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CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo

Nature Methods volume 12, pages 982988 (2015) | Download Citation

Abstract

CRISPR-Cas9 technology provides a powerful system for genome engineering. However, variable activity across different single guide RNAs (sgRNAs) remains a significant limitation. We analyzed the molecular features that influence sgRNA stability, activity and loading into Cas9 in vivo. We observed that guanine enrichment and adenine depletion increased sgRNA stability and activity, whereas differential sgRNA loading, nucleosome positioning and Cas9 off-target binding were not major determinants. We also identified sgRNAs truncated by one or two nucleotides and containing 5′ mismatches as efficient alternatives to canonical sgRNAs. On the basis of these results, we created a predictive sgRNA-scoring algorithm, CRISPRscan, that effectively captures the sequence features affecting the activity of CRISPR-Cas9 in vivo. Finally, we show that targeting Cas9 to the germ line using a Cas9-nanos 3′ UTR led to the generation of maternal-zygotic mutants, as well as increased viability and decreased somatic mutations. These results identify determinants that influence Cas9 activity and provide a framework for the design of highly efficient sgRNAs for genome targeting in vivo.

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Acknowledgements

We thank E. Fleming and H. Codore for technical help; D. Cifuentes, A. Bazzini and M. Lee for discussions; all the members of the Giraldez laboratory for intellectual and technical support; and S. Lau, M. Lee and M. Fernandez-Fuertes for cloning of nanos 3′ UTR, help with MNase analysis and help with pictures of the adult fish, respectively. We thank C. Takacs, M. Lee and K. Divito for manuscript editing. Supported by the Swiss National Science Foundation (grant P2GEP3_148600 to C.E.V.), Programa de Movilidad en Áreas de Investigación priorizadas por la Consejería de Igualdad, Salud y Políticas Sociales de la Junta de Andalucía (M.A.M.-M.), the Fonds de Recherche du Québec (grant 29818 to J.-D.B.) and the US National Institutes of Health (grants R21 HD073768, R01 GM103789, R01 GM102251, R01 GM101108 and GM081602 to A.J.G. and grant R01 HD081379 to E.K.M. and M.K.K.). M.K.K. is supported by the Edward Mallinckrodt Jr. Foundation.

Author information

Author notes

    • Miguel A Moreno-Mateos
    •  & Charles E Vejnar

    These authors contributed equally to this work.

Affiliations

  1. Department of Genetics, Yale University School of Medicine, New Haven, Connecticut, USA.

    • Miguel A Moreno-Mateos
    • , Charles E Vejnar
    • , Jean-Denis Beaudoin
    • , Juan P Fernandez
    • , Emily K Mis
    • , Mustafa K Khokha
    •  & Antonio J Giraldez
  2. Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut, USA.

    • Emily K Mis
    •  & Mustafa K Khokha
  3. Yale Stem Cell Center, Yale University School of Medicine, New Haven, Connecticut, USA.

    • Antonio J Giraldez

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Contributions

M.A.M.-M., C.E.V. and A.J.G. designed the project, performed experiments and data analysis and wrote the manuscript. J.-D.B. created the sgRNA libraries, performed G-quadruplex experiments and helped write part of the manuscript. J.P.F. performed F0 phenotype analysis and Xenopus phenotype analysis with M.A.M.-M. E.K.M. carried out Xenopus injections and phenotype analysis. M.K.K. provided reagents and materials.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Antonio J Giraldez.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figure 1–9

  2. 2.

    Supplementary Data Set 1

    Schematic representation of deletions and insertions found on 128 targeted loci by canonical sgRNAs

  3. 3.

    Supplementary Data Set 2

    Schematic representation of deletions and insertions found on 64 targeted loci by alternative sgRNAs

Excel files

  1. 1.

    Supplementary Table 1

    Oligo sequences

  2. 2.

    Supplementary Table 2

    Formation of G-quadruplex in sgRNAs

  3. 3.

    Supplementary Table 3

    Parameters of CRISPRscan model

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DOI

https://doi.org/10.1038/nmeth.3543

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