一个全基因组规模的CRISPR干扰向导文库能够在酵母中实现全面的表型分析。
A genome-scale CRISPR interference guide library enables comprehensive phenotypic profiling in yeast.
作者信息
McGlincy Nicholas J, Meacham Zuriah A, Reynaud Kendra K, Muller Ryan, Baum Rachel, Ingolia Nicholas T
机构信息
Department of Molecular and Cell Biology, Berkeley, CA, 94720, USA.
Biophysics Graduate Group, University of California, Berkeley, CA, 94720, USA.
出版信息
BMC Genomics. 2021 Mar 23;22(1):205. doi: 10.1186/s12864-021-07518-0.
BACKGROUND
CRISPR/Cas9-mediated transcriptional interference (CRISPRi) enables programmable gene knock-down, yielding loss-of-function phenotypes for nearly any gene. Effective, inducible CRISPRi has been demonstrated in budding yeast, and genome-scale guide libraries enable systematic, genome-wide genetic analysis.
RESULTS
We present a comprehensive yeast CRISPRi library, based on empirical design rules, containing 10 distinct guides for most genes. Competitive growth after pooled transformation revealed strong fitness defects for most essential genes, verifying that the library provides comprehensive genome coverage. We used the relative growth defects caused by different guides targeting essential genes to further refine yeast CRISPRi design rules. In order to obtain more accurate and robust guide abundance measurements in pooled screens, we link guides with random nucleotide barcodes and carry out linear amplification by in vitro transcription.
CONCLUSIONS
Taken together, we demonstrate a broadly useful platform for comprehensive, high-precision CRISPRi screening in yeast.
背景
CRISPR/Cas9介导的转录干扰（CRISPRi）能够实现可编程的基因敲低，几乎可为任何基因产生功能丧失表型。有效的、可诱导的CRISPRi已在芽殖酵母中得到证实，并且基因组规模的向导文库能够进行系统的全基因组遗传分析。
结果
我们基于经验设计规则展示了一个全面的酵母CRISPRi文库，其中大多数基因包含10个不同的向导。混合转化后的竞争性生长揭示了大多数必需基因存在强烈的适应性缺陷，证实该文库提供了全面的基因组覆盖。我们利用针对必需基因的不同向导所导致的相对生长缺陷，进一步完善酵母CRISPRi设计规则。为了在混合筛选中获得更准确、更可靠的向导丰度测量结果，我们将向导与随机核苷酸条形码相连，并通过体外转录进行线性扩增。
结论
综上所述，我们展示了一个在酵母中进行全面、高精度CRISPRi筛选的广泛有用的平台。
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