Each insert screening assay performed using the Guide-it Knockin Screening Kit involves a set of oligos designed to detect seamless, error-free incorporation of a designated insert sequence at a specific genomic target site. You will need to input (in the 5'→3' orientation) four sequence fragments that correspond to the 5' and 3' junctions of the insert and genomic target site following successful editing. (Please see figure below for further clarification.)

• To generate the oligos to detect the 5' junction:
  • Enter the wild-type sequence 5' upstream of the insertion (1)
  • Enter the 5' terminal sequence of the insert (2)

• To generate the oligos to detect the 3' junction:
  • Enter the 3' terminal sequence of the insert (3)
  • Enter the wild-type sequence 3' downstream of the insertion (4)

Please note that the following conditions must be satisfied in order to use the tool successfully:

• Input sequences cannot exceed 1,000 bases
• Input sequences should consist of at least 35 bases

Following submission of the four sequences, the oligo design tool will output displacement, flap-probe, wild-type control, and knockin control oligos for analysis of both the 5' and 3' junctions.

Users can return to the input screen by refreshing the page or clicking the [Back] button of the web browser.

Generation of the final edited 5' and 3' junctions

The inputs are first validated to ensure they meet all the requirements mentioned above. The top of the results page will display the sequences of both 5' and 3' junctions with the insert sequences depicted in lowercase. The user can check if the introduced sequences are correct. An example of this output is shown below:

Probe and control oligos

Sequences of the following four pairs of oligos are provided in a ready-to-order format:

• 2 x Displacement oligos (presented below in purple with the noncomplementary base indicated by underline)
• 2 x Flap-probe oligos (presented below in green for the 5' junction and orange for the 3' junction, with the 5' fixed sequences indicated by underline and the hexanediol modifications indicated by "3C6" in black font)
• 2 x Knockin control oligos (presented in blue with the insert sequence indicated in dark blue and in lowercase)
• 2 x Wild-type control oligos (presented in blue)

Depending on the input sequences, the tool will design oligos according to either Design 1 or Design 2. At the bottom of each output, the tool will indicate which design was employed and present a color-coded diagram indicating the orientations of the design at the 5' and 3' junctions. For more detailed information about the oligo design process for the Guide-it Knockin Screening Kit, please refer to the product user manual. For more insights about the Guide-it Knockin Screening Kit assay, please refer to our FAQs page.

While we have attempted to make the oligo design tool as simple and straightforward as possible, users may run into difficulties that are technical or biological in nature. In either case, please reach out to our technical support team for assistance with your designs. Providing additional information such as any error messages encountered, and snapshots of the corresponding inputs and outputs will be extremely helpful in these instances. For all support questions, please contact a Technical Support Scientist through our support page or the Live Chat option on the bottom right corner of our web pages.