flyCRISPR Optimal Target Finder

In CRISPR/Cas9-mediated genome engineering, a chimeric guide RNA (gRNA) directs the Cas9 nuclease to a user-specified DNA sequence through basepairing to 20-nt CRISPR target sites. CRISPR target sites must be located next to a 3-nt protospacer adjacent motif (PAM) required for DNA cleavage. The S. pyogenes CRISPR/Cas9 system currently in use in Drosophila recognizes PAM sequences in the form NGG. Since the CRISPR/Cas9 system can tolerate mismatches between the guide RNA and 20-nt CRISPR target sequence, avoiding CRISPR targets with similar sequences elsewhere in the genome is key to minimizing the potential for off-target cleavage. flyCRISPR Target Finder is a web tool for identifying CRISPR target sites and evaluating their specificity using transparent rules based on empirical data in the literature so you can easily select the best target(s) for your project. 

flyCRISPR Target Finder uses TagScan [1] and algorithms based on the large-scale analyses of CRISPR/Cas9 specificity in cell lines [2-6] and animals [7,8] published to date to identify potential off-target cleavage sites for a given CRISPR target. These studies demonstrate that the PAM-proximal region of the CRISPR target sequence (also referred to as the “seed”) is more critical for specificity than the distal 8 nucleotides (Fig. 1). Our algorithms consider both the number and location of mismatches to evaluate all potential off-target cleavage sites.

Example target sequence:

Distal   Proximal     PAM

In transformed cell lines, CRISPR sequences adjacent to an NAG PAM sequence can also be cleaved at ~20% efficiency [4]. This has not been observed in animals to date, so the program allows the user to choose whether or not NAG-adjacent sites are considered in the evaluation of CRISPR target sequence specificity.

The rules behind our algorithms are detailed in the user manual and will be updated as new data becomes available.


1. Iseli, C., Ambrosini, G., Bucher, P. & Jongeneel, C.V. Indexing strategies for rapid searches of short words in genome sequences. PLoS ONE 2, e579 (2007).

2. Cradick, T.J., Fine, E.J., Antico, C.J. & Bao, G. CRISPR/Cas9 systems targeting beta-globin and CCR5 genes have substantial off-target activity. Nucleic Acids Res (2013).

 3. Fu, Y. et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol (2013).

4. Hsu, P.D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol (2013).

5. Mali, P. et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol 31, 833-8 (2013).

6. Pattanayak, V. et al. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat Biotechnol (2013).

7. Chiu, H., Schwartz, H.T., Antoshechkin, I. & Sternberg, P.W. Transgene-Free Genome Editing in Caenorhabditis elegans Using CRISPR-Cas. Genetics (2013).

8. Yang, H. et al. One-Step Generation of Mice Carrying Reporter and Conditional Alleles by CRISPR/Cas-Mediated Genome Engineering. Cell 154, 1370-9 (2013).