Design sgRNAs for CRISPRko (S. pyogenes NGG PAM)
This tool ranks and picks candidate sgRNA sequences for the targets provided, while attempting to maximize on-target activity and minimizing off-target activity. For more information about the inputs and outputs of this tool, see How to use the sgRNA Designer (CRISPRko).
On-target scoring is performed using the "Rule Set 2" method described in Doench, Fusi et al., Nature Biotechnology 2016. The current Microsoft implementation of this scoring model is Azimuth 2.0. Off-target sites are evaluated using the CFD (Cutting Frequency Determination) score. Please see How the sgRNA Designer Works for more details on these annotation strategies. For general discussion on sgRNA design, see Addgene. The Brunello and Brie human and mouse libraries using this design methodology and an earlier version of the Rule Set 2 model are available from Addgene.
The scope of this tool is currently limited to the S. pyogenes (NGG) PAM--i.e. only NGG on-target sites are considered, and off-target CFD scores are computed relative to a preferred NGG PAM.