Pooled clone collections by multiplexed CRISPR-Cas12a-assisted gene tagging in yeast
Pooled clone collections by multiplexed CRISPR-Cas12a-assisted gene tagging in yeast
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DOI：
10.1038/s41467-019-10816-7
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发表时间：
2019-07-04
影响因子：
16.6
通讯作者：
Knop, Michael
中科院分区：
文献类型：
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作者：
Buchmullen, Benjamin C.;Herbst, Konrad;Knop, Michael
修饰菌株的克隆集合（“文库”）是用酵母酿酒酵母进行系统研究的主要资源。这种文库的构建是耗时的，昂贵的，并且局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-Cas 12 a（Cpf 1）辅助的标签库工程（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工程来通过同源重组编程长DNA构建体的基因组插入。一个简单的转化产生具有>90%的正确标记的克隆的合并文库。在一个步骤中可以标记多达数百个基因，并且在基因组规模上，大约一半的基因仅以类似于10倍的过采样进行标记。我们报告了影响标记成功的几个参数，并提供了一个定量的有针对性的下一代测序方法来分析这种汇集的集合。因此，CASTLING开启了增加功能基因组学和细胞生物学研究通量的途径。
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.