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Genome Editing using Nucleofector™ Technology - A Technical Reference Guide:
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The reference guide contains:
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:
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).
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.
1) Gaj T et al (2013) ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends in Biotechnology 31(7):397-405 (review)