Fig. 2

Genome editing with engineered nucleases. Genome editing involves two steps: i) a nuclease is engineered to cleave a specific (target) sequence in the DNA creating a double strand break (DSB); ii) the cell’s ability to repair the DSB by non-homologous end-joining (NHEJ) causes a deletion in the target gene that can result in gene mutation or complete knockout whereas homology-directed repair (HDR) by homologous recombination using a homologous DNA template results in gene correction or insertion depending on the DNA donor structure. There are three main classes of engineered nucleases. a Zinc finger nucleases (ZFNs) consist of a DNA-binding macro-domain designed to target the sequence of interest that is composed of several zinc-fingers each one recognising three nucleotides in the target sequence and linked to the nuclease domain of the FokI restriction enzyme. After dimerisation of two ZFNs in inverse orientation and with an optimal spacing of 5–7 nucleotides, the dimeric FokI cleaves the DNA between the binding sites. b Transcription activator-like effector nucleases (TALENs) have a similar structure to that of ZFNs. The TALEN DNA-binding macro-domain is composed of a tandem array of 34 aminoacids each recognising a single nucleotide. Similarly to ZFNs, TALENs also depend on FoKI activity and dimerisation to create a DSB between the binding sites. c In the CRISPR-Cas9 system, a site-specific DNA cleavage is performed by nuclease Cas9 directed by complementary between an engineered single guide RNA (gRNA) and the target sequence