Table 1 Comparison of ZFN, TALEN and CRISPR/Cas9 platforms.
| Â | ZFN | TALEN | CRISPR/Cas9 |
|---|---|---|---|
Recognition site | Zinc-finger protein | RVD tandem repeat region of TALE protein | Single-strand guide RNA |
Modification pattern | Fok1 nuclease | Fok1 nuclease | Cas9 nuclease |
Target sequence size | 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 | Typically 20 bp guide sequence + PAM sequence |
Specificity | Tolerating a small number of positional mismatches | Tolerating a small number of positional mismatches | Tolerating positional/multiple consecutive mismatches |
Targeting limitations | Difficult to target non-G-rich sites | 5ʹ targeted base must be a T for each TALEN monomer | Targeted site must precede a PAM sequence |
Difficulties of engineering | Requiring substantial protein engineering | Requiring complex molecular cloning methods | Using standard cloning procedures and oligo synthesis |
Difficulties of delivering | Relatively easy as the small size of ZFN expression elements is suitable for a variety of viral vectors | Difficult due to the large size of functional components | Moderate as the commonly used SpCas9 is large and may cause packaging problems for viral vectors such as AAV, but smaller orthologs exist |
