Hi there!
We have previously implemented a CRISPR/Cas9 design in Hek293 with great success (more than 60% cutting efficiency), but now we want to move the experiments to human adult fibroblasts from a skin biopsy.
We have tried to transfect the fibroblasts with px458 (a 9,2kb GFP reporter plasmid with Cas9 and our specific gRNA) with different methods: Nucleofector, PEI, Viromer, Lipofectamine 3000 and Lipofectamine LTX. Unfortunately, we had 0% transfection efficiency.
We use 3ug of plasmid for 0,2-0,5x10^6 cells.
Recently we also tried using RNPs using thermofisher's CRISPRmax kit, with the concentrations suggested on the manual. But again, 0% success.
Has anyone tried any transfection method for this type of fibroblasts?
Thanks!
Hi Carla,
Take a look at MTI-GlobalStem's new PluriQ G9 Gene Editing System. EditPro™ Stem, when used to transfect iPS or hES cells as part of the G9™ Gene Editing System, results in the highest efficiency of transfection regardless of what type of substrate is being transfected.
We also have DNA-In CRISPR Transfection Reagent that may be of interest.
https://www.mti-globalstem.com/pluriq-g9-gene-editing-system?search=crispr
https://www.mti-globalstem.com/DNAIn-CRISPR-Cas9?search=crispr
Let me know if you have any questions.
Hey Carla,
I am also facing the same kind of problem which is mentioned in detail at following link: https://www.researchgate.net/post/Why_there_is_low_nHDF_cell_viability_after_transfection_with_more_than_4ug_plasmid_12kb_using_Neon_Electroporation_system
I was just wondering that did you optimize your protocol? Please share your experience!
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