Plasmid Donor Design and Ordering
The process of designing your donor template begins with the guide RNA sequence directing the DSB. If you have not already created a guide RNA and tested for efficient CRISPR Cas9 cutting, we recommend utilizing the CRISPR Design Tool before continuing.
In the Edit-R HDR Donor Designer, enter the following information to specify your target region for HDR:
- Organism: Select from over 30 species from the drop-down menu
- Gene target: Enter a gene ID or symbol, select the correct entry from the drop-down
- The gene’s primary transcript will be displayed by default. You may select a different transcript from the drop-down if necessary
- Select a Sequence to Insert; either mKate2, EGFP, or Custom. If you select Custom, you will be prompted to enter the DNA sequence of your custom insert for the purpose of primer design. The DNA insert itself will not be provided (synthesized) as part of your order.
- Enter or paste the 20 nt Guide RNA targeting sequence
- Click Show Insert Region to display the genomic DNA region surrounding the specified gRNA target site
Within the displayed target region of the genomic DNA, you will see the relative position and alignment of the gRNA (grey bar), the PAM sequence (grey arrow tip), the cut site (vertical bold line within grey bar), and a slider to indicate the site of insertion.
- Drag the slider to the desired insertion position
- Click on Generate primers to generate the primer pairs for amplification of the homology arms
- Use checkboxes to select the Forward and Reverse primer pairs for the 5ʹ and 3ʹ homology arms.
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- Note: Only one primer design will be available for the insert-adjacent end of the homology arm. The resulting homology arm length is indicated for each of the primer pairs.
Schematic of HDR plasmid donor assembly
Figure 2 - a visualization of the custom donor plasmid generation workflow. First, custom designed homology arm primers are used to PCR amplify homology arms (with flanking adapters for directional assembly). Second, the homology arms, mKate2 insert and the vector backbone are assembled into the desired donor plasmid.