Sorbonne Université, CNRS, Institut de Biologie Paris-Seine, Laboratory of Computational and Quantitative Biology, Paris, France.
Université de Strasbourg, CNRS, GMGM UMR7156, Strasbourg, France.
PLoS Genet. 2019 Aug 29;15(8):e1008332. doi: 10.1371/journal.pgen.1008332. eCollection 2019 Aug.
Genome engineering is a powerful approach to study how chromosomal architecture impacts phenotypes. However, quantifying the fitness impact of translocations independently from the confounding effect of base substitutions has so far remained challenging. We report a novel application of the CRISPR/Cas9 technology allowing to generate with high efficiency both uniquely targeted and multiple concomitant reciprocal translocations in the yeast genome. Targeted translocations are constructed by inducing two double-strand breaks on different chromosomes and forcing the trans-chromosomal repair through homologous recombination by chimerical donor DNAs. Multiple translocations are generated from the induction of several DSBs in LTR repeated sequences and promoting repair using endogenous uncut LTR copies as template. All engineered translocations are markerless and scarless. Targeted translocations are produced at base pair resolution and can be sequentially generated one after the other. Multiple translocations result in a large diversity of karyotypes and are associated in many instances with the formation of unanticipated segmental duplications. To test the phenotypic impact of translocations, we first recapitulated in a lab strain the SSU1/ECM34 translocation providing increased sulphite resistance to wine isolates. Surprisingly, the same translocation in a laboratory strain resulted in decreased sulphite resistance. However, adding the repeated sequences that are present in the SSU1 promoter of the resistant wine strain induced sulphite resistance in the lab strain, yet to a lower level than that of the wine isolate, implying that additional polymorphisms also contribute to the phenotype. These findings illustrate the advantage brought by our technique to untangle the phenotypic impacts of structural variations from confounding effects of base substitutions. Secondly, we showed that strains with multiple translocations, even those devoid of unanticipated segmental duplications, display large phenotypic diversity in a wide range of environmental conditions, showing that simply reconfiguring chromosome architecture is sufficient to provide fitness advantages in stressful growth conditions.
基因组工程是一种强大的方法，可以研究染色体结构如何影响表型。然而，迄今为止，独立于碱基替换的混杂效应来量化易位的适合度影响仍然具有挑战性。我们报告了一种新型的 CRISPR/Cas9 技术的应用，该技术可以高效地在酵母基因组中产生独特靶向和多个同时发生的相互易位。靶向易位是通过在不同染色体上诱导两个双链断裂，并通过嵌合供体 DNA 迫使跨染色体修复通过同源重组来构建的。多个易位是通过在 LTR 重复序列中诱导几个 DSB 并使用未切割的内源性 LTR 拷贝作为模板促进修复而产生的。所有工程化的易位都是无标记和无痕的。靶向易位是在碱基对分辨率下产生的，可以一个接一个地连续产生。多个易位导致大量不同的核型，并且在许多情况下与意想不到的片段重复的形成相关。为了测试易位的表型影响，我们首先在实验室菌株中重复了 SSU1/ECM34 易位，该易位为葡萄酒分离株提供了更高的亚硫酸盐抗性。令人惊讶的是，在实验室菌株中相同的易位导致亚硫酸盐抗性降低。然而，添加存在于抗性葡萄酒菌株 SSU1 启动子中的重复序列在实验室菌株中诱导了亚硫酸盐抗性，但水平低于葡萄酒分离株，这表明其他多态性也有助于表型。这些发现说明了我们的技术的优势，可以将结构变异的表型影响与碱基替换的混杂效应区分开来。其次，我们表明，即使没有意外的片段重复，具有多个易位的菌株在广泛的环境条件下表现出很大的表型多样性，表明仅仅重新配置染色体结构就足以在压力生长条件下提供适合度优势。
