CRISPR Efficiency Prediction Tool Help Page

The tool reports a predicted efficiency score based on nucleotide sequence. The score reflects cumulative p-value for high efficiency based on in vitro data generated using a Drosophila cell line with higher values representing higher efficiency. Scores above 7.5 indicate high efficiency gRNAs. This tool does not evaluate potential off-target effects or gene annotation-related aspects of design.

Detailed information about the algorithm is published in the following paper:
Housden BE, Valvezan AJ, Kelley C, Sopko R, Hu Y, Roesel C, Lin S, Buckner M, Tao R, Yilmazel B, Mohr SE, Manning BD, Perrimon N. (2015) Identification of potential drug targets for tuberous sclerosis complex by synthetic screens combining CRISPR-based knockouts with RNAi. Sci Signal. 2015 Sep 8;8(393):rs9. doi: 10.1126/scisignal.aab3729.

Input Page

• Step 1: Provide sequences by entering them directly into the text box or by uploading a file (text or excel file). Users may provide sequences with identifiers or sequences alone. An example of input format is available here.


• Step 2: Indicate sequence details by choosing one of the options below:
  
  
  
  • PAM is excluded from input sequence.
    The tool analyzes the full input sequences (up to 20 bases).
    
  
  
  • PAM is included - ignore last three bases.
    The last 3 bases are excluded from the analysis.
    
  
  
  • Use partial sequence for analysis.
    This option allows users to calculate scores based on a particular region of the input sequences, such as the seed region of the gRNA.
    

Note that if the input sequence is longer than 20bp without PAM or 23 bp with PAM, only the first 20 nucleotides are analyzed.

Results Page

Predicted efficiency scores for the input sequences, as well as the region used for score calculation, are provided in table format. The table can be viewed on the results page or downloaded. Comments will be included in the results table if there are any ambiguous bases or illegal characters present in the input sequence. The results table also indicates if the U6 terminator sequence (TTTT) is present. If the U6 promoter is used to drive expression of the short guide RNA and the U6 terminator is present, this would result in a nonfunctional gRNA, and thus should be avoided. The results table also indicates if a translational stop codon is present in any of the 3 frames in the input sequence. This can be important for assays involving CRISPR-mediated disruption of a reporter gene. Please note that this tool does not check for potential off-targeted sequences.

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