Highly efficient CRISPR gene editing in yeast enabled by double selection
Highly efficient CRISPR gene editing in yeast enabled by double selection
复制标题
通过双重选择实现酵母中高效 CRISPR 基因编辑
DOI：
10.1101/262808
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发表时间：
2018
期刊：
影响因子：
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通讯作者：
Landry Christian R
中科院分区：
文献类型：
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作者：
Despres Philippe C;Dube Alexandre K;Nielly-Thibault Lou;Yachie Nozomu;Landry Christian R
CRISPR-Cas9功能丧失（LOF）筛选是一种强大的系统生物学工具，可以进行全基因组干扰研究。虽然目前在许多模式物种中是可能的，但这种方法尚未在酵母中开发，因为在没有供体DNA用于同源定向修复的情况下诱变率低。在这里，我们开发了一种基于共选择的双重选择策略，以显着提高CRISPR-Cas9和Target-AID碱基编辑器的LOF突变率。我们构建了pDYSCKO载体，其适合于通过CRISPR-Cas9和Target-AID诱变对LOF进行高通量双重选择。我们开发并使用了CRISPR-LOF条形码测序竞争筛选模型，以表明这种改进在试图检测突变的适应性效应时提供了统计功效的重要增加。我们还表明，在酵母中使用Cas9进行多重基因组编辑会导致高频率的可编程染色体易位，这表明应谨慎进行多重编辑。该工具将允许开发新的S. graesystems生物学工作流程，并应证明与大多数酵母资源兼容。
CRISPR-Cas9 loss of function (LOF) screens are powerful systems biology tools that allow genome-wide perturbation studies. While currently possible in many model species, such an approach has yet to be developed in yeast because of low mutagenesis rates without donor DNA for homology directed repair. Here we develop a double selection strategy based on co-selection to dramatically increase LOF mutation rates, both for CRISPR-Cas9 and the Target-AID base editor. We constructed the pDYSCKO vector, which is amenable to high throughput double selection both for LOF by CRISPR-Cas9 and Target-AID mutagenesis. We develop and use a model of CRISPR-LOF barcode sequencing competition screens to show that this improvement provides an important increase in statistical power when attempting to detect the fitness effects of mutations. We also show that multiplex genome editing with Cas9 in yeast causes programmable chromosomal translocations at high frequency, suggesting that multiplex editing should be performed with caution. This tool will allow the development of new S.cerevisiaesystems biology workflows and should prove compatible with most resources already available for yeast.