Table 1.

Comparison of ZFNs, TALENs, and CRISPR/Cas9 Nuclease Systems for Genome Editing

Genome Editing Tool	ZFNs	TALENs	CRISPR/Cas9
Features	ZF-FokI nuclease protein fusion	TALE-FokI nuclease protein fusion	Cas9 nuclease and sgRNA
Target site identification	protein-DNA interaction, capable of targeting virtually any site	protein-DNA interaction, capable of targeting virtually any site	RNA-DNA interaction, site selection restricted by the NGG motif, which occurs statistically every 8 nt in a random DNA sequence
Genome altering	DSBs directed by the FokI domain, repaired through NHEJ or HDR	DSBs directed by the FokI domain, repaired through NHEJ or HDR	DSBs directed by Cas9, repaired through NHEJ or HDR
Design	+	++	+++
	custom design based on the target sequence, labor intensive and time-consuming	custom design based on the target sequence, labor intensive, less time-consuming than ZFN	very simple design by altering the crRNA sequence of the sgRNA
Efficiency	++	++	+++
Biallelic targeting	++	++	+++
Off-target effects	++	+++	+
	specific	more specific than ZFN	low compared with ZFN and TALEN as a result of allowed mismatches by the Cas9 nuclease between sgRNA and the DNA target sequence
Multiplexing	+	+	+++
	rarely used	rarely used	yes, capable of targeting multiple sites simultaneously
Size and delivery	+++	++	+
	the ZFN monomer is significantly smaller than Cas9	TALEN monomers are situated in the middle of Cas9 and ZFN nucleases in terms of size	massive Cas9 protein encoded by a 4.2-kb sequence