Table 2.

Applications of the technology in ZFN, TALEN, and CRISPR/Cas9 gene editing.

	ZFN	TALEN	CRISPR/Cas9
Recognition/target site	18–36 bp/zinc finger pair; guanine-rich region	30–40 bp per TALEN pair	22 bp; followed immediately by 5′-NGG-3′ PAM sequence
Targeting specificity	18 bp ZFN can confer specificity within 418 bases [10]	TALEN plasmid library developed can target 18,742 human genes	Unknown; theoretically any genomic site that precedes PAM sequence
Off-target mutagenesis	Unknown and hard to determine mutagenic sites due to many possible indiscriminate protein-DNA interactions that can occur	Unknown and hard to determine mutagenic sites due to many possible indiscriminate protein-DNA interactions that can occur	Easier to predict possible mutagenic sites by utilizing Watson–Crick base-pairing rules
Ease of Delivery	Difficult due to extensive cloning needed to link zinc finger modules together	Difficult due to extensive TALE repeat sequences	Easy, facile design of gRNA and standard cloning techniques
Methods employed to deliver editing systems in vivo	AAV	AAV	AAV Lentivirus
Multiplexing ability	No	No	Yes
Clinical or pre-clinical stage	Clinical trial application for HIV and Hunter’s syndrome	Pre-clinical	Pre-clinical
Advantages	Small protein size (<1 kb) allows packaging into a single AAV	High specificity with each module recognizing 1 bp; no need to engineer linkage between repeats	Enables multiplexing (targeting multiple genes)
Limitations	Length of target sequence confined to the multiples of three; cumbersome cloning methods that needs additional linker sequences to fuse modules together	Large protein size makes it challenging to utilize viral system; repetitive sequences may induce undesirable recombination events within the TALE array	Limited PAM sequences in human genome; Cas9 nuclease (~4.2 kb) is large for packaging into AAV