Brief Communication | Published:

On the design of CRISPR-based single-cell molecular screens

Nature Methods volume 15, pages 271274 (2018) | Download Citation

Abstract

Several groups recently coupled CRISPR perturbations and single-cell RNA-seq for pooled genetic screens. We demonstrate that vector designs of these studies are susceptible to 50% swapping of guide RNA–barcode associations because of lentiviral template switching. We optimized a published alternative, CROP-seq, in which the guide RNA also serves as the barcode, and here confirm that this strategy performs robustly and doubled the rate at which guides are assigned to cells to 94%.

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Acknowledgements

We thank all members of the Shendure and Trapnell labs for feedback on our manuscript and helpful discussions, particularly S. Srivatsan, G. Findlay, A. McKenna, R. Daza, B. Martin, M. Kircher, D. Cusanovich, X. Qiu, and V. Ramani. We thank J. Bloom and D. Fowler for discussions about lentivirus, and K. Han, J. Ousey, and M. Bassik for experimental advice and reagents for CRISPRi experiments. A.J.H. thanks Stella the cat for support. This work was supported by the following funding: NIH DP1HG007811 and UM1HG009408 (to J.S.), DP2HD088158 (to C.T.), and the W.M. Keck Foundation (to C.T. and J.S.). A.J.H. and M.J.G. are funded by the National Science Foundation Graduate Research Fellowship. J.L.M. is supported by the NIH Genome Training Grant (5T32HG000035) and the Cardiovascular Research Training Grant (4T32HL007828). C.T. is partly supported by an Alfred P. Sloan Foundation Research Fellowship. J.S. is an Investigator of the Howard Hughes Medical Institute.

Author information

Author notes

    • Andrew J Hill
    •  & José L McFaline-Figueroa

    These authors contributed equally to this work.

    • Jay Shendure
    •  & Cole Trapnell

    These authors jointly directed this work.

Affiliations

  1. Department of Genome Sciences, University of Washington, Seattle, Washington, USA.

    • Andrew J Hill
    • , José L McFaline-Figueroa
    • , Lea M Starita
    • , Molly J Gasperini
    • , Kenneth A Matreyek
    • , Jonathan Packer
    • , Dana Jackson
    • , Jay Shendure
    •  & Cole Trapnell
  2. Howard Hughes Medical Institute, Seattle, Washington, USA.

    • Jay Shendure

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Contributions

A.J.H., J.L.M.-F., J.S., and C.T. devised the project. A.J.H., J.L.M.-F., L.M.S., and M.J.G. performed experiments. D.J. optimized cloning strategies and provided substantial technical support. A.J.H., J.L.M.-F., and J.P. performed analysis. K.A.M. provided critical input on mechanisms of template switching in lentivirus. A.J.H., J.L.M., J.S. and C.T. wrote the manuscript with input from other authors.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Jay Shendure or Cole Trapnell.

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DOI

https://doi.org/10.1038/nmeth.4604

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