Zhang Changyi, Whitaker Rachel J
Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA
Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.
Appl Environ Microbiol. 2017 Dec 15;84(1). doi: 10.1128/AEM.02167-17. Print 2018 Jan 1.
is rapidly emerging as a model system for studying the biology and evolution within the TACK lineage of the archaeal domain. As the tree of life grows, identifying the cellular functions of genes within this lineage will have significant impacts on our understanding of the evolution of the ast rchaeal ukaryote ommon ncestor (LEACA) and the differentiation of archaea from eukaryotes during the evolution of the modern-day cell. To increase our understanding of this key archaeal organism, we report a novel high-throughput method for targeted gene inactivation in through one-step microhomology-directed homologous recombination (HR). We validated the efficacy of this approach by systematically deleting 21 individual toxin-antitoxin gene pairs and its application to delete chromosomal regions as large as 50 kb. Sequence analysis of 96 ArgD transformants showed that can effectively incorporate donor markers as short segments through HR in a continuous or discontinuous manner. We determined that the minimal size of homology allowing native marker replacement was as few as 10 bp, whereas marker replacement was frequently observed when increasing the size of homology to 30 to 50 bp. The microhomology-mediated gene inactivation system developed here will greatly facilitate isolation of gene deletion strains, making generation of a collection of genome-wide targeted mutants feasible and providing a tool to investigate homologous recombination in this organism. Current procedures for the construction of deletion mutants of are still tedious and time-consuming. We developed a novel procedure based on microhomology-mediated HR, allowing for rapid and efficient removal for genetic regions as large as 50 kb. Our work will greatly facilitate functional genomic studies in this promising model organism. Additionally, we developed a quantitative genetic assay to measure HR properties in , providing evidence that the ability to incorporate short, mismatched donor DNA into the genome through HR was probably a common trait for members of the genus that are recombinogenic.
正迅速成为研究古菌域TACK谱系内生物学和进化的模型系统。随着生命之树的不断发展，确定该谱系内基因的细胞功能将对我们理解古核生物共同祖先（LEACA）的进化以及现代细胞进化过程中古菌与真核生物的分化产生重大影响。为了增进我们对这种关键古菌的了解，我们报告了一种通过一步微同源性定向同源重组（HR）在[具体古菌名称未提及]中进行靶向基因失活的新型高通量方法。我们通过系统删除21个单独的毒素 - 抗毒素基因对验证了该方法的有效性，并将其应用于删除长达50 kb的染色体区域。对96个ArgD转化体的序列分析表明，[具体古菌名称未提及]能够通过HR以连续或不连续的方式有效地将供体标记作为短片段整合进去。我们确定允许天然[具体古菌名称未提及]标记替换的最小同源性大小低至10 bp，而当将同源性大小增加到30至50 bp时，经常观察到[具体古菌名称未提及]标记替换。在此开发的微同源性介导的基因失活系统将极大地促进[具体古菌名称未提及]基因缺失菌株的分离，使得全基因组靶向突变体文库的构建变得可行，并为研究该生物体中的同源重组提供工具。目前构建[具体古菌名称未提及]缺失突变体的程序仍然繁琐且耗时。我们基于微同源性介导的HR开发了一种新程序，允许快速有效地去除长达50 kb的遗传区域。我们的工作将极大地促进对这种有前景的模型生物体的功能基因组学研究。此外，我们开发了一种定量遗传测定法来测量[具体古菌名称未提及]中的HR特性，提供了证据表明通过HR将短的、错配的供体DNA整合到基因组中的能力可能是具有重组能力的[具体古菌名称未提及]属成员的共同特征。
