Reverse transfection for high-throughput CRISPR studies
Arrayed crRNA libraries allow for functional genetic screens using a wide range of phenotypic readouts, including high-content imaging, therefore further facilitating the discovery of genes with roles in different biological processes. Dharmacon Edit-R predesigned crRNA libraries enable rapid, high-throughput analysis of editing events in hundreds of genes with multiple target sites per gene, but the identification of relevant hits and successful screening outcomes from these types of assays depends on high transfection efficiency. Transfection conditions that induce changes in cell viability and/or provide insufficient delivery of the targeting agent can mislead researchers toward false interpretations of data, which in the long run, are time consuming and costly.
Optimize crRNA:tracrRNA delivery with a reverse transfection method
Forward transfection is a widely used method that works well for adherent cell types; however, if one is using suspension cells or a high-throughput format, a more appropriate approach is often reverse transfection, where cells are added to the plated transfection reagent complex. The reverse transfection method can be used to increase the throughput and reproducibility of a CRISPR-Cas9 screen by optimizing gene editing efficiency as demonstrated in our recently published application note: “Optimization of reverse transfection of Dharmacon Edit-R synthetic crRNA and tracrRNA components with DharmaFECT transfection reagent in a Cas9-expressing cell line.”
In this application note, we present an example of transfection optimization in reverse transfection format using a recombinant U2OS reporter cell line stably expressing Cas9 nuclease under the CAG promotor (Ubi[G76V]-EGFP-Cas9 cells). An un-cleavable ubiquitin moiety (Gly76Val) fused to EGFP allows constitutive degradation of the EGFP protein (and low basal fluorescence), while disruption of the proteasome components by functional protein knockout leads to accumulation of EGFP and detectable fluorescence. This functional knockout data shows that determining optimal transfection conditions prior to arrayed screening leads to high gene editing efficiency with minimal effect on cell viability.
- This protocol is for reverse transfection of Dharmacon Edit-R crRNA arrayed libraries
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