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Pooled clone collections by multiplexed CRISPR/Cas12a-assisted gene tagging in yeast

Benjamin C. Buchmuller, Konrad Herbst, Matthias Meurer, Daniel Kirrmaier, Ehud Sass, Emmanuel D. Levy, Michael Knop
doi: https://doi.org/10.1101/476804
Benjamin C. Buchmuller
1Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany.
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Konrad Herbst
1Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany.
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Matthias Meurer
1Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany.
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Daniel Kirrmaier
1Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany.
3Cell Morphogenesis and Signal Transduction, German Cancer Research Center (DKFZ), DKFZ-ZMBH Alliance, Heidelberg, Germany.
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Ehud Sass
2Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel.
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Emmanuel D. Levy
2Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel.
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Michael Knop
1Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany.
3Cell Morphogenesis and Signal Transduction, German Cancer Research Center (DKFZ), DKFZ-ZMBH Alliance, Heidelberg, Germany.
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Abstract

Clone collections of modified strains (‘libraries’) are a major resource for systematic studies with the yeast Saccharomyces cerevisiae. Construction of such libraries is time-consuming, costly and confined to the genetic background of a specific yeast strain. To overcome these limitations, we developed CRISPR/Cas12a (Cpf1)-assisted tag library engineering (CASTLING) for multiplexed strain construction. CASTLING uses microarray-synthesized oligonucleotide pools and in vitro recombineering to program the genomic insertion of long DNA constructs via homologous recombination. One simple transformation yields pooled libraries with >90% of correctly tagged clones. Up to several hundred genes can be tagged in a single step and, on a genomic scale, approximately half of all genes are tagged with only ~10-fold oversampling. We report several parameters that affect tagging success and provide a quantitative targeted next-generation sequencing method to analyze such pooled collections. Thus, CASTLING unlocks new avenues for increased throughput in functional genomics and cell biology research. (max 150 words)

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  • Competing financial interest statement. The authors declare no competing financial interests.

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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-NC 4.0 International license.
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Posted November 22, 2018.
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Pooled clone collections by multiplexed CRISPR/Cas12a-assisted gene tagging in yeast
Benjamin C. Buchmuller, Konrad Herbst, Matthias Meurer, Daniel Kirrmaier, Ehud Sass, Emmanuel D. Levy, Michael Knop
bioRxiv 476804; doi: https://doi.org/10.1101/476804
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Pooled clone collections by multiplexed CRISPR/Cas12a-assisted gene tagging in yeast
Benjamin C. Buchmuller, Konrad Herbst, Matthias Meurer, Daniel Kirrmaier, Ehud Sass, Emmanuel D. Levy, Michael Knop
bioRxiv 476804; doi: https://doi.org/10.1101/476804

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