Synonymous edits in the Escherichia coli genome have substantial and condition-dependent effects on fitness.
Synonymous edits in the Escherichia coli genome have substantial and condition-dependent effects on fitness.
复制标题
DOI：
10.1073/pnas.2316834121
复制
发表时间：
2024-01
影响因子：
11.1
通讯作者：
Dong-Dong Yang-Dong;Leo M. Rusch;Karl A Widney;A. Morgenthaler;S. Copley
中科院分区：
文献类型：
--
作者：
Dong-Dong Yang-Dong;Leo M. Rusch;Karl A Widney;A. Morgenthaler;S. Copley
基于CRISPR-Cas的基因组编辑广泛用于细菌中，其规模从单个突变体的构建到大规模平行文库。该程序依赖于引导RNA指导的基因组切割，然后用模板修复，所述模板引入期望的突变沿着同义“免疫”突变以防止编辑后基因组的再切割。由于免疫突变不改变蛋白质序列，它们通常被认为是中性的。然而，同义突变可以改变mRNA结构的方式，改变编码蛋白质的水平。我们已经通过构建一个包含超过50，000个编辑的文库来测试免疫突变是中性的假设，该文库仅由大肠杆菌中的同义突变组成。在E.大肠杆菌对乙酸盐的依赖性很强，乙酸盐是一种贫碳源，在高浓度下是有毒的。高影响编辑的百分比在基因之间和基因内的不同位置上变化很大。我们用高影响力的编辑重建了克隆，发现69%确实对乙酸盐中的生长有显著影响。有趣的是，在葡萄糖（一种优选的碳源）中生长期间，较少的编辑影响了适应性，这表明当生物体遇到挑战性条件时，同义突变引起的蛋白质表达变化可能是最重要的。最后，我们发现同义编辑可以产生广泛的影响; ptsI 5'端的同义编辑改变了数百个基因的表达。我们的研究结果表明，在基于CRISPR-Cas的基因组编辑过程中引入的同义免疫编辑不应该被认为是无害的。
CRISPR-Cas-based genome editing is widely used in bacteria at scales ranging from construction of individual mutants to massively parallel libraries. This procedure relies on guide RNA-directed cleavage of the genome followed by repair with a template that introduces a desired mutation along with synonymous "immunizing" mutations to prevent re-cleavage of the genome after editing. Because the immunizing mutations do not change the protein sequence, they are often assumed to be neutral. However, synonymous mutations can change mRNA structures in ways that alter levels of the encoded proteins. We have tested the assumption that immunizing mutations are neutral by constructing a library of over 50,000 edits that consist of only synonymous mutations in Escherichia coli. Thousands of edits had substantial effects on fitness during growth of E. coli on acetate, a poor carbon source that is toxic at high concentrations. The percentage of high-impact edits varied considerably between genes and at different positions within genes. We reconstructed clones with high-impact edits and found that 69% indeed had significant effects on growth in acetate. Interestingly, fewer edits affected fitness during growth in glucose, a preferred carbon source, suggesting that changes in protein expression caused by synonymous mutations may be most important when an organism encounters challenging conditions. Finally, we showed that synonymous edits can have widespread effects; a synonymous edit at the 5' end of ptsI altered expression of hundreds of genes. Our results suggest that the synonymous immunizing edits introduced during CRISPR-Cas-based genome editing should not be assumed to be innocuous.