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Increase the throughput and reproducibility of your gene editing screen
Predesigned crRNA libraries enable rapid analysis of editing events in hundreds of genes or sites at once, but the identification of relevant hits and successful screening outcomes from these types of assays depends on transfection that has been optimized to have high efficiency and low toxicity. 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.
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, reverse transfection, where cells are added to the plated transfection reagent complex, is a more appropriate approach. This 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.”
An un-cleavable ubiquitin moiety fused to EGFP allows constitutive degradation of the EGFP protein, 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.
Figure 1. PPIB gene editing efficiency assessed with DNA mismatch assay using T7EI 72 hours post-transfection (duplicate wells are shown). The amount of transfection reagent required for successful gene editing varies with cell density. FastRuler™ Low Range DNA Ladder (Thermo Scientific, Cat #SM1103).
The following is a general workflow for reverse transfection of arrayed crRNA libraries using stable Cas9-expressing mammalian cells. Find more details in the full protocol for “Reverse Transfection of Arrayed crRNA”.
Note: Optimal plating density will depend on growth characteristics of specific cell lines and assay requirements and these parameters should be determined experimentally. Exact parameters for crRNA:tracrRNA transfection in your cells of interest should be empirically determined through careful optimization prior to experimentation. Catalog crRNA library plates are supplied with columns 1 and 12 empty to allow addition of researcher-defined controls. We suggest including the following controls:
Arrayed collections of predesigned synthetic crRNA for high-throughput gene editing studies across entire gene families for human and mouse.
DharmaFECT transfection reagents provide efficient and reliable transfection at low siRNA/microRNA reagent concentrations with minimal cellular toxicity.