Design sgRNAs for CRISPRko (S. pyogenes and S. aureus)

For notes about revisions, updates, and bug fixes please see our sgRNA Design Tool Changelog.

This tool provides designs for CRISPRko applications only. For designs specific to CRISPRa/i applications see our CRISPRa/i Design Tool.

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.

Looking for a downloadable tool to compute CFD scores for existing sgRNA designs? Go here.
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 100 Human or Mouse RefSeq Transcript IDs (e.g., NM_014911, NM_014911.3, etc.), NCBI Gene IDs or Gene Symbols (e.g., 988, CDC5L, etc.), or a single nucleotide sequence of at least 30 bases.

Please refer to our sgRNA Designer Help Page for details on how a transcript is chosen for a gene input.
File inputs must be smaller than 10kb in size, and any sequences submitted via file must be in FASTA format.
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.