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Table 1 Comparison of the three main currently used genome engineering platforms: ZFN, TALEN, and CRISPR/Cas9*

From: The genome editing revolution: review

Aspect of comparison ZFN TALEN CRISPR/Cas9
Origin Eukaryotes Bacteria Bacteria/archaea
Structure Dimer Dimer Monomer
Design simplicity Moderate (ZFNs need customized protein for every DNA sequence) Slightly complex (identical repeats are multiple, which creates technical issues of engineering and delivery into cells) Simpler (available versions for crRNA can be easily designed)
Engineering feasibility/affordability Low/limited Moderate/affordable but resource intensive High
Popularity/affordability Low/limited Moderate/affordable but resource intensive High/highly affordable
DNA-binding molecule/DNA recognition mechanism Zinc finger protein/protein-DNA interactions that introduce DSB Transcription activator-like effectors/protein-DNA interactions that introduce DSB crRNA or sgRNA/RNA-guided protein-DNA interactions that introduce DSB
Modification pattern FokI nuclease FokI nuclease Cas9 nuclease
Specificity-determining length of recognition site Typically 9–18 bp per ZFN monomer, 18–36 bp per ZFN pair Typically 14–20 bp per TALEN monomer, 28–40 bp per TALEN pair 22 bp (20-bp guide sequence C 2-bp protospacer adjacent motif (PAM) for Cas9; up to 44 bp for double nicking
Targeting/target specificity Low/difficult to target non-G-rich sequences/high; G-rich sequence preference; only small positional mismatches are tolerated; re-targeting requires protein engineering Higher/for each TALEN monomer targeted base sequence must start (5′) with a T and end with an A (3’) end.
High, requires a T at each 5’-end of its target; small positional mismatches are tolerated; re-targeting requires complex molecular cloning
Highest/targeted sequence end with an NGG or NAG (lower activity) sequence (that is, PAM)
Moderate: RNA-targeted sequence must precede the 2 base pairs recognized by PAM. Only small positional and multiple consecutive mismatches are tolerated. Re-targeting requires new RNA guide. Protein engineering is not required.
Mechanism of action Introduction of double-strand breaks (DSBs) in target DNA Introduction of double-strand breaks (DSBs) in target DNA Introduction of DSBs in target DNA by wtCas9 or single-strand nicks by Cas9 nickase
Cleavage efficacy Efficient Efficient Highly efficient
Multiplex genome editing Not easy (few models) Not easy (few models) Easy (high-yield multiplexing available (no need for obtaining embryonic stem cells))
Delivery vehicle Easy via electroporation and viral vectors transduction Easy in vitro delivery; difficult in vivo due to the large size of TALEN DNA and their high probability of recombination Easy in vitro; moderate difficulty of delivery in vivo due to poor packaging of the large Cas9 by viral vectors.
Use as gene activator Yes; activation of endogenous genes; minimal off-target effects; may require engineering to target particular sequences Yes; activation of endogenous genes; minimal off-target effects; no sequence limitations Yes; activation of endogenous genes; minimal off-target effects; requires “NGG” PAM next to the target sequence
Use as gene inhibitor Yes; works by blocking transcription elongation via chromatin repression; minimal off-target effects; may require engineering to target particular sequences Yes; works by blocking transcription elongation via chromatin repression; minimal off-target effects; no sequence limitations Yes; works by blocking transcription elongation via chromatin repression; minimal off-target effects; requires “NGG” PAM next to target sequence.
Success rate‡ Low (~ 24%) High (> 99%) High (~ 90%)
Average mutation rate§ Low or variable (~ 10%) High (~ 20%) High (~ 20%)
Off-target effects Highly possible off-target activities Low possible off-target activities Variable; limited off-target activities, not fully studied in plants
Programmable Highly difficult Difficult Easy
Cytotoxicity Variable to high Low Low
Cost Low High Low
Online resources for nuclease design • The Zinc Finger Consortium includes software tools and protocols genome-wide tag scanner for nuclease off-sites
• The Segal Laboratory software site
• ZFN target site algorithm for identifying sites compatible with the Lawson-Wolfe modular assembly system
• Zinc finger tools
• ZiFiT Targeter software
• E-TALEN
• Genome engineering resources
• Scoring algorithm for predicting TALE(N) activity
• ToolGen TALEN designer
• ZiFiT Targeter software
• E-CRISP
• Genome engineering resources
• RGEN tools
• ZiFiT Targeter software
Suppliers Non-profit organizations
*Companies
- Addgene (https://www.addgene.org/)
*Sigma-Aldrich/ToolGen
- Addgene/TALEN library resource
*Cellectis Bioresearch/Life Technologies/ToolGen/Transposagen Biopharmaceuticals
- Addgene
*Life Technologies/Sigma-Aldrich/System Biosciences/ToolGen/Transposagen Biopharmaceuticals
  1. A wide range of success rates and mutation rates (which depend on factors such as the methods used to construct these nucleases, delivery methods, and cell lines or organisms) have been reported. Mutation frequencies are higher in K562 cells and HeLa cells than in HEK293 cells
  2. *Abbreviations: Cas9 CRISPR (clustered regularly interspaced short palindromic repeat)-2 associated protein 9, crRNA CRISPR RNA, N any nucleotide, PAM protospacer adjacent motif, RGEN RNA-guided engineered nuclease, sgRNA single-chain guide RNA, TALEN transcription activator-like effector nuclease, ZFN zinc finger nuclease
  3. ‡The success rate is defined as the proportion of nucleases that induce mutations at frequencies > 0.5% in HEK293 cells
  4. §The average mutation rate is based on the frequency of non-homologous end-joining-mediated insertions and deletions obtained at the nuclease target site [1, 39, 48, 78]. The Innovative Genomics Institute (https://innovativegenomics.org/) is another excellent source of background information, explainers, and a terrific glossary with fun animations (https://innovativegenomics.org/resources/educational-materials/)