We present GuideScan software for the design of CRISPR guide RNA libraries that can be used to edit coding and noncoding genomic regions. GuideScan produces high-density sets of guide RNAs (gRNAs) for single- and paired-gRNA genome-wide screens. We also show that the trie data structure of GuideScan enables the design of gRNAs that are more specific than those designed by existing tools.
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We thank members of the Ventura and the Leslie laboratories for comments and suggestions. We thank L. Fairchild and R. Pelossof for providing source code for the SplashRNA web server to serve as the backbone for the GuideScan website. This work was supported in part by NIH: grants P30-CA008748 (MSK Core), U01-HG007033 (C.S.L.), U01-HG007893 (C.S.L.), and by grants from the Geoffrey Beene Cancer Research Foundation (A.V.), the Uniting Against Lung Cancer Foundation (A.V.), the Cycle for Survival Foundation (A.V.), the Pershing Square Sohn Cancer Research Alliance (A.V.), and the Lung Cancer Research Foundation (J.A.V.). The GuideScan source code and all associated documentation are deposited at guidescan.com.
The authors declare no competing financial interests.
Integrated supplementary information
(a) Genome-wide density of guides in GuideScan’s murine Cas9 database (blue), compared to the UCSC genome track from mit.edu (red). (b) Dot plot showing specificity scores and number of perfect off-target sites for promiscuous guides designed by the mit.edu gRNA web design tool. Data points are color-coded based on the specificity scores of the corresponding gRNAs following the guidelines of mit.edu web portal (red = low specificity, score 1-19; yellow = medium specificity, score 20-49; green = high specificity, score 50-100).
(a) Uncropped gel of T7 cleavage assay shown in Figure 2d. First lane shows the separations of the 1kb+ (Invitrogen) molecular size ladder. The size of selected bands of the ladder is shown on the left. Numbers above gel refer to gel lanes on which T7 assays were run. Red and Blue bars below gel highlight assays unrelated to this work (red; lanes 1—4), and those shown in Fig. 2 (blue; lanes 5—10); (b) Uncropped gel of PCR shown on Figure 2f; (c) Uncropped gel of PCRs shown in Fig. 2e. Bars below gels in panels b—c highlight the chromosomal region amplified.
Supplementary Figures 1,2 and Supplementary Note 1 (PDF 673 kb)
Off-target reporting by competitor tools for a subset of promiscuous gRNAs. (XLSX 66 kb)
Number of gRNAs with off-targets with 0 or 1 mismatches. (XLSX 78 kb)
Genomic Coordinates used for tool comparison experiment. (XLSX 50 kb)
Supplementary Code (ZIP 27314 kb)
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Perez, A., Pritykin, Y., Vidigal, J. et al. GuideScan software for improved single and paired CRISPR guide RNA design. Nat Biotechnol 35, 347–349 (2017). https://doi.org/10.1038/nbt.3804
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