Protocol | Published:

Integrated design, execution, and analysis of arrayed and pooled CRISPR genome-editing experiments

Nature Protocols volume 13, pages 946986 (2018) | Download Citation

Abstract

CRISPR (clustered regularly interspaced short palindromic repeats) genome-editing experiments offer enormous potential for the evaluation of genomic loci using arrayed single guide RNAs (sgRNAs) or pooled sgRNA libraries. Numerous computational tools are available to help design sgRNAs with optimal on-target efficiency and minimal off-target potential. In addition, computational tools have been developed to analyze deep-sequencing data resulting from genome-editing experiments. However, these tools are typically developed in isolation and oftentimes are not readily translatable into laboratory-based experiments. Here, we present a protocol that describes in detail both the computational and benchtop implementation of an arrayed and/or pooled CRISPR genome-editing experiment. This protocol provides instructions for sgRNA design with CRISPOR (computational tool for the design, evaluation, and cloning of sgRNA sequences), experimental implementation, and analysis of the resulting high-throughput sequencing data with CRISPResso (computational tool for analysis of genome-editing outcomes from deep-sequencing data). This protocol allows for design and execution of arrayed and pooled CRISPR experiments in 4–5 weeks by non-experts, as well as computational data analysis that can be performed in 1–2 d by both computational and noncomputational biologists alike using web-based and/or command-line versions.

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Acknowledgements

M.C.C. was supported by a National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Award (F30DK103359). M.H. was funded by National Institutes of Health (NIH)/National Human Genome Research Institute (NHGRI) grant 5U41HG002371-15 and NIH/National Cancer Institute (NCI) grant 5U54HG007990-02 and by a grant from the California Institute of Regenerative Medicine, CIRM GC1R-06673C. D.E.B. was supported by the National Heart, Lung, and Blood Institute (NHLBI) (DP2OD022716, P01HL032262), the Burroughs Wellcome Fund, and a Doris Duke Charitable Foundation Innovations in Clinical Research Award. S.H.O. was supported by an award from the NHLBI (P01HL032262) and an award from the NIDDK (P30DK049216, Center of Excellence in Molecular Hematology). N.E.S. was supported by the NIH through the NHGRI (R00-HG008171). L.P. was supported by an NHGRI Career Development Award (R00HG008399) and the Defense Advanced Research Projects Agency (HR0011-17-2-0042).

Author information

Author notes

    • Matthew C Canver
    •  & Maximilian Haeussler

    These authors contributed equally to this work.

Affiliations

  1. Molecular Pathology Unit and Cancer Center, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA.

    • Matthew C Canver
    •  & Luca Pinello
  2. Santa Cruz Genomics Institute, University of California, Santa Cruz, Santa Cruz, California, USA.

    • Maximilian Haeussler
  3. Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, USA.

    • Daniel E Bauer
    •  & Stuart H Orkin
  4. Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Stem Cell Institute, Harvard University, Boston, Massachusetts, USA.

    • Daniel E Bauer
    •  & Stuart H Orkin
  5. Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, USA.

    • Daniel E Bauer
    •  & Stuart H Orkin
  6. Howard Hughes Medical Institute, Boston, Massachusetts, USA.

    • Stuart H Orkin
  7. New York Genome Center and Department of Biology, New York University, New York, New York, USA.

    • Neville E Sanjana
  8. Raymond G. Perelman Center for Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.

    • Ophir Shalem
  9. Department of Genetics, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

    • Ophir Shalem
  10. Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute and Harvard T.H. Chan School of Public Health, Boston, Massachusetts, USA.

    • Guo-Cheng Yuan
  11. The Broad Institute, Cambridge, Massachusetts, USA.

    • Feng Zhang
    •  & Luca Pinello
  12. McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Feng Zhang
  13. Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Feng Zhang
  14. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Feng Zhang
  15. INSERM U1154, CNRS UMR 7196, Muséum National d'Histoire Naturelle, Paris, France.

    • Jean-Paul Concordet

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Contributions

M.C.C., M.H., and L.P. conceived this project. M.H. and J.-P.C. created CRISPOR. L.P., M.C.C., D.E.B., and G.-C.Y. created CRISPResso. M.C.C. and D.E.B. performed the experiments. M.C.C., D.E.B., S.H.O., N.E.S., O.S., G.-C.Y., F.Z., and L.P. analyzed the experimental data. M.C.C., M.H., and L.P. wrote the manuscript with input from all authors.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Luca Pinello.

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https://doi.org/10.1038/nprot.2018.005

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