CRISPR/Cas9–based gene knockouts (KOs) enable precise perturbation of target gene function in human cells, which is ideally assessed in an unbiased fashion by molecular omics readouts. Typically, this requires the lengthy process of isolating KO subclones. We show here that KO subclones are phenotypically heterogenous, regardless of the guide RNA used. We present an experimental strategy that avoids subcloning and achieves fast and efficient gene silencing on cell pools, based on the synergistic combination of two guide RNAs mapping at close (40–300 bp) genomic proximity. Our strategy results in better predictable indel generation with a low allelic heterogeneity, concomitant with low or undetectable residual target protein expression, as determined by MS3 mass spectrometry proteomics. Our method is compatible with nondividing primary cells and can also be used to study essential genes. It enables the generation of high confidence omics data which solely reflect the phenotype of the target ablation.

中文翻译：

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基于串联引导 RNA 的策略，可对编辑等位基因异质性低的细胞群进行高效 CRISPR 基因编辑。


基于 CRISPR/Cas9 的基因敲除 (KO) 能够精确扰动人类细胞中的靶基因功能，理想情况下可以通过分子组学读数以公正的方式对其进行评估。通常，这需要分离 KO 亚克隆的漫长过程。我们在此表明​​，无论使用何种指导 RNA，KO 亚克隆在表型上都是异质的。我们提出了一种实验策略，该策略可避免亚克隆并在细胞池上实现快速有效的基因沉默，该策略基于两个基因组邻近（40-300 bp）定位的向导RNA的协同组合。我们的策略导致插入缺失生成具有更好的可预测性，且等位基因异质性较低，同时残留靶蛋白表达较低或无法检测到（由 MS3 质谱蛋白质组学测定）。我们的方法与非分裂原代细胞兼容，也可用于研究必需基因。它能够生成仅反映目标消融表型的高置信度组学数据。