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In the SureGuide design wizard, SureDesign searches for potential gRNA sequences that are complimentary to suitable sequences within your defined target window. The gRNA sequences that are returned in the gRNA search job meet the following criteria.
· 20 nucleotides in length
· does not contain more mismatches in the seed and non-seed regions than the allowed number of mismatches specified by the user
· if required based on user input, has the sequence GG at nucleotide positions 19 and 20
After locating gRNA sequences that meet this criteria, SureDesign ranks the gRNA sequences according to specificity. The gRNAs are first ranked based on the number of secondary hits within the target window (gRNAs with fewest hits are ranked highest). Then, for gRNAs with the same number of secondary hits, the gRNAs are ranked based on their distance from the start of the target window (gRNAs closest to the start are ranked highest).