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


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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Figure 1: Template switching decreases the sensitivity of CRISPR-based single-cell molecular screens that employ linked barcodes.
Figure 2: CROP-seq with PCR enrichment offers improvements over alternate screen designs in a tumor suppressor knockout screen.

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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.

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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.

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Correspondence to Jay Shendure or Cole Trapnell.

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Hill, A., McFaline-Figueroa, J., Starita, L. et al. On the design of CRISPR-based single-cell molecular screens. Nat Methods 15, 271–274 (2018).

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