Abstract

CRISPR/Cas9 has revolutionized our ability to engineer genomes and conduct genome-wide screens in human cells1,2,3. Whereas some cell types are amenable to genome engineering, genomes of human pluripotent stem cells (hPSCs) have been difficult to engineer, with reduced efficiencies relative to tumour cell lines or mouse embryonic stem cells3,4,5,6,7,8,9,10,11,12,13. Here, using hPSC lines with stable integration of Cas9 or transient delivery of Cas9-ribonucleoproteins (RNPs), we achieved an average insertion or deletion (indel) efficiency greater than 80%. This high efficiency of indel generation revealed that double-strand breaks (DSBs) induced by Cas9 are toxic and kill most hPSCs. In previous studies, the toxicity of Cas9 in hPSCs was less apparent because of low transfection efficiency and subsequently low DSB induction3. The toxic response to DSBs was P53/TP53-dependent, such that the efficiency of precise genome engineering in hPSCs with a wild-type P53 gene was severely reduced. Our results indicate that Cas9 toxicity creates an obstacle to the high-throughput use of CRISPR/Cas9 for genome engineering and screening in hPSCs. Moreover, as hPSCs can acquire P53 mutations14, cell replacement therapies using CRISPR/Cas9-enginereed hPSCs should proceed with caution, and such engineered hPSCs should be monitored for P53 function.

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Acknowledgements

The authors thank F. Sigoillot for access to the list of sgRNAs with multiple perfect binding sites, M. Morris and A. Hill for help with interactome analysis, and M. Hild for constructive feedback on the project.

Author information

Affiliations

  1. Department of Neuroscience, Novartis Institutes for Biomedical Research, Cambridge, MA, USA

    • Robert J. Ihry
    • , Kathleen A. Worringer
    • , Max R. Salick
    • , Daniel Ho
    • , Kraig Theriault
    • , Sravya Kommineni
    • , Ranjit Randhawa
    • , Tripti Kulkarni
    • , Ricardo Dolmetsch
    •  & Ajamete Kaykas
  2. Department of Chemical Biology and Therapeutics, Novartis Institutes for Biomedical Research, Cambridge, MA, USA

    • Elizabeth Frias
    • , Zinger Yang
    • , Gregory McAllister
    • , Carsten Russ
    • , John Reece-Hoyes
    • , William Forrester
    •  & Gregory R. Hoffman
  3. Department of Oncology, Novartis Institutes for Biomedical Research, Cambridge, MA, USA

    • Julie Chen
  4. Abbvie, Cambridge, MA, USA

    • Marie Sondey
  5. Blueprint Medicine, Cambridge, MA, USA

    • Chaoyang Ye

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Contributions

R.J.I. and A.K. designed all the experiments and wrote the manuscript. R.D. revised the manuscript. R.J.I. designed iCas9 constructs. R.J.I. and S.K. made transgenic cell lines and characterized them. D.H. and C.Y. developed and performed indel analysis of mutated DNA samples. M.S. packaged the 47 individual sgRNAs, and K.A.W. tested them. K.T. helped with live cell imaging of confluence. E.F., G.R.H. and G.M. helped with the design of the pooled screen, execution and analysis. J.R.-H. generated sgRNA libraries. C.R. sequenced pooled screen samples. G.R.H., G.M., Z.Y. and W.F. provided access and analysed non-targeting control data across transformed cell lines. T.K. identified sgRNAs with SNPs in the H1-hESC genome. J.C. prepped RNA samples for RNA-seq experiments. R.R. performed RNA-seq and interactome analysis. T.K. analysed SNP and multicutter data. M.R.S. conducted high-content image analysis. K.A.W. helped design and performed the OCT4 HDR assay

Competing interests

All authors were employees of Novartis Institutes for Biomedical Research when the research was conducted.

Corresponding author

Correspondence to Ajamete Kaykas.

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figures 1–6 and Supplementary Tables 1–4

  2. Reporting Summary

  3. Supplementary Data 1

    pAAVS1-iCas9 plasmid map

  4. Supplementary Data 2

    pAAVS1-ieCas9 plasmid map

  5. Supplementary Data 3

    pB-iNgn2-cDDCas9 plasmid map

  6. Supplementary Data 4

    13K pooled screen in hPSCs

  7. Supplementary Data 5

    DSB day 2 differential expression analysis

  8. Supplementary Data 6

    hOCT4-C-LinkHA-tdTomato-Donor plasmid map

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DOI

https://doi.org/10.1038/s41591-018-0050-6

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