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Table 1 Difference between the modern gene-editing tools ZFNs, TALENs, and CRISPR–Cas9

From: Genetic tool development and systemic regulation in biosynthetic technology

No. Gene-editing Features Advantages Limitations References
1. ZFNs Restriction nuclease Fok1 fused to multiple zinc finger peptides, each target triplet codon of genomic DNA
Target site length 18–36 base pair
Binding specificity–3 nucleotide
Nuclease design success rate low
Effect of CpG methylation not known
Adequate flexibility
Easy gene delivery to the desired target
Targeting efficiency variable
Recognizing specific long target sequences
Can have high off-target frequency
No high-throughput targeting
High cost
Low specificity and can be influenced by neighboring protein domain easily
[76,77,78] [81] [83]
2. TALENs Non-specific DNA nuclease fused to a domain specific for genomic loci
Target site length 30–40 base pair
Binding specific-1 nucleotide
Nuclease design success rate high
Sensitive to CpG methylation
High specific and easy to design
High targeting efficiency
Heavier to deliver to the targets
Repetitive sequence may cause unintended cuts to the DNA sequence
Low off-target effect
Limited/low high-throughput targeting
[79, 80] [82]
3. CRISPR–Cas9 20 nucleotide crRNA fused to Cas9 nuclease and tracrRNA
Target site length 20–22 base pair
Binding specific—1:1 nucleotide
Nuclease design success rate high
No effect of CpG methylation
High specific and easy multiplexed gene editing
High targeting efficiency
Some/variable off-target effect
No limitation in high-throughput targeting
[87, 88]