@article{4f82009a51bf4922a96ace27902eefcf,
title = "Pooled clone collections by multiplexed CRISPR-Cas12a-assisted gene tagging in yeast",
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 similar to 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.",
author = "Buchmullen, {Benjamin C.} and Konrad Herbst and Matthias Meurer and Daniel Kirrmaier and Ehud Sass and Levy, {Emmanuel D.} and Michael Knop",
note = "The authors wish to thank Ilia Kats, Cyril Mongis, and Krisztina Gubicza for help with IT infrastructure and experiments. We acknowledge support by the Deutsche Forschungsgemeinschaft (DFG KN498/12–1), the state of Baden-W{\"u}rttemberg through bwHPC for high-performance computing and SDS@hd for data storage (grant INST 35/1314–1 FUGG), and the Dietmar Hopp foundation. K.H. was supported by a HBIGS graduate school fellowship. We also acknowledge help from the Flow Cytometry Core Facility at ZMBH, and the Deep Sequencing Core Facility of the University of Heidelberg, both of which are supported by the CellNetworks cluster of excellence. E.D.L. acknowledges support from A.-M. Boucher, from the Estelle Funk Foundation, the Estate of Fannie Sherr, the Estate of Albert Delighter, the Merle S. Cahn Foundation, Mrs. Mildred S. Gosden, the Estate of Elizabeth Wachsman, the Arnold Bortman Family Foundation. E.D.L. is incumbent of the Recanati Career Development Chair of Cancer Research. Contributions - M.K. conceived the project. M.K., B.C.B., K.H., and M.M. designed the experiments and B.C.B., K.H., M.M., and D.K. performed the experiments. E.D.L. and E.S. contributed methods. K.H., B.C.B., M.K., and M.M. analyzed the data. M.K. and B.C.B. wrote the manuscript. All authors read and approved the final manuscript.",
year = "2019",
month = jul,
day = "4",
doi = "https://doi.org/10.1038/s41467-019-10816-7",
language = "الإنجليزيّة",
volume = "10",
journal = "Nature Communications",
issn = "2041-1723",
publisher = "Nature Publishing Group",
}