Table 1.

Comparison of the three programmable nucleases

	ZFN	TALEN	CRISPR/Cas9
Components	DNA binding domain is composed of multiple zinc finger proteins and it is fused to FokI nuclease	DNA binding domain is based on TALE proteins and it is fused to FokI nuclease	gRNA and Cas9 endonuclease
Length of recognition site	Typically 18-36 bp per ZFN pair	Typically 30-40 bp per TALEN pair	Total length of 23 bp: 20 bp target sequence + 3 bp PAM sequence (for Streptococcus pyogenes Cas9)
Basis of target recognition	Protein-DNA interaction	Protein-DNA interaction	RNA-DNA interaction
Target site requirements	Preference for G-rich sequences	5’-T preceding the target site is preferred although not necessary	The target sequence must be followed by PAM sequence
Size of coding DNA sequence	1 kb for each monomer	3 kb for each monomer	4.3 kb in total
Modularity	Low modularity since adjacent zinc finger proteins might affect each other's binding	High modularity	High modularity
Ease of construction	It may require extensive protein engineering	Easier than ZFNs although it requires special cloning methods and a library of sequences coding for subdomains of the DNA binding domain	It is much simpler than others since it only requires standard cloning of a synthesized oligonucleotide