PDF(Pubmed)
Abstract：
Technologies that generate precise combinatorial genome modifications are well suited to dissect the polygenic basis of complex phenotypes and engineer synthetic genomes. Genome modifications with engineered nucleases can lead to undesirable repair outcomes through imprecise homology-directed repair, requiring non-cleavable gene editing strategies. Eukaryotic multiplex genome engineering (eMAGE) generates precise combinatorial genome modifications in Saccharomyces cerevisiae without generating DNA breaks or using engineered nucleases. Here, we systematically optimize eMAGE to achieve 90% editing frequency, reduce workflow time, and extend editing distance to 20 kb. We further engineer an inducible dominant negative mismatch repair system, allowing for high-efficiency editing via eMAGE while suppressing the elevated background mutation rate 17-fold resulting from mismatch repair inactivation. We apply these advances to construct a library of cancer-associated mutations in the ligand-binding domains of human estrogen receptor alpha and progesterone receptor to understand their impact on ligand-independent autoactivation. We validate that this yeast model captures autoactivation mutations characterized in human breast cancer models and further leads to the discovery of several previously uncharacterized autoactivating mutations. This work demonstrates the development and optimization of a cleavage-free method of genome editing well suited for applications requiring efficient multiplex editing with minimal background mutations.
摘要：
产生精确组合基因组修饰的技术非常适合于解剖复杂表型的多基因基础并对合成基因组进行工程改造。用工程核酸酶进行的基因组修饰可通过不精确的同源性定向修复导致不良的修复结果。需要不可切割的基因编辑策略。真核多重基因组工程（eMAGE）在酿酒酵母中产生精确的组合基因组修饰，而不会产生DNA断裂或使用工程核酸酶。这里,我们系统地优化eMAGE以实现90%的编辑频率，减少工作流时间，并将编辑距离扩展到20kb。我们进一步设计了一个可诱导的显性负错配修复系统，允许通过eMAGE进行高效编辑，同时抑制由错配修复失活导致的背景突变率升高17倍。我们应用这些进展来构建人类雌激素受体α和孕激素受体的配体结合域中的癌症相关突变文库，以了解它们对配体非依赖性自激活的影响。我们验证了该酵母模型捕获了人类乳腺癌模型中表征的自激活突变，并进一步导致发现了几种先前未表征的自激活突变。这项工作证明了基因组编辑的无切割方法的开发和优化，非常适合于需要高效多重编辑和最少背景突变的应用。
