Design sgRNAs for CRISPRa and CRISPRi (S. pyogenes and S. aureus)

October 17, 2017: S. aureus Update

Support for S. aureus sgRNA designs has been re-enabled after correcting a bug in its on-target scoring methodology. This bug affected all sgRNAs designed for S. aureus Cas9 using this tool through October 12, 2017. Any S. aureus sgRNAs that had already been produced using this tool prior to that date do have the correct PAM sequences and therefore will generally have some activity, but they are not the optimized designs that are predicted to produce best activity. Therefore, they should not be used for new experiments, but any experiments that may already have been performed should not automatically be discarded.

We sincerely apologize for any inconvenience this may have caused.

NOTE: The S. pyogenes scoring method was not affected by this bug.

This tool ranks and picks candidate CRISPRa/i sgRNA sequences for the gene 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 (CRISPRa/i).

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.

The scope of this tool is currently limited to the S. pyogenes (NGG PAM) and S. aureus (NNGRR PAM) CRISPR Cas9 enzyme families; i.e. only on-target sites that include the appropriate PAM are considered, and off-target CFD scores are reduced for sites that depart from this PAM.
Enter up to 10 Human or Mouse NCBI Gene IDs or Gene Symbols (e.g., 988, CDC5L, etc.). Transcript identifier and sequence inputs are not accepted.
NOTE: you must select a Target Taxon above.

This tool not only ranks all candidate sgRNA sequences for each target, but it will also select (or "pick") the top N such candidates according the raw ranking, as well as other criteria such as cut position and mutual spacing. If you don't care about this, you can safely ignore the "Pick Order" and related columns in the output file, and just use the raw ranking columns as desired.
If checked, then all possible candidates for each target will be returned (the picked ones will still be marked). If unchecked, then the result file will be limited to the sgRNA sequences actually picked to fulfill your quota.