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. Author manuscript; available in PMC: 2015 Jul 6.
Published in final edited form as: Nat Med. 2015 Feb;21(2):121–131. doi: 10.1038/nm.3793

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