacteria. TALE proteins connect to specific squences on the target DNA and enhance the expression of some genes of targetted cell in order to supply spreading of the pathogen. As ZFs can be modified to detect user defined sequences, TALEs are able to be done, too.

TALEs can be combined with an activator, repressor , histone modifier , DNA demethylase , recombinase and certainly FokI nuclease. Almost any sequence can be targetted via TALEs.

TALENs have the advatage, of providing more genome editing activity while causing less damage, over ZFNs. Till the invention of CRISPR mediated genome engineering happens, TALENs were the general preference of the gene engineering researchers as ZFNs were not commonly chosen for the editing. Though it seems like that TALENs are fairly more target specific than CRISPR/Cas, ease of CRISPR/Cas system make it dominant among other targetable nuclease tools.