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Table 2 Main features of the different genome-editing nucleases

From: Current strategies employed in the manipulation of gene expression for clinical purposes

 

ZFNs

TALENs

CRISPR/Cas

Phylogenetic origin

artificial restriction enzyme [252, 253]

Xanthomonas bacteria [254]

Streptococcus pyogenes [255]

DNA binding domain

zinc finger protein [253, 256]

TALE protein [16, 257, 258]

guide RNA [259,260,261]

DNA cleavage

FokI [262]

FokI [257, 258, 262]

Cas9 [259, 260]

DNA recognition range

18–36 bp (3 bp per module) [253]

30–40 bp (1 bp per module) [257, 258, 263]

22 bp (DNA-RNA base pairing) [261]

DNA cut

dsDNA as a dimer [264]

dsDNA as a dimer [265]

dsDNA complex protein-gRNA [259]

Recognition sequence

5'-GNNGNNGNN-3’ [256]

sequence with 5'-T and A-3' [16, 254, 263]

sequence immediately followed by 5'-NGG-3' [259, 266]

Advantages

Small protein size (< 1 Kb), sequence-based module engineering [267]

High specificity, easy selection of target region [268]

Easy to multiplex, simple synthesis of gRNA, easy selection of target region [269]

Disadvantages

Difficult sequence selection and protein engineering,, expensive and time consuming [267]

Large protein size (> 3 Kb), expensive and time-consuming [269, 270]

Large protein size (> 4 Kb) [269]

Safety concerns

off-target effects: genome mutagenesis and GCRs [271]

off-target effects: genome mutagenesis and GCRs [270]

off-target effects: genome mutagenesis and GCRs [271]