期刊
NATURE COMMUNICATIONS
卷 10, 期 -, 页码 -出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/s41467-019-10816-7
关键词
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资金
- Deutsche Forschungsgemeinschaft [DFG KN498/12-1]
- state of Baden-Wurttemberg through bwHPC [INST 35/1314-1 FUGG]
- Dietmar Hopp foundation
- HBIGS graduate school fellowship
- CellNetworks cluster of excellence
- Estelle Funk Foundation
- Estate of Fannie Sherr
- Estate of Albert Delighter
- Merle S. Cahn Foundation
- Mrs. Mildred S. Gosden
- Estate of Elizabeth Wachsman
- Arnold Bortman Family Foundation
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.
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