Validating genome-wide CRISPR-Cas9 function improves screening in the oleaginous yeast Yarrowia lipolytica
Validating genome-wide CRISPR-Cas9 function improves screening in the oleaginous yeast Yarrowia lipolytica
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DOI：
10.1016/j.ymben.2019.06.007
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发表时间：
2019-09-01
影响因子：
8.4
通讯作者：
Wheeldon, Ian
中科院分区：
文献类型：
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作者：
Schwartz, Cory;Cheng, Jan-Fang;Wheeldon, Ian
全基因组突变筛选对于理解进化和工程表型的遗传基础至关重要。CRISPR-Cas9基因组编辑的广泛采用使这种筛选在许多生物体中成为可能，但鉴定功能性sgRNA仍然是一个挑战。在这里，我们开发了一种方法来量化基因组规模库中每个sgRNA的切割效率，并通过这样做来改善生物技术上重要的酵母耶氏酵母的筛选。在存在和不存在天然DNA修复的情况下进行筛选使得能够高通量定量sgRNA功能，从而鉴定出覆盖94%基因的高效sgRNA。文库验证通过识别产生假阴性和掩盖成功破坏影响的非活性指导来增强必需基因的分类。指导有效性的量化还创建了一个数据集，从中可以鉴定CRISPR-Cas9的决定因素。最后，应用该文库鉴定了用于高脂质积累的代谢工程的新突变。
Genome-wide mutational screens are central to understanding the genetic underpinnings of evolved and engineered phenotypes. The widespread adoption of CRISPR-Cas9 genome editing has enabled such screens in many organisms, but identifying functional sgRNAs still remains a challenge. Here, we developed a methodology to quantify the cutting efficiency of each sgRNA in a genome-scale library, and in doing so improve screens in the biotechnologically important yeast Yarrowia kpolytica. Screening in the presence and absence of native DNA repair enabled high-throughput quantification of sgRNA function leading to the identification of high efficiency sgRNAs that cover 94% of genes. Library validation enhanced the classification of essential genes by identifying inactive guides that create false negatives and mask the effects of successful disruptions. Quantification of guide effectiveness also creates a dataset from which determinants of CRISPR-Cas9 can be identified. Finally, application of the library identified novel mutations for metabolic engineering of high lipid accumulation.