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Flexible guide-RNA design for CRISPR applications using Protospacer Workbench

Nature Biotechnology volume 33, pages 805–806 (2015)Cite this article

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The CRISPR-Cas9 endonuclease system permits genome editing at nucleotide resolution, but ensuring high efficiency while minimizing off-target cleavage requires careful design of the single guide RNA (sgRNA). Currently most researchers use online tools (e.g., E-CRISP1, CRISPR2, ZiFiT3) for sgRNA design. However, these tools have several drawbacks: the number of target-sgRNA mismatches allowed is limited; they are unable to detect every potential off-target site; they are generally limited in flexibility and throughput; and they are available for only a few organisms. Offline solutions such as sgRNAcas9 (ref. 4) provide greater flexibility and speed but require knowledge of programming and/or command-line interfaces. Neither online nor offline software facilitates sharing of sgRNA libraries, their putative off-target sites or the statistical reports that lead to successful designs.

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Figure 1: Typical Protospacer Workbench users first obtain a useful Protospacer database from www.protospacer.com or collaborators, or build a custom database.

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Acknowledgements

The authors would like to thank M. Ghorbal for extensive testing and recommendations. This work was supported by the French Parasitology consortium ParaFrap (ANR-11-LABX0024) ERC Advanced grant (PlasmoEscape 250320).

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Authors and Affiliations

  1. Biology of Host-Parasite Interactions Unit, Institut Pasteur, Paris, France

    Cameron Ross MacPherson & Artur Scherf

  2. CNRS, Paris, ERL 9195, France

    Cameron Ross MacPherson & Artur Scherf

  3. INSERM, Unit U1201, Paris, France

    Cameron Ross MacPherson & Artur Scherf

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  1. Cameron Ross MacPherson
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  2. Artur Scherf
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Correspondence to Cameron Ross MacPherson or Artur Scherf.

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MacPherson, C., Scherf, A. Flexible guide-RNA design for CRISPR applications using Protospacer Workbench. Nat Biotechnol 33, 805–806 (2015). https://doi.org/10.1038/nbt.3291

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