Table 1.

Comparison of three engineered nucleases-ZFN, TALEN, and CRISPR/Cas9

	ZFN	TALEN	CRISPR/Cas9
Backbone origin	Highly prevalent in eukaryotes	Bacteria (Xanthomonas spp.)	Bacteria (S. pyogenes)
Specificity module	ZFP	TALE	sgRNA (crRNA + tracrRNA complex)
Cleavage module	FokI	FokI	Cas9
Target site	18–36 bp (3 nt per zinc finger module)	30–40 bp (1 nt per RVD; TALE binding sites should start with a T)	20 bp + PAM (NGG) sequence (Cas9 binding sites should end with G-rich PAM)
Working mechanism	DNA/protein recognition, DSB, and its repair pathway	DNA/protein recognition, DSB, and its repair pathway	DNA/RNA recognition, DSB, and its repair pathway
Reprogramming efficiency	Relatively low	Relatively low	High (easier to design, faster to synthesize, and cost-effective; furthermore, multiplex genome editing is available)

ZFN zinc finger nuclease, ZFP zinc finger protein, TALE transcription activator-like effector, TALEN TALE nuclease, RVD repeat-variable di-residue, CRISPR clustered regularly interspaced short palindromic repeat, Cas9 CRISPR-associated enzyme 9, sgRNA single guide RNA, crRNA CRISPR RNA, tracrRNA trans-activating crRNA, PAM protospacer adjacent motif