How can I find guide RNAs and their efficiency scores?
Adding CRISPR and SNP Tracks
The CRISPR and SNP track export is handy if you would like to design guide RNAs and check how they score while ensuring there are no important SNPs to consider.
You can do this by accessing the genome browser from the top navigation banner. Then on the left side choose a CRISPR and SNP track from the tracks dropdown. Note: if you do not have one in your library you can select the gear icon to open the tracks modal and download a track.
Importing Tracks into Photo51
Once you have downloaded and opened SNP and CRISPR tracks, you will see them in the browser view. You then have three options for exporting both back into Photo51. The first option is to click on any gene to select it (this will be indicated by the gene turning red) and then pressing the save button that appears in the information box. Second option is to again select it and then press the “Save gene” in the top banner. For the third option instead of selecting a gene you can select a region by holding down shift and dragging across the desired region. Then select the “Save sequence” button from the top banner.
After clicking save you have the option to select, which guides, SNPs or genes to include in the downloaded sequence. Additionally, you can add flanking sequence to the 5’ and 3’ ends, revers complement the sequence and choose if you want the introns and exons automatically numbered. Once your selection is made select save and you will be prompted to give the sequence a name. By default we autofill the name to be genome assembly followed by the selected gene when downloading genes and simply the coordinates when downloading a region.
Examining CRISPR Cas guide RNAs
Once you have exported the gene/sequence along with a CRISPR track, you can open the file, go to sequence view and you will see all the guides across the sequence color coded according to their scoring. By default the Doench ’16 efficiency score is displayed by the color coding however you can use the View > guide RNAs dropdown or the CRISPR > Show Spacers dropdown to select which of the scorings are displayed by the coloring. These include MIT specificity, Doench ’16 efficiency, Moreno-Mateos efficiency and Out-of-frame. You can hover over each guide to see its exact score.
You can also click on any guide, this will unstack all the guides in that region.
Click on any of the guides and the guide overview modal will open. Here you can change the name of the guide, select which type of Cas system you would be using (only NGG guides are accepted and not all have scores), view the cut site and the seed region, view all the efficiency, specificity and out of frame scores, see if there is a SNP somewhere on the guide (only if SNP track is downloaded) and finally select the whole sgRNA, crRNA or tracrRNA sequence to copy it. If you wish you can save the guide this will display it in a different visual manner and indicate that it is “picked” instead of “candidate” so you can easily find it back later in the guide RNAs table view.
If you would like to get one large overview of all the guideRNAs in one place, then you can navigate in the bottom bar to the Guide RNAs table overview. Here you can see a list of all the guides both candidate and picked and filter by any desired column.
