Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, 69120, Heidelberg, Germany.
Cell Morphogenesis and Signal Transduction, German Cancer Research Center (DKFZ), DKFZ-ZMBH Alliance, 69120, Heidelberg, Germany.
Nat Commun. 2019 Jul 4;10(1):2960. doi: 10.1038/s41467-019-10816-7.
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 ~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.
经过修饰的菌株（“文库”）的克隆集合是进行系统性酵母酿酒酵母研究的主要资源。此类文库的构建耗时、昂贵，且仅限于特定酵母菌株的遗传背景。为了克服这些限制，我们提出了 CRISPR-Cas12a（Cpf1）辅助标签文库工程（CASTLING），用于多重菌株构建。CASTLING 使用微阵列合成的寡核苷酸池和体外重组酶来通过同源重组编程长 DNA 构建体的基因组插入。一次简单的转化就可以得到 >90%的正确标记克隆的混合文库。在单个步骤中可以标记多达数百个基因，并且在基因组规模上，大约一半的基因仅通过约 10 倍的过采样进行标记。我们报告了影响标记成功率的几个参数，并提供了一种定量靶向下一代测序方法来分析此类混合文库。因此，CASTLING 为功能基因组学和细胞生物学研究的通量增加开辟了途径。
