In CRISPR genome engineering, a gRNA directs the Cas9 nuclease to an experimenter-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) that is absolutely required for DNA cleavage. The broadly used S. pyogenes CRISPR system recognizes PAM sequences in the form of NGG. Since the CRISPR system can tolerate mismatches between the gRNA and the genomic target sequence, avoiding CRISPR targets with similar sequences elsewhere in the genome is key to minimizing the potential for off-target cleavage. CRISPR Target Finder is a web tool for identifying CRISPR target sites and evaluating their specificity. Note that Target Finder searches user-supplied sequences rather than reference genomes. In Drosophila, we highly recommend that you sequence the region you are targeting in the line you will be engineering prior to designing your experiment as the presence of SNPs in gRNA target sites may impair cleavage.CRISPR Target Finder uses TagScan (Iseli et al., 2007) and algorithms based on the large-scale analyses of CRISPR-Cas9 specificity in cell lines and animals 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 GATCGATG|TTGAATGCCGAT TGG
In transformed cell lines, CRISPR sequences adjacent to an NAG PAM sequence can also be cleaved at ~20% efficiency. 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.
Gratz, S.J.*, Ukken, F.P.*, Rubinstein, C.D., Thiede, G., Donohue, L.K., Cummings, A.M. and O’Connor-Giles, K.M. (2014). Highly specific and efficient CRISPR/Cas9-catalyzed homology-directed repair in Drosophila. Genetics 196, 961-971.*These authors contributed equally.
Iseli, C., Ambrosini, G., Bucher, P. and Jongeneel, C.V. (2007). Indexing strategies for rapid searches of short words in genome sequences. PLoS ONE 2, e579.