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[Protocol] Optimizing transfection for genome editing

Jun 10, 2016 2:25:54 PM 1 Comment

Transfection or electroporation is used to efficiently introduce the nucleic acids required for CRISPR cell line engineering into a cell line. The type and number of nucleic acids (usually plasmids) being introduced into a cell line will depend on the engineering event being undertaken. At its most simple, gene knockouts can be achieved by transfection of a plasmid expressing wild-type Cas9 along with a guide RNA (gRNA) to the gene that is being knocked out.

Even if you're able to select for cells that have been successfully transfected (by antibiotic or cell sorting), optimizing transfection can have a big impact on the success of your gene editing experiment - potentially yielding cells that recover faster and are then able to withstand the process of single cell dilution. If you are unable to enrich for transfected cells then a transfection efficiency of 50% compared to 99% means there will be a need to screen twice as many cells to find the same number of targeting events.

For every cell line the optimal transfection conditions will be different, and so it is often worth trying a few different lipid reagents and electroporation. At Horizon, we have found the Amaxa™ Nucleofector™ electroporation system from Lonza suitable and effective for most of our experiments - but it will vary. Details of Lonza’s protocols can be found on the cell and transfection database on the Lonza website (http://bio.lonza.com).

To assess transfection efficiency at Horizon, we assess and optimize based on analysis of fluorescence levels measured by FACS following introduction of a plasmid expressing GFP.

Below are some pulse codes for common cell lines recommended for transfections using the Amaxa™ Nucleofector™ electroporation system.

Cell line

Buffer

Pulse code

HCT116

SE

FP100

HEK293

SF

FF132

MCF10a

SE

FF130

K562

SF

FF120

 

#Cell lines, #Gene editing, #Protocols

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