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On the design of CRISPR-based single cell molecular screens

View ORCID ProfileAndrew J. Hill, José L. McFaline-Figueroa, Lea M. Starita, View ORCID ProfileMolly J. Gasperini, View ORCID ProfileKenneth A. Matreyek, Jonathan Packer, Dana Jackson, View ORCID ProfileJay Shendure, View ORCID ProfileCole Trapnell
doi: https://doi.org/10.1101/254334
Andrew J. Hill
1Department of Genome Sciences, University of Washington, Seattle, WA, USA
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José L. McFaline-Figueroa
1Department of Genome Sciences, University of Washington, Seattle, WA, USA
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Lea M. Starita
1Department of Genome Sciences, University of Washington, Seattle, WA, USA
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Molly J. Gasperini
1Department of Genome Sciences, University of Washington, Seattle, WA, USA
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Kenneth A. Matreyek
1Department of Genome Sciences, University of Washington, Seattle, WA, USA
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Jonathan Packer
1Department of Genome Sciences, University of Washington, Seattle, WA, USA
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Dana Jackson
1Department of Genome Sciences, University of Washington, Seattle, WA, USA
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Jay Shendure
1Department of Genome Sciences, University of Washington, Seattle, WA, USA
2Howard Hughes Medical Institute, Seattle, WA, USA
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Cole Trapnell
1Department of Genome Sciences, University of Washington, Seattle, WA, USA
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Abstract

Several groups recently reported coupling CRISPR/Cas9 perturbations and single cell RNA-seq as a potentially powerful approach for forward genetics. Here we demonstrate that vector designs for such screens that rely on cis linkage of guides and distally located barcodes suffer from swapping of intended guide-barcode associations at rates approaching 50% due to template switching during lentivirus production, greatly reducing sensitivity. We optimize a published strategy, CROP-seq, that instead uses a Pol II transcribed copy of the sgRNA sequence itself, doubling the rate at which guides are assigned to cells to 94%. We confirm this strategy performs robustly and further explore experimental best practices for CRISPR/Cas9-based single cell molecular screens.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-ND 4.0 International license.
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Posted February 12, 2018.
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On the design of CRISPR-based single cell molecular screens
Andrew J. Hill, José L. McFaline-Figueroa, Lea M. Starita, Molly J. Gasperini, Kenneth A. Matreyek, Jonathan Packer, Dana Jackson, Jay Shendure, Cole Trapnell
bioRxiv 254334; doi: https://doi.org/10.1101/254334
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On the design of CRISPR-based single cell molecular screens
Andrew J. Hill, José L. McFaline-Figueroa, Lea M. Starita, Molly J. Gasperini, Kenneth A. Matreyek, Jonathan Packer, Dana Jackson, Jay Shendure, Cole Trapnell
bioRxiv 254334; doi: https://doi.org/10.1101/254334

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