TABLE 1.
Systematic comparison of meganuclease, Zinc Finger Nuclease (ZFN), Transcription Activator Like Effector Nuclease (TALEN), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) genome editing platforms.
| Features | Meganuclease | ZFN | TALEN | CRISPR/Cas9 |
| Source | Organellar DNA, bacteria, phage | Bacteria, eukaryotes | Bacteria (Xanthomonas sp.) | Bacteria (S. pyogenes) |
| Polymeric state | Dimers (two identical subunits) | Dimers (two FokI domains) | Dimers (two FokI domains) | Monomer (only sgRNA-Cas9 complex) |
| Type of recognition | Protein-DNA | Protein-DNA | Protein-DNA | RNA-DNA |
| Recognition site | Between 18 and 44 bp | Between 18 and 36 bp | Between 24 and 40 bp | Between 17 and 23 bp |
| DSB pattern | Staggered (3′ overhang) | Staggered (5′ overhang) | Staggered (heterogenous overhang) | Staggered (5′ overhang, Cpf1 system); blunt (SpCas9) |
| Specificity | High | Low to moderate | Moderate | Low to moderate |
| Ease of design and engineering | Difficult | Difficult | Moderate | Easy |
| Immunogenicity | Unknown | Low | Unknown | Unknown |
| Ex vivo delivery | Easy using electroporation and viral vector | Easy using electroporation, viral vector and lipofection | Easy using electroporation, viral vector and lipofection | Easy using electroporation, viral vector and lipofection |
| In vivo delivery | Easy to difficult (depending on size of nuclease) | Easy to difficult (depending on size of nuclease) | Difficult (large size of TALEN) | Moderate (S. pyogenes is large) |
| Multiplexing | Low | Low | Moderate to high | High |
| Cost (USD) | 4,000–5,000 | 5–10,000 | Less than 1,000 | Less than 100 |
| Success rate | Low | Low (∼24%) | High (>99%) | High (∼90%) |
| Targeting constraints | Targeting novel sequencing | Targeting non-G-rich sequence | 5′ targeted base must be a T for each TALEN monomer | Targeted sequence must precede a PAM sequence |
| Advantages | Possible to edit various types of genome editing (knockout, reporter, specific alleles) | Designed to target any DNA sequence; targeting of biallelic genes | Designed to target any DNA sequence; targeting of biallelic genes | Targeting of biallelic genes and multiplexing |
| Disadvantages | Lacks DNA-binding domains; inefficient for inadequate knowledge on designing construct; time-consuming | Binding capacity of ZFN depends on neighboring ZFs; decreased specificity can lead to off-target cleavage | Cloning of TALE repeats is troublesome and error prone | Target sites limited for PAM motif; higher chance of off-target cleavage |