Table 1.

Comparison of Different Programmable Nuclease Platforms.

	Zinc Finger Nuclease	TALEN	Cas9	Meganuclease
Recognition site	Typically 9 to 18 bp per ZFN monomer, 18 to 36 bp per ZFN pair	Typically 14 to 20 bp per TALEN monomer, 28 to 40bp per TALEN pair	22bp (20bp guide sequence + 2bp PAM sequence for S. pyognes Cas9); up to 44 bp for double nicking	Between 14 and 40 bp
Specificity	Small number of positional mismatches tolerated	Small number of positional mismatches tolerated	Positional and multiple consecutive mismatches tolerated	Small number of positional mismatches tolerated
Targeting constraints	Difficult to target non-G-rich sequences	5’ targeted base must be a T for each TALEN monomer	Targeted sequence must precede a PAM [AU: please define]	Targeting novel sequences often results in low efficiency
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	Difficult, may require substantial protein engineering
Immunogenicity	Likely low, as ZFs are based on human protein scaffold. Fokl is derived from bacteria and may be immunogenic	Unknown, protein derived from Xanthamonas sp.	Unknown, protein derived from various bacterial species	Unknown, meganucleases may be derived from many organisms including eukaryotes
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	Relatively easy through methods such as electroporation and viral transduction
Ease of in vivo delivery	Relatively easy due to 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, leanding 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.	Relatively easy due to small size of meganucleases, allows use in a variety of viral vectors.
Ease of multiplexing	Low	Low	High	Low