Deep mutational scanning can provide significant insights into the function of essential genes in bacteria. Here, we developed a high-throughput method for mutating essential genes of Escherichia coli in their native genetic context. We used Cas9-mediated recombineering to introduce a library of mutations, created by error-prone PCR, within a gene fragment on the genome using a single gRNA pre-validated for high efficiency. Tracking mutation frequency through deep sequencing revealed biases in the position and the number of the introduced mutations. We overcame these biases by increasing the homology arm length and blocking mismatch repair to achieve a mutation efficiency of 85% for non-essential genes and 55% for essential genes. These experiments also improved our understanding of poorly characterized recombineering process using dsDNA donors with single nucleotide changes. Finally, we applied our technology to target rpoB, the beta subunit of RNA polymerase, to study resistance against rifampicin. In a single experiment, we validate multiple biochemical and clinical observations made in the previous decades and provide insights into resistance compensation with the study of double mutants.

中文翻译：

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CRISPR / Cas9重组介导的大肠杆菌基本基因的深度突变扫描。

深度突变扫描可以为细菌中必需基因的功能提供重要见解。在这里，我们开发了一种高通量方法，可用于在自然遗传背景下诱变大肠杆菌的必需基因。我们使用Cas9介导的重组技术，通过使用预先经过高效验证的单个gRNA，在基因组的基因片段内引入由易错PCR产生的突变库。通过深度测序追踪突变的频率揭示了引入的突变的位置和数量存在偏差。我们通过增加同源臂长度和阻断错配修复来克服这些偏见，从而使非必需基因的突变效率达到85％，而必需基因的突变效率达到55％。这些实验还提高了我们对使用具有单核苷酸变化的dsDNA供体的特征较差的重组过程的理解。最后，我们将技术应用于rpoB（RNA聚合酶的β亚基），以研究对利福平的耐药性。在单个实验中，我们验证了过去几十年的多项生化和临床观察结果，并通过双突变体的研究提供了对耐药性补偿的见识。