Zinc-Finger non-homologous end joining or homologous directed

Zinc-Finger Nucleases (ZFNs) arehighly specific, they can recognise a sequence between 9-18 basepairs. ZFNs aremade up of two domains, the binding domain and the cleavage domain (Horizon). The cleavage domain is made up of thenon-specific, restriction endonuclease, Fokl, which is responsible for introducingthe double strand break. To target a specific gene, Fokl must dimerise (combine with smaller molecules) with zinc fingerswhich make up the binding domain. There is normally between 3 and 6 zincfingers which recognise 3 basepairs each, zinc fingers are syntheticallysynthesised to recognise larger specific sequences by combining smaller zincfinger modules of known sequence (Gene Therapy Net, n.

d.). This method ishigly complex and costly (Nemudryi, et al., 2014)TALE proteins were discoveredwhen studying the bacteria genus Xanthomonas,they have a central domain capable of DNA binding and and a domain thatactivates target gene transcription.

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The discovery of TALE proteins lead to thedevelopment of the TALENs genome editing technique by the combination of the TALEproteins binding domain and the FokI cleavagedomain which as in ZFNs introduces the double stranded break. The DNA bindingdomain of TALE proteins consist of multiple monomers which bind one nucleotidein the target sequence, meaning that many different sequences can be targetedby combining monomers in different orders (Nemudryi, et al., 2014).The versatility when creating the binding sequences makes TALENs artificialnucleases favourable over Zinc-Finger Nucleases because they easier to engineer(Yeadon, n.

d.)CRISPR-Cas9 is a veryversatile RNA guided nuclease it has two core components, the Cas9 nuclease anda single guided RNA (sgRNA), which are in complex together. The sgRNA has a20-nucleotide guide sequence which is used to specifically target a gene andCas9 is used to create a double stranded break (DBS). The DBS is repaired byeither non-homologous end joining or homologous directed repair.

CRISPR-Cas9 iscapable of inducing gene loss of function and gene gain of function (Liu, etal., 2016).

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