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.
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 . 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.
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