Genome Editing Technology
Over the past several years, the advances in genome editing technologies such as Zinc Finger Nucleases (ZFN), Transcription Activator-like Effector Nuclease (TALENs) and most recently the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 Nuclease have vastly expanded a researcher’s toolkit for exploring gene function and shown potential for therapeutics.
One thing all the technologies have in common is the need to get the functional parts of the tools delivered into the cells in which the editing will take place. The optimal tool for facilitating this delivery can vary dependent on what is to be delivered, i.e. DNA, RNA, protein, size of the substrate, and to what cell type it is to be delivered. The goal is to deliver effectively such that a high percentage of the cells treated effect the edit and yet remain viable for downstream screening and analysis in the case of research or potentially to be returned to a patient in the case of therapeutics. The delivery method itself should have little to no effect on the viability and functionality of the cell.
We have 2 offerings to support gene editing in adherent primary cells and cells lines, DNA-In® CRISPR Transfection Reagent for delivery of large CRISPR/Cas9 plasmid and EditPro™ Transfection Reagent for delivery of Cas9 mRNA or Cas9 protein (RNP) with the associated guide RNA and if desired ssDNA constructs or single-stranded oligodeoxynucleotides (ssODN) can be mixed with the Cas9mRNA/gRNA or Cas9 protein/gRNA to promote homology-directed repair (HDR) to introduce targeted substitutions.
DNA-In® CRISPR Transfection Reagent is optimized for large plasmid delivery and can be used to deliver CRISPR/Cas9 vectors into a range of cell types. This reagent is especially well suited for hard-to-transfect primary cells.
Fig. 8 DNA-In® CRISPR delivers Cas9-GFP expression vector with maximum efficiency - Human primary fibroblasts, human primary keratinocytes, primary human endothelial cells (HuVEC), human skeletal muscle cells, HeLa cells and C2C12 mouse myoblasts were plated in 24-well plates in complete medium without antibiotics at the optimal plating density for each cell type to yield 50-70% confluency at the time of transfection. Cells were transfected with the pSpCas9(BB) -2A-GFP plasmid (9.3kb )(source: Addgene), using DNA-In® CRISPR reagent and incubated overnight at 37°C. Cells were then fixed and stained with anti-GFP and anti-Cas9 antibodies to better observe the transfection efficiency.
EditPro™ Transfection Reagent has been optimized to deliver Cas9 mRNA or RNP into a wide range of adherent cell types. It is well suited for delivery of gene editing tools, therefore, into many primary cells and cell lines.
The table below illustrates the optimal uses of our different reagents that may be used for gene editing. Click to the product pages for additional information and data:
Genome Editing Reagents
|mRNA||Protein (RNP)||Cell Type|
|XX||XX||adherent primary cells and cell lines|
|XX||XX|| primary fibroblasts, muscles cells, keratinocytes and other adherent primary cells |