Description

This track displays the subset of the "CRISPR/Cas9 targets" track that begin with a "GG" at the 5' end, making them compatible with T7/T3 promoters or the SP6 promoter. These targets are "in vivo ready" in the sense that no additional bases need to be added to the guide RNA in order to make them compatible with a promoter. All other conventions of the track remain the same: all targets have at least three mismatches relative to all other potential CRISPR/Cas9 sites, and these sites are not specific to the NHGRI-1 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. All targets on this track begin with a GG.

Methods

Potential CRISPR/Cas9 targets were identified by using Bowtie (Langmead et al., 2009) to find all PAM sites in the zebrafish genome, and examining the 20 bp 5' of the PAM. There is some slight cutting activity at PAM sites of the form NAG, in addition to the main NGG PAMs (Hsu et al. 2013). Because of this, targets with NAG PAMs were considered as potential off-targets, but are not reported as main targets. 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. Next, the 20-mers were filtered so those that remained are all at least three mismatches different from all other PAM-adjacent 20-mers. Finally, all 20-mers that did not begin with GG were filtered out.

Credits

These data were produced by the Developmental Genomics Section at the National Human Genome Research Institute. For questions, please email Shawn Burgess or Matthew LaFave.

References

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

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