Zinc-Finger targeted by combining monomers in different orders

Zinc-Finger Nucleases (ZFNs) are
highly specific, they can recognise a sequence between 9-18 basepairs. ZFNs are
made up of two domains, the binding domain and the cleavage domain (Horizon). The cleavage domain is made up of the
non-specific, restriction endonuclease, Fokl, which is responsible for introducing
the double strand break. To target a specific gene, Fokl must dimerise (combine with smaller molecules) with zinc fingers
which make up the binding domain. There is normally between 3 and 6 zinc
fingers which recognise 3 basepairs each, zinc fingers are synthetically
synthesised to recognise larger specific sequences by combining smaller zinc
finger modules of known sequence (Gene Therapy Net, n.d.). This method is
higly complex and costly (Nemudryi, et al., 2014)

TALE proteins were discovered
when studying the bacteria genus Xanthomonas,
they have a central domain capable of DNA binding and and a domain that
activates target gene transcription. The discovery of TALE proteins lead to the
development of the TALENs genome editing technique by the combination of the TALE
proteins binding domain and the FokI cleavage
domain which as in ZFNs introduces the double stranded break. The DNA binding
domain of TALE proteins consist of multiple monomers which bind one nucleotide
in the target sequence, meaning that many different sequences can be targeted
by combining monomers in different orders (Nemudryi,
et al., 2014).
The versatility when creating the binding sequences makes TALENs artificial
nucleases favourable over Zinc-Finger Nucleases because they easier to engineer

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CRISPR-Cas9 is a very
versatile RNA guided nuclease it has two core components, the Cas9 nuclease and
a single guided RNA (sgRNA), which are in complex together. The sgRNA has a
20-nucleotide guide sequence which is used to specifically target a gene and
Cas9 is used to create a double stranded break (DBS). The DBS is repaired by
either non-homologous end joining or homologous directed repair. CRISPR-Cas9 is
capable of inducing gene loss of function and gene gain of function (Liu, et
al., 2016).


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