PDF(Pubmed)
Abstract：
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.
摘要：
基于CRISPR-Cas的基因组编辑在细菌中广泛使用，范围从构建单个突变体到大规模平行文库。该程序依赖于指导RNA指导的基因组切割，然后用模板修复，该模板引入所需的突变以及同义的“免疫”突变，以防止编辑后基因组的重新切割。因为免疫突变不会改变蛋白质序列，他们通常被认为是中立的。然而,同义突变可以改变mRNA结构，从而改变编码蛋白质的水平。我们已经通过构建超过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。成千上万的编辑对大肠杆菌在乙酸盐上生长期间的适应性有重大影响，高浓度时有毒的不良碳源。高影响力编辑的百分比在基因之间以及基因内不同位置有很大差异。我们重建了具有高影响力编辑的克隆，发现69％的克隆确实对乙酸盐的生长产生了显着影响。有趣的是,在葡萄糖生长过程中，受影响的编辑较少，优选的碳源，这表明，当生物体遇到具有挑战性的条件时，由同义突变引起的蛋白质表达变化可能是最重要的。最后,我们表明同义编辑可以产生广泛的影响;在ptsI的5'末端的同义编辑改变了数百个基因的表达。我们的结果表明，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被认为是无害的。
