The World of Nucleases

Nuclease-based genome editing tools can induce mutagenesis faster and more economically than traditional ES cell gene targeting technologies. Therefore, nuclease-based tools are more accessible to the scientific community. However, despite the impressive results obtained so far, these tools are still limited by significant shortcomings such as low reliability and low flexibility in model design, transgene size and off-target effects. Such deficiencies mean that the use of robust ES cell gene targeting methods is still preferred for sophisticated and complex model creation.

genOway's strategy is to use the most appropriate technology (nuclease-based or ES cell-based genome editing tools) in terms of time and risk for each project.

Nuclease Mechanisms of Action

Nucleases such as Meganucleases, Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs) and CRISPR/Cas9 systems create specific double-strand breaks at the target locus that trigger DNA repair mechanisms. These corrections result in two types of genome modifications: constitutive Knockouts (KO) through non-homologous end joining and Knockins (KI) through homologous recombination.

 

nuclease-based genome editing

 

Using Nucleases to Create Constitutive Knockout (KO) Models

Directly injecting a nuclease into zygotes creates a constitutive KO mutation in founder animal genes by non-homologous end joining, whereas the ES cell technology requires additional, time-consuming steps like vector construction and ES cell targeting. Consequently, the nuclease-based approach is 8 to 10 weeks shorter.

See: Knockout models developed by genOway.

 

Using Nucleases to Create High Value Knockin (KI) Models

A nuclease enables homologous recombination to occur directly in zygotes, eliminating the ES cell-targeting step. Consequently, the nuclease-based approach should be 6 to 8 weeks shorter.

However, the timesaving is only valid if the nuclease technology is robust and reliable enough to achieve homologous recombination in the first set of injected zygotes.

The current genOway strategy is to use either CRISPR/Cas9 or ES cell-based technologies according to project objectives.

See: Knockin models developed by genOway.

 

Different Nuclease Systems

Four types of engineered nucleases are being used today: Meganucleases, Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs) and the CRISPR/Cas9 system.

The following table recapitulates the main steps in the history of nuclease-based model creation over the last six years, published by the research community (white boxes) and achieved by genOway (grey boxes).

genOway and published nuclease achievements

(1) Menoret S et al., FASEB J, 2013; (2) Remy S et al., Genome Res, 2014

 

Each of these technologies has strengths and weaknesses.

a) Comparison of intrinsic technical performance for each nuclease technology (e.g., flexibility in target sequence identification).

a - Comparison Meganuclease, ZFN, TALEN, CRISPR-Cas9, ES cell targeting

b) Reliability of each nuclease technology in model creation.

b - Comparison Meganuclease, ZFN, TALEN, CRISPR-Cas9, ES cell targeting

 

Conclusion

CRISPR/Cas9 represents the best nuclease-based tool whereas the ES cell-based technology is the most successful and reliable tool, particularly for high value and complex models.

genOway has chosen to offer a complete platform containing ES cell-based and the CRISPR/Cas9 nuclease technologies. The most appropriate technology can be chosen for each genetically modified model to be developed.