Pooled clone collections by multiplexed CRISPR/Cas12a-assisted gene tagging in yeast
Clone collections of modified strains (‘libraries’) are a major resource for systematic studies with the yeast <i>Saccharomyces cerevisiae</i>. Construction of such libraries is time-consuming, costly and confined to the genetic background of a specific yeast strain. To overcome these li...
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| Main Authors: | , , , , , , |
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| Format: | Article (Journal) Chapter/Article |
| Language: | English |
| Published: |
November 22, 2018
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| In: |
bioRxiv beta
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| DOI: | 10.1101/476804 |
| Online Access: | Verlag, Volltext: https://doi.org/10.1101/476804 Verlag, Volltext: https://www.biorxiv.org/content/10.1101/476804v1 
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| Author Notes: | Benjamin C. Buchmuller, Konrad Herbst, Matthias Meurer, Daniel Kirrmaier, Ehud Sass, Emmanuel D. Levy, Michael Knop |
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| 520 | |a Clone collections of modified strains (‘libraries’) are a major resource for systematic studies with the yeast <i>Saccharomyces cerevisiae</i>. 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 <i>in vitro</i> 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)</p> | ||
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