Engineered nuclease platform technologies
| Nuclease . | Properties . | Advantages . | Disadvantages . |
|---|---|---|---|
| ZFN | Heterodimeric protein with multimeric sequence-specific zinc finger–binding domains and the Fok1 endonuclease | Most clinically advanced platform. | De novo design of effective and specific ZFNs is difficult |
| Can iteratively improve activity and specificity | |||
| Homing endonuclease (HE) | Homo- or dimeric protein containing site- specific DNA recognition domains and endonucleolytic activity | Smallest coding sequence; compatible with all vector platforms | Have been challenging to direct to new specific target sequences |
| High activity and specificity from complex protein structure | |||
| Unique 3′ overhang may have unique in vivo biochemistry | |||
| TALEN | Heterodimeric protein with multimeric site-specific TAL effector domains and the Fok1 endonuclease | Relatively easy to design and produce | Variable activity and specificity. May be able to evolve in vitro to higher efficacy; repetitive structure and large size render vectorization more complex |
| CRISPR | Combines a protein (with both RNA- directed DNA sequence-specific binding and endonuclease activity) and a short RNA guide sequence that recognizes the target site and directs the DNA binding of the nuclease | Easy to design to target new sequences; use as ribonucleoprotein particle may allow efficient modification of cells where translation rates are low | Need further knowledge of rare off-target events, frequency, and potential genotoxic effects; need for 2 biochemically different components complicates vectorization for some applications |
| Mega-TAL | Monomeric protein with TAL effector DNA-binding domain fused to a homing (mega-) endonuclease cleavage domain | Monomeric, compact albeit larger than HE alone, high activity and specificity from HE domain | Requires engineering a HE cleavage domain |
| Nuclease . | Properties . | Advantages . | Disadvantages . |
|---|---|---|---|
| ZFN | Heterodimeric protein with multimeric sequence-specific zinc finger–binding domains and the Fok1 endonuclease | Most clinically advanced platform. | De novo design of effective and specific ZFNs is difficult |
| Can iteratively improve activity and specificity | |||
| Homing endonuclease (HE) | Homo- or dimeric protein containing site- specific DNA recognition domains and endonucleolytic activity | Smallest coding sequence; compatible with all vector platforms | Have been challenging to direct to new specific target sequences |
| High activity and specificity from complex protein structure | |||
| Unique 3′ overhang may have unique in vivo biochemistry | |||
| TALEN | Heterodimeric protein with multimeric site-specific TAL effector domains and the Fok1 endonuclease | Relatively easy to design and produce | Variable activity and specificity. May be able to evolve in vitro to higher efficacy; repetitive structure and large size render vectorization more complex |
| CRISPR | Combines a protein (with both RNA- directed DNA sequence-specific binding and endonuclease activity) and a short RNA guide sequence that recognizes the target site and directs the DNA binding of the nuclease | Easy to design to target new sequences; use as ribonucleoprotein particle may allow efficient modification of cells where translation rates are low | Need further knowledge of rare off-target events, frequency, and potential genotoxic effects; need for 2 biochemically different components complicates vectorization for some applications |
| Mega-TAL | Monomeric protein with TAL effector DNA-binding domain fused to a homing (mega-) endonuclease cleavage domain | Monomeric, compact albeit larger than HE alone, high activity and specificity from HE domain | Requires engineering a HE cleavage domain |