Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, Colorado, USA.
Department of Chemical Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania, USA.
Nat Biotechnol. 2015 Jun;33(6):631-7. doi: 10.1038/nbt.3177. Epub 2015 Mar 23.
Multiplexed genome engineering approaches can be used to generate targeted genetic diversity in cell populations on laboratory timescales, but methods to track mutations and link them to phenotypes have been lacking. We present an approach for tracking combinatorial engineered libraries (TRACE) through the simultaneous mapping of millions of combinatorially engineered genomes at single-cell resolution. Distal genomic sites are assembled into individual DNA constructs that are compatible with next-generation sequencing strategies. We used TRACE to map growth selection dynamics for Escherichia coli combinatorial libraries created by recursive multiplex recombineering at a depth 10(4)-fold greater than before. TRACE was used to identify genotype-to-phenotype correlations and to map the evolutionary trajectory of two individual combinatorial mutants in E. coli. Combinatorial mutations in the human ES2 ovarian carcinoma cell line were also assessed with TRACE. TRACE completes the combinatorial engineering cycle and enables more sophisticated approaches to genome engineering in both bacteria and eukaryotic cells than are currently possible.
多重基因组工程方法可用于在实验室时间范围内对细胞群体产生靶向遗传多样性，但缺乏跟踪突变并将其与表型联系起来的方法。我们提出了一种通过单细胞分辨率同时对数百万个组合工程基因组进行映射来跟踪组合工程文库 (TRACE) 的方法。远端基因组位点被组装成单个 DNA 构建体，这些构建体与下一代测序策略兼容。我们使用 TRACE 来映射通过递归多重重组酶工程创建的大肠杆菌组合文库的生长选择动力学，其深度比以前增加了 104 倍。TRACE 用于识别基因型与表型的相关性，并绘制大肠杆菌中两个组合突变体的进化轨迹。还使用 TRACE 评估了人 ES2 卵巢癌细胞系中的组合突变。TRACE 完成了组合工程周期，使细菌和真核细胞中的基因组工程能够采用比目前更复杂的方法。
