Univ. Bordeaux, INRAE, Biologie du Fruit et Pathologie, UMR 1332, F-33140 Villenave d'Ornon, France.
CIRAD, UMR ASTRE, F-34398 Montpellier, France.
ACS Synth Biol. 2023 Nov 17;12(11):3252-3266. doi: 10.1021/acssynbio.3c00248. Epub 2023 Oct 16.
The genetic engineering of genome fragments larger than 100 kbp is challenging and requires both specific methods and cloning hosts. The yeast is considered as a host of choice for cloning and engineering whole or partial genomes from viruses, bacteria, and algae. Several methods are now available to perform these manipulations, each with its own limitations. In order to extend the range of yeast cloning strategies, a new approach combining two already described methods, Fusion cloning and CReasPy-Cloning, was developed. The CReasPy-Fusion method allows the simultaneous cloning and engineering of megabase-sized genomes in yeast by the fusion of bacterial cells with yeast spheroplasts carrying the CRISPR-Cas9 system. With this new approach, we demonstrate the feasibility of cloning and editing whole genomes from several species belonging to different phylogenetic groups. We also show that CReasPy-Fusion allows the capture of large genome fragments with high efficacy, resulting in the successful cloning of selected loci in yeast. We finally identify bacterial nuclease encoding genes as barriers for CReasPy-Fusion by showing that their removal from the donor genome improves the cloning efficacy.
将大于 100 kbp 的基因组片段进行基因工程是具有挑战性的，需要特定的方法和克隆宿主。酵母被认为是克隆和工程病毒、细菌和藻类的完整或部分基因组的首选宿主。现在有几种方法可以进行这些操作，每种方法都有其自身的局限性。为了扩展酵母克隆策略的范围，开发了一种新方法，将两种已描述的方法（Fusion cloning 和 CReasPy-Cloning）结合起来。CReasPy-Fusion 方法通过融合携带 CRISPR-Cas9 系统的酵母原生质体与细菌细胞，允许在酵母中同时克隆和工程处理兆碱基大小的基因组。通过这种新方法，我们证明了从属于不同系统发育群的多个物种克隆和编辑整个基因组的可行性。我们还表明，CReasPy-Fusion 可以高效地捕获大片段基因组，从而成功地在酵母中克隆了选定的基因座。最后，我们通过显示从供体基因组中去除细菌核酸酶编码基因可以提高克隆效率，确定这些基因是 CReasPy-Fusion 的障碍。
