| Rapid Genome Engineering of Pseudomonas Assisted by Fluorescent Markers and Tractable Curing of Plasmids
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Author:
Date:
2021-02-20
[Abstract] Precise genome engineering has become a commonplace technique for metabolic engineering. Also, insertion, deletion and alteration of genes and other functional DNA sequences are essential for understanding and engineering cells. Several techniques have been developed to this end (e.g., CRISPR/Cas-assisted methods, homologous recombination, or λ Red recombineering), yet most of them rely on the use of auxiliary plasmids, which have to be cured after the editing procedure. Temperature-sensitive replicons, counter-selectable markers or repeated passaging of plasmid-bearing cells have been traditionally employed to circumvent this hurdle. While these protocols work reasonably well in some bacteria, they are not applicable for other species or are time consuming and laborious. Here, we present ...
[摘要] [摘要]精确的基因组工程已成为代谢工程的一种普遍技术。同样，基因和其他功能性DNA序列的插入，缺失和改变对于理解和改造细胞也是必不可少的。几种技术已经发展到该端部（例如，CRISPR / CAS-辅助方法，同源重组，或 λ 红色重组），但其中大多数依赖于辅助质粒的使用，必须在编辑程序后将其固化。传统上已采用对温度敏感的复制子，反向选择标记或带有质粒的细胞的重复传代来规避这一障碍。尽管这些协议在某些细菌中可以很好地发挥作用，但它们不适用于其他物种，或者既费时又费力。在这里，我们提出了快速和通用的荧光假单胞菌荧光标记辅助基因组编辑协议，然后通过用户控制的质粒复制干净固化辅助质粒。一种荧光标记有助于鉴定基因组编辑的菌落，而第二种报道分子能够检测无质粒的细菌克隆。该协议不仅是用于假单胞菌物种的最快方法，而且可以轻松地适应任何类型的基因组修饰，包括序列删除，插入和替换。
图形概要：
带有可治愈质粒的假单胞菌的快速基因组工程
[背景]靶向，精确的基因组操纵技术已经大大推进了微生物工程领域。这样的方法不仅允许评估基因型与表型的关系，而且使微生物细胞工厂的复杂工程化成为可能。近年来，CRISPR / Cas9方法为真核生物的精确基因组工程铺平了道路。在细菌中，CRISPR / ...
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| Single-step Precision Genome Editing in Yeast Using CRISPR-Cas9
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Author:
Date:
2018-03-20
[Abstract] Genome modification in budding yeast has been extremely successful largely due to its highly efficient homology-directed DNA repair machinery. Several methods for modifying the yeast genome have previously been described, many of them involving at least two-steps: insertion of a selectable marker and substitution of that marker for the intended modification. Here, we describe a CRISPR-Cas9 mediated genome editing protocol for modifying any yeast gene of interest (either essential or nonessential) in a single-step transformation without any selectable marker. In this system, the Cas9 nuclease creates a double-stranded break at the locus of choice, which is typically lethal in yeast cells regardless of the essentiality of the targeted locus due to inefficient non-homologous end-joining ...
[摘要] 芽殖酵母中的基因组修饰已经非常成功，主要归功于其高度同源性的DNA修复机制。之前已经描述了几种用于修饰酵母基因组的方法，其中许多方法涉及至少两个步骤：插入选择标记并用该标记取代预期的修饰。在这里，我们描述了CRISPR-Cas9介导的基因组编辑方案，用于在没有任何选择标记的情况下在单步转化中修饰任何感兴趣的酵母基因（基本或非必需）。在该系统中，Cas9核酸酶在选择的基因座处产生双链断裂，这在酵母细胞中通常是致死的，而不管由于无效的非同源末端连接修复导致的靶基因座的重要性。该致死性通过使用源自PCR的修复模板的同源重组导致有效的修复。在涉及必需基因的情况下，用功能性等位基因编辑基因组病变的必要性作为额外的选择层。作为一个激励性的例子，我们描述了使用这种策略替代HEM2，一种必需的酵母基因，以及相应的人类直向同源物ALAD。
【背景】酿酒酵母（Baccharomyces cerevisiae，Baker's酵母）作为一种遗传易处理的生物体具有悠久的历史，并且有许多操作酵母基因组的方法。然而，直到最近，有必要应用选择以分离具有所需遗传改变的克隆（Kearse等人，2012; DiCarlo等人，2013; Lee等人，等，2015; ...
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| Library Construction for Genome-wide Bisulfite Sequencing in Plants
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Author:
Date:
2013-12-20
[Abstract] DNA methylation is the most studied epigenetic modification, which involves the addition of a methyl group to the carbon-5 position of cytosine residues in DNA. DNA methylation is important for the regulation of gene expression. Bisulfite sequencing is the gold standard technique for determining genome-wide DNA methylation profiles in eukaryotes. This protocol describes how to prepare libraries of genomic DNA for whole-genome bisulfite sequencing in Arabidopsis, which could be adapted for use in other plant species.
[摘要] DNA甲基化是研究最多的表观遗传修饰，其涉及将甲基添加到DNA中胞嘧啶残基的碳-5位置。 DNA甲基化对于基因表达的调节是重要的。 亚硫酸氢盐测序是确定真核生物全基因组DNA甲基化谱的金标准技术。 该协议描述了如何在拟南芥中制备用于全基因组亚硫酸氢盐测序的基因组DNA文库，其可以适用于其他植物物种。
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