Table 1.

Comparison of different engineered nuclease platforms.

	ZFNs	TALENs	Cas9	Meganuclease
Recognition location	Typically 9–18 bp per monomer, 18–36 bp per pair	Typically 14–20 bp per monomer, 28–40 bp per pair	Typically 20 bp guide sequence + PAM sequence	Between 14 and 40 bp
Targeting restrictions	Difficult for non-G-rich sites	5′ targeted base must be a T	Targeted site should precede a PAM sequence	Typically low efficiency for targeting novel sites
Specificity	Tolerating few positional mismatches	Tolerating few positional mismatches	Tolerating positional and multiple consecutive mismatches	Tolerating few positional mismatches
Difficulties of engineering	Requiring substantial protein engineering	Requiring complex molecular cloning methods	Using easy cloning methods and oligo synthesis	Requiring substantial protein engineering
Difficulties of in vivo delivery	Relatively easy as small size of expression elements suitable for varieties of viral vectors	Difficult due to the large size of functional components	Commonly used SpCas9 with large size may cause packaging problems for viral vectors such as AAV	Relatively easy as small size of expression elements suitable for varieties of viral vectors