PT  - JOURNAL ARTICLE
AU  - Buchmuller, Benjamin C.
AU  - Herbst, Konrad
AU  - Meurer, Matthias
AU  - Kirrmaier, Daniel
AU  - Sass, Ehud
AU  - Levy, Emmanuel D.
AU  - Knop, Michael
TI  - Pooled clone collections by multiplexed CRISPR/Cas12a-assisted gene tagging in yeast
AID  - 10.1101/476804
DP  - 2018 Jan 01
TA  - bioRxiv
PG  - 476804
4099  - http://biorxiv.org/content/early/2018/11/22/476804.short
4100  - http://biorxiv.org/content/early/2018/11/22/476804.full
AB  - 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)