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

Author

Listed:
  • Benjamin C. Buchmuller

    (DKFZ-ZMBH Alliance)

  • Konrad Herbst

    (DKFZ-ZMBH Alliance)

  • Matthias Meurer

    (DKFZ-ZMBH Alliance)

  • Daniel Kirrmaier

    (DKFZ-ZMBH Alliance
    DKFZ-ZMBH Alliance)

  • Ehud Sass

    (Weizmann Institute of Science)

  • Emmanuel D. Levy

    (Weizmann Institute of Science)

  • Michael Knop

    (DKFZ-ZMBH Alliance
    DKFZ-ZMBH Alliance)

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 present 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 avenues for increasing throughput in functional genomics and cell biology research.

Suggested Citation

  • Benjamin C. Buchmuller & Konrad Herbst & Matthias Meurer & Daniel Kirrmaier & Ehud Sass & Emmanuel D. Levy & Michael Knop, 2019. "Pooled clone collections by multiplexed CRISPR-Cas12a-assisted gene tagging in yeast," Nature Communications, Nature, vol. 10(1), pages 1-13, December.
    • Handle: RePEc:nat:natcom:v:10:y:2019:i:1:d:10.1038_s41467-019-10816-7
    DOI: 10.1038/s41467-019-10816-7
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