DRSC CRISPRs Search Page

Step 1. Enter a gene identifier or genome coordinates at the search box. Gene identifiers: FlyBase gene id (FBgn), CG number or gene symbol (not full name). Genome coordinates: to search with a specific sequence, please first use BLAST at FlyBase to get the genome coordinates, then enter the coordinates at the search box (eg. X:3,028,903..3,066,254 [+]).

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 re not experimentally validated).

Step 3. Select PAM sequences to include.

Step 4. Select stringency level for off-target analysis.

Step 5. Zoom in to the region of interest.

Step 6. Click a CRISPR [e.g. "Efficiency:3.99 OT:0 RE:BfuCI FBgn0026379"] to view detailed information e.g. CRISPR sequence, genome coordinate and predicted off-target gene information.

Step 7. Right click each CRISPR of interest to add to a work list

Step 8. To save the information on worklist, copy/paste to another file eg txt file then save.

CRISPR Efficiency Prediction Tool

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.

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.

• 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 three bases are excluded from the analysis.

• Use partial sequence for 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.