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

• DOI: 10.1038/s41467-019-10816-7
• Publication Date: 2019-07-04T14:18:10Z
• Authors: Benjamin C. Buchm...; Konrad Herbst; Matthias Meurer; Daniel Kirrmaier; Ehud Sass; Emmanuel D. Levy; Michael Knop
• Keywords: clone (Java method)

Abstract Clone collections of modified strains (“libraries”) are a major resource for systematic studies with the yeast Saccharomyces cerevisiae . Construction such libraries is time-consuming, costly and confined to genetic background specific strain. To overcome these limitations, we present CRISPR-Cas12a (Cpf1)-assisted tag library engineering (CASTLING) multiplexed strain construction. CASTLING uses microarray-synthesized oligonucleotide pools in vitro recombineering program genomic insertion long DNA constructs via homologous recombination. One simple transformation yields pooled >90% correctly tagged clones. Up several hundred genes can be single step and, on scale, approximately half all only ~10-fold oversampling. We report parameters that affect tagging success provide quantitative targeted next-generation sequencing method analyze collections. Thus, unlocks avenues increasing throughput functional genomics cell biology research.