Genome Editing - 4D-Nucleofector Visual

Genome Editing using Nucleofector™ Technology - A Technical Reference Guide:

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The reference guide contains:

  • A brief introduction to genome editing tools
  • Use of Nucleofector™ Technology for transfection of ZFN, TALEN or CRISPR/Cas9 System
  • Technical tips for ZFN/CRISPR usage and Nucleofector™ Technology

 


Archived Webinar – Watch on Demand:
CRISPR/Cas9 Cell Therapeutics – The NextGeneration of Cures

Presented by GEN, sponsored by Lonza

Topics include: opportunities and challenges for the use of CRISPR/Cas9 in gene/cell therapeutic applications and its impact on next generation disease model systems and drug screening.

Speakers:

  • Matt Porteus, M.D., Ph.D.
    Associate Professor of Pediatrics, Stanford University
  • Gregory Alberts, Ph.D.
    Global Subject Matter Expert Transfection and Genome Editing, Lonza

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Genome editing using engineered nucleases is a powerful tool to delete, insert or replace a gene at a targeted genomic location1. The nuclease is generating a double-strand break in the genomic DNA and thus inducing one of two possible cellular repair processes. Non-homologous end-joining (NHEJ) can lead to a deletion of the functional gene since it is often accompanied with mutations when closing the break. If a partially homologous donor sequence is available an insertion or replacement of a gene can take place via homology dependent repair (HDR). To perform such a modification at a specific target sequence, the nuclease is fused to or interacts with sequence-specific DNA-binding component directing the nuclease to the target sequence.

Genome Editing Tools - Brief Overview

The typically used tools for site-specific genome editing tools are:

  • ZFN (Zinc Finger Nucleases)
  • TALEN (Transcriptional Activator-Like Effector Nucleases)
  • CRISPR/Cas9 (Clustered Regularly Interspaced Palindromic Repeats and CRISPR-Associated nuclease 9).

  ZFN TALEN CRISPR/Cas9 
Nuclease Fok1 Fok1 Cas9
DNA binding molecule ZF Protein TALE protein GuideRNA (gRNA)
Type Fusion protein
→ High effort to modify for new targeting site
Fusion protein
→ High effort to modify for new targeting site
Protein + RNA
→ Easy to modify
→ Multiple targeting possible
Binding sites 2 sites (15 or 18 bp each)
→ High specificity
→ Low risk for off-target effects
2 sites (≥ 13 bp each)
→ High specificity
→ Low risk for off-target effects
1 site (18-20 bp + 3bp PAM)
→ Lower specificity
→ Higher risk for off-target effects

 

Genome Editing – Successful Transfection of CRISPR & Co. using Nucleofector™ Technology

All genome editing tools require co-transfer of several substrates into the cell type of interest for successful modification of genomic DNA. Lonza’s non-viral Nucleofector™ Technology has been shown to work as a reliable and sufficient method for transferring the required DNA- or RNA-based components into various cell lines and primary or stem cells, e.g., primary T cells, human embryonic stem cells (hESC) or induced pluripotent stem cells (iPSCs). 

  • High transfection efficiencies for a broad range of cell types, including iPSCs
  • Efficient co-transfection of various substrates
  • Same conditions for transfecting plasmids, DNA, mRNA or PCR cassettes, ssODN
  • Proven for ZFN, TALEN and CRISPR by more than 30 publications, including high ranking journals 

 

If you need any technical guidance on using the Nucleofector™ Technology for genome editing contact Lonza Scientific Support.

Besides using the Nucleofector™ Technology for genome editing in iPSCs or ESCs, it also offers a reliable method for iPSC generation. Thus, together with our new L7™ hPSC Culture System, Lonza can support various stages of the workflow in pluripotent stem cell research - from iPSC generation, growth and expansion to modification of those cells via genome editing.

References

1) Gaj T et al (2013) ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends in Biotechnology 31(7):397-405 (review)