Description

This track displays the C. jejuni CRISPR/Cas9 targets with at least three mismatches relative to all other potential C. jejuni CRISPR/Cas9 sites. It is based on the Zv9/danRer7 assembly, and is not specific to the NHGRI-1 fish line.

Display Conventions and Configuration

CRISPR targets are represented as the 20 bp adjacent to the protospacer adjacent matrix (PAM). The orientation of the target is indicated by the arrows in the span. If viewed in "pack" or "full" view, the sequence of the target will be presented in 5' to 3' orientation, as well as an integer that gives an indirect measure of the off-target activity (see below). The shading of the targets corresponds to this integer, such that darker targets are less likely to have off-target activity.

Methods

Potential CRISPR/Cas9 targets were identified by using crisprTrack. crisprTrack used Bowtie (Langmead et al., 2009) to find all PAM sites in the zebrafish genome, and the 20 bp 5' of the PAM were examined. Only the 'NNNNACA' PAM was considered, as there was no information on off-target PAMs with sub-optimal cutting efficiency (Fonfara et al., 2014). The "seed" region - the 12 bp directly adjacent to the PAM - of each site was evaluated to determine the number of other PAM-adjacent seeds that it matched with fewer than two mismatches. This value is the integer visible in "pack" or "full" view. While this isn't a direct measure of the number of off-targets of the 20-mer as a whole, a lower number suggests fewer off-targets overall. Finally, the 20-mers were filtered so those that remained are all at least three mismatches different from all other PAM-adjacent 20-mers. The mismatch cutoffs were derived from Hsu et al., 2013.

Credits

These data were produced by the Developmental Genomics Section at the National Human Genome Research Institute of the National Institutes of Health. For questions, please email Shawn Burgess.

References

Fonfara I, Le Rhun A, Chylinski K, Makarova KS, Lecrivain AL, Bzdrenga J, Koonin EV, Charpentier E. Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems. Nucleic Acids Res. 2014 Feb;42(4):2577-90. PMID: 24270795; PMC: PMC3936727

Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu X, Shalem O, Cradick TJ, Marraffini LA, Bao G, Zhang F. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol. 2013 Sep;31(9):827-32. PMID: 23873081; PMC: PMC3969858

LaFave MC, Burgess SM. crisprTrack version 1.0.0. Zenodo. 2015. DOI: 10.5281/zenodo.17607

Langmead B, Trapnell C, Pop M, Salzberg SL. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 2009;10(3):R25. PMID: 19261174; PMC: PMC2690996