TABLE 2.
Comparison of engineered endonucleases.
| Specificities, advantages, limitations | Meganucleases | ZFN | TALEN | CRISPR-Cas |
| DNA binding determinant | Protein | ZF protein | TAL protein | crRNA/sgRNA |
| Binding specificity | Long sequences of nucleotidesa | 3 nucleotides | 1 nucleotideb | 1/1 nucleotide pairing |
| Endonuclease | I-CreI and I-SceIa | FokIc | FokIc | Cas9 |
| Function specificity | Monomer | Dimer | Dimer | Monomer |
| Design/Engineering | Very difficult | Difficult | Simple | Very simple |
| Restriction in target site | Chromatin compaction | G-rich sequence | Start with T and end with A | End with a NGG sequence |
| Target site length | 18–44 bp | 18–36 bpd | 24–40 bp | 22–25 bp |
| Targeting frequency | Low | High (one/100 bp) | High (one/bp) | High (one/4 or 8 bp) |
| Specificity | High | Moderatee | High | High |
| Sensitivity to DNA methylation | Yes | Yes | Yes | Nof |
| Off-targets | Variable | Lowe | Very low | Variable |
| Size | Small size | Small size (∼1 kb/monomer) | Large size (∼3 kb/monomer) | Large size (4.2 kb Cas9) |
| Commercially available, Cost | Yes, high | Yes, high | Yes, moderate | Yes, low |
| Patents concern | Yes | Yes | Yes | Yes |
| Type of editing | ||||
| Gene KO (Indels and frameshift) | Yes | Yes | Yes | Yes |
| Multiplex KO | No datah | Very limited | Limited | Yes (up to eight alleles)g |
| Gene correction/point mutagenesis (repaired basepairs) | No datah | Yes | Yes | Yes |
| Gene addition/sequence replacement (integrated gene cassette) | No datah | Yes | Yes | Yes |
| Gene deletion (deleted gene fragments) | No datah | No data | No data | Yes |
| Prime and base editing | No datah | No data | No data | Yes |
aDNA-binding specificities and cleavage mechanism combined in the same protein (Galetto et al., 2009). I-CreI and I-SceI are the main endonucleases used but a few others have been applied to genome editing. bTALE protein consist of 34 amino acid repeat domains, each one recognizing a single DNA nucleotide; highly conserved, excepting two hypervariable residues at positions 12 and 13, which confer the specificity of TALE. cFokI cleaves only in its dimeric form dAssociation of 3–6 ZF DNA binding domains fused to the FokI catalytic domain. Binding of two ZFN-FokI heterodimers to two contiguous DNA sequences and separated by a 5–7 bp gap. eSpecificity depends on number and selected ZF modules. fNo direct effect of methylation on Cas9 binding or effectivity (Verkuijl and Rots, 2019). gDifficult on same chromosome. Limitations overcome by Prime and base editing (cf Table 3). hThe difficulty in designing meganucleases has limited their application in creating new model organisms.