Review Article
Gene Editing in Pluripotent Stem Cells and Their Derived Organoids
Table 1
Characteristics of current gene editing technologies and their advantages and limitation.
| | Identifying patterns | Cleavage domain | Recognition length | Identification conditions | Minimum identification unit | Accuracy | Molecular weight size of editing tools | Operational difficulty | Off-target level | Cytotoxicity | Advantages | Limitations |
| | MegNs | Binds specific DNA through protein-DNA interactions | 4 bp | Double-stranded DNA sequences of 12 to 40 base pairs | Monomer, target DNA | Indeterminate | +++ | 200-400 aa | +++ | + | + | Higher specificity | Limited variety; difficult to retrofit | | ZFN | Binds specific DNA through protein-DNA interactions | 5-7 bp | 9-18 bp per ZFN | Dimers, 3 bp units of target DNA | 3 bp | ++ | 500-1300 aa | ++ | ++ | ++ | Mature platform; more efficient than homologous recombination | High off-target rate; low specificity; design dependent on upstream and downstream sequences; only for in vitro operations | | TALEN | Binds specific DNA through protein-DNA interactions | 5-7 bp | 14-20 bp per TALEN | Dimer, transcription activator-like effector or transcription activating effector nuclease 5 preceded by a central structural domain of T | 1 bp | ++ | 900-1100 aa | ++ | ++ | +++ | Unrestricted target sites; easier design than ZFN; higher specificity | Cumbersome module assembly; requires large sequencing effort; high cost | | CRISPR | Binding of specific DNA through base complementary pairing and protein-DNA interactions | 0 bp | 20 bp | Monomer, 3 sequence for NGG’s guide RNA | 1 bp | ++ | 1300-1500 aa | + | + | + | High rate of gene modification; diverse gene regulation; allows simultaneous knockout of multiple target loci; precise targeting inexpensive | No PAM in the pretarget region cannot be cut; transfection difficulties |
|
|
PAM: protospacer adjacent motif.
|
