Step 1. Enter a gene identifier or genome coordinates at the search box.
Step 2. Select relevant track(s) eg. "RNA" and "CRISPRs target CDS/without any off-target". "Off-target" or OT refers to predicted off-targets (the designs presented here are not experimentally validated).
Step 3. Select stringency level for off-target analysis.
Step 4. Once JBrowse is available, zoom in to the region of interest.
Step 5. Click a CRISPR [e.g. "Efficiency:3.99 OT:0 RE:BfuCI FBgn0026379"] to view detailed information e.g. CRISPR sequence, genome coordinates, and predicted off-target gene information.
This 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 sgRNAs. This tool does not evaluate potential off-target effects or gene annotation-related aspects of design.
Input Page
Step 1. Provide sequences by entering them in the text box or by uploading a file (text or Excel file). Users may provide only sequences or sequences with identifiers. An example of the input format is available here.
Step 2. Indicate sequence details by choosing one of the options below.
The tool analyzes the full input sequences (up to 20 bases).
The last three bases are excluded from the analysis.
This option allows users to calculate scores based in a particular region of the input sequences, such as the seed region of the gRNA.
Note: If the input sequence is longer than 20 bp 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. Please note that this tool does not check for potential off-targeted sequences.
The CRISPR/Cas9 system generates double strand breaks (DSBs) that can be used effectively to generate mutations or for genome engineering approaches in flies (Bassett et al. 2013; Gratz et al. 2013; Kondo and Ueda 2013; Ren et al. 2013; Yu et al. 2013; Sebo et al. 2014). The vectors and reagents for transgenic production platform of shRNA and sgRNA lines have been described previously (Perkins et al. 2015; Zirin et al. 2020) and a general overview of our approach is available at https://fgr.hms.harvard.edu/protocols. We also provide the website for scientist to nominate genes for knock-out and/or over-expression gRNA stocks (https://www.flyrnai.org/tools/grna_tracker/web/).
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