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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] [84,85,86]
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]