CRISPR/Cas9 recombineering-mediated deep mutational scanning of essential genes in Escherichia coli
CRISPR/Cas9 recombineering-mediated deep mutational scanning of essential genes in Escherichia coli
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DOI：
10.15252/msb.20199265
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发表时间：
2020-03-01
影响因子：
9.9
通讯作者：
Gill, Ryan T.
中科院分区：
文献类型：
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作者：
Choudhury, Alaksh;Fenster, Acob A.;Gill, Ryan T.
深度突变扫描可以为细菌中必需基因的功能提供重要的见解。在这里，我们开发了一种高通量的方法，用于突变大肠杆菌在其天然遗传背景下的必需基因。我们使用Cas9介导的重组工程引入了一个突变文库，该文库由容易出错的PCR创建，在基因组上的一个基因片段中使用一个预先验证为高效率的gRNA。通过深度测序跟踪突变频率，发现引入突变的位置和数量存在偏见。我们通过增加同源臂长度和阻断错配修复来克服这些偏差，使非必需基因的突变效率达到85%，必需基因的突变效率达到55%。这些实验也提高了我们对使用具有单核苷酸变化的dsDNA供体的不良重组工程过程的理解。最后，我们将我们的技术应用于RNA聚合酶的β亚基rpoB，以研究其对利福平的耐药性。在一个实验中，我们验证了在过去几十年中所做的多个生化和临床观察，并通过对双突变的研究提供了对耐药性补偿的见解。
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.