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Design and order a plasmid DNA donor kit for insertion of a fluorescent marker
Recent studies have resulted in greater understanding of many of the key requirements for achieving precise CRISPR-Cas9 gene engineering using homology-directed repair (HDR). Following a double-strand break (DSB) by CRISPR-Cas9, HDR is a repair pathway that requires a donor template with sequence homology adjacent to the desired genomic modification site. A well-designed donor template can introduce precise alterations to the genomic sequence to facilitate nucleotide insertion, removal or replacement, and even tagging with a fluorescent reporter.
The Edit-R HDR plasmid donor is created through plasmid cloning and assembly of a fluorescent protein insert sequence, the cut vector backbone, and two homology arms generated with custom PCR primers to geneomc regions flanking the insertion site.
One of the significant remaining challenges is the design and construction of the DNA donor; especially when a plasmid donor is required for a larger change. Donor plasmid construction requires generation of the design of the insert, creation of the homology arms, and assembly of the final donor. Greater understanding of this process has enabled the development of a new design tool that significantly speed up and simplify the process of generating plasmid DNA donor templates to enable knocking in the mKate2 reporter into almost any position in the human, mouse, and rat genomes. The Edit-R HDR Donor Designer enables you to easily design the necessary components for rapid assembly of donor plasmids and automatically determines which silent mutations would be most appropriate for the crRNA being employed.
The process of designing your donor template begins with the guide RNA sequence directing the double strand break. If you have not already created a guide RNA and tested for efficient CRISPR Cas9 cutting, we recommend utilizing the CRISPR RNA Configurator to seamlessly integrate with the design of your donor template. Once the gRNA is selected, this sequence can be used to direct the precise location for design of the donor plasmid. The Edit-R HDR Donor Designer will help you quickly design and order the primers necessary for generation of your custom homology arms and the HDR Plasmid Donor Kit contains everything you need to construct and verify the plasmid for introduction of the mKate2 fluorescent reporter into your cells.
Considerations for successful HDR experimental design
Performing endogenous gene tagging using Dharmacon Edit-R Cas9 mRNA, synthetic crRNA-tracrRNA, and an EGFP donor plasmid.
Quickly and easily design and order a donor template for precise CRISPR-Cas9 gene editing!
Design and order a plasmid DNA donor kit for insertion of an mKate2 fluorescent marker