Find CRISPRs

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 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.


CRISPR Efficiency Prediction Tool

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.

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.


Making Transgenic Fly

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/).

Gratz SJ, Cummings AM, Nguyen JN, Hamm DC, Donohue LK, Harrison MM, Wildonger J, O'Connor-Giles KM. 2013. Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease. Genetics 194: 1029-1035. doi:10.1534/genetics.113.152710

Kondo S, Ueda R. 2013. Highly improved gene targeting by germline-specific Cas9 expression in Drosophila. Genetics 195: 715-721. doi:10.1534/genetics.113.156737

Perkins LA, Holderbaum L, Tao R, Hu Y, Sopko R, McCall K, Yang-Zhou D, Flockhart I, Binari R, Shim HS et al. 2015. The Transgenic RNAi Project at Harvard Medical School: Resources and Validation. Genetics 201: 843-852. doi:10.1534/genetics.115.180208

Ren X, Sun J, Housden BE, Hu Y, Roesel C, Lin S, Liu LP, Yang Z, Mao D, Sun L et al. 2013. Optimized gene editing technology for Drosophila melanogaster using germ line-specific Cas9. Proc Natl Acad Sci U S A 110: 19012-19017. doi:10.1073/pnas.1318481110

Sebo ZL, Lee HB, Peng Y, Guo Y. 2014. A simplified and efficient germline-specific CRISPR/Cas9 system for Drosophila genomic engineering. Fly (Austin) 8: 52-57. doi:10.4161/fly.26828

Yu Z, Ren M, Wang Z, Zhang B, Rong YS, Jiao R, Gao G. 2013. Highly efficient genome modifications mediated by CRISPR/Cas9 in Drosophila. Genetics 195: 289-291. doi:10.1534/genetics.113.153825

Zirin J, Hu Y, Liu L, Yang-Zhou D, Colbeth R, Yan D, Ewen-Campen B, Tao R, Vogt E, VanNest S et al. 2020. Large-Scale Transgenic Drosophila Resource Collections for Loss- and Gain-of-Function Studies. Genetics 214: 755-767. doi:10.1534/genetics.119.302964