Table 1.

Comparison of different programmable nuclease platforms used in livestock genome editing [adapted from Cox et al. (22) with permission from the Publisher].

	Zinc finger nuclease	TALEN	Cas9
Recognition site	Typically 9–18 bp per ZFN monomer, 18–36 bp per ZFN pair	Typically 14–20 bp per TALEN monomer, 28–40 bp per TALEN pair	22 bp [20-bp guide sequence + 2-bp protospacer adjacent motif (PAM) for Streptococcus pyogenes Cas9]; up to 44 bp for double nicking
Specificity	Small number of positional mismatches tolerated	Small number of positional mismatches tolerated	Positional and multiple consecutive mismatches tolerated
Targeting constraints	Difficult to target non-G-rich sequences	Five targeted base must be a T for each TALEN monomer	Targeted sequence must precede a PAM
Ease of engineering	Difficult; may require substantial protein engineering	Moderate; requires complex molecular cloning methods	Easily re-targeted using standard cloning procedures and oligo synthesis
Immunogenicity	Likely low, as zinc fingers are based on human protein scaffold; FokI is derived from bacteria and may be immunogenic	Unknown; protein derived from Xanthamonas sp.	Unknown; protein derived from various bacterial species
Ease of ex vivo delivery	Relatively easy through methods such as electroporation and viral transduction	Relatively easy through methods such as electroporation and viral transduction	Relatively easy through methods such as electroporation and viral transduction
Ease of in vivo delivery	Relatively easy as small size of ZFN expression cassettes allows use in a variety of viral vectors	Difficult due to the large size of each TALEN and repetitive nature of DNA encoding TALENs, leading to unwanted recombination events when packaged into lentiviral vectors	Moderate: the commonly used Cas9 from S. pyogenes is large and may impose packaging problems for viral vectors such as AAV, but smaller orthologs exist
Ease of multiplexing	Low	Low	High