Target A Gene
This feature is used to generate all potential guide RNA sequences for a selected gene. The user selects run parameters, runs the program and is presented with a table of scored sgRNA sequences and a genome track view of where the guide RNAs land on the selected gene.
- Select a Strain from the Strain dropdown. The list of supported genes will change based on the strain selected.
- Select a Gene to target from the Gene dropdown.
- Optionally select a PAM sequence to target
- Optionally select a Target Type from the Target Type dropdown. This will change depending on what is supported based on the selected strain.
- Optionally select a spacer length. This defaults to 20.
*** If the genomic track doesn't center on the correct gene, click one of the green Go buttons
*** to center the view on a specific guide RNA. IGV viewer has some quirks about coordinate formatting,
*** sometimes having to do with targets on extreme ends of chromosomes.
Search for off target hits
This feature is used to search a guide RNA sequence against a reference genome to see if any off target hits are predicted.
- Paste a sgRNA sequence into the text field. This requires that the PAM sequence be present.
- Select the PAM sequence used.
- Select the reference genome with which to search for off target hits.
Target a custom sequence
This feature is used to generate guide RNA sequences for a User supplied sequence. This feature can also search for off target hits in the context of a selected genome. If no genome is selected, no off target searching is performed.
- Paste a custom sequence target into the text field. This does not support sequences with ambiguity codes or other formatting, only ATCG's are permitted. Remove any header lines as well.
- Optional: Select the PAM sequence or define desired spacer length.
- Optional: Select the reference genome with which to search for off target hits.
The IGV browser creates indexed files of the gene tracks, and this can result in updated gene tracks not being displayed properly.
If gene tracks do not display gene strand orientation, try clearing your browser's cache. On Chrome this is done by:
- Settings -> Advanced - > Privacy and Security
- Clear browsing data -> Cached images & files