An innovative workflow to guide the insertion of 10-12 nucleotides into a gene of interest

Genome engineering using CRISPR-Cas9 requires expression of the Cas9 nuclease with the crRNA and tracrRNA. This can be achieved by co-transfection of a plasmid expressing Cas9 and crRNA:tracrRNA, or by creation of a cell line in which the Cas9 cassette is delivered using lentiviral particles and stably integrated and expressed prior to transfection with crRNA:tracrRNA. The Dharmacon Edit-R™ CRISPR-Cas9 product line represents the most comprehensive portfolio of tools for gene editing available, but tools are useless without an understanding how to use them. The Edit-R CRISPR-Cas9 genome engineering platform employing chemically synthesized RNAs is a relatively quick and easy method to test multiple guide sequences for optimizing % indel formation through non homologous end joining (NHEJ) and achieving functional gene knockouts.

One topic that comes up frequently when speaking with researchers and discussing their experiments is the need for help with performing homology-directed repair, or HDR. For HDR and creation of knock-ins, it is important to optimize experimental conditions to obtain the maximal levels of double-strand breaks (DSBs). To address these variables, we have created an application note; not only does it explain and demystify HDR is, it also provides guidance on how the Edit-R products can be used for common HDR applications.

In this application note we demonstrate the ability to utilize the same experimental workflow to rapidly create knockins through the HDR pathway simply by including a single-strand DNA oligo donor template. Therefore, experiments to optimize DSBs without a donor template should be performed to assess and maximize levels of gene editing. These experiments should be performed prior to experiments using the donor template and be used as a preliminary assessment for overall levels of DSB generation. We further demonstrate the use of lipid-based transfection using DharmaFECT Duo Transfection Reagent to achieve precise sequence insertion through the HDR pathway, whereas, in the literature, this is commonly achieved through expensive electroporation methods and use of costly Cas9 nuclease reagents. Recommended experimental conditions for performing similar successful sequence insertion experiments using these Edit-R CRISPR-Cas9 reagents and workflows are also shown.

Check out the HDR application note now! »

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