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 and Michael Knop ()
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Benjamin C. Buchmuller: DKFZ-ZMBH Alliance
Konrad Herbst: DKFZ-ZMBH Alliance
Matthias Meurer: DKFZ-ZMBH Alliance
Daniel Kirrmaier: DKFZ-ZMBH Alliance
Ehud Sass: Weizmann Institute of Science
Emmanuel D. Levy: Weizmann Institute of Science
Michael Knop: DKFZ-ZMBH Alliance

Nature Communications, 2019, vol. 10, issue 1, 1-13

Abstract: 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.

Date: 2019
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DOI: 10.1038/s41467-019-10816-7

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