TY - JOUR T1 - Pooled clone collections by multiplexed CRISPR/Cas12a-assisted gene tagging in yeast JF - bioRxiv DO - 10.1101/476804 SP - 476804 AU - Benjamin C. Buchmuller AU - Konrad Herbst AU - Matthias Meurer AU - Daniel Kirrmaier AU - Ehud Sass AU - Emmanuel D. Levy AU - Michael Knop Y1 - 2018/01/01 UR - http://biorxiv.org/content/early/2018/11/22/476804.abstract N2 - 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) ER -