Dual sgRNA-based Targeted Deletion of Large Genomic Regions and Isolation of Heritable Cas9-free Mutants in Arabidopsis
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Author:
Date:
2020-10-20
[Abstract] CRISPR/Cas9 system directed by a gene-specific single guide RNA (sgRNA) is an effective tool for genome editing such as deletions of few bases in coding genes. However, targeted deletion of larger regions generate loss-of-function alleles that offer a straightforward starting point for functional dissections of genomic loci. We present an easy-to-use strategy including a fast cloning dual-sgRNA vector linked to efficient isolation of heritable Cas9-free genomic deletions to rapidly and cost-effectively generate a targeted heritable genome deletion. This step-by-step protocol includes gRNA design, cloning strategy and mutation detection for Arabidopsis and may be adapted for other plant species.
[摘要] [摘要] CRISPR/Cas9由基因特异性单导RNA(sgRNA)引导的系统是一种有效的基因组编辑工具,如编码基因中少部分碱基的删除。然而,大区域的靶向缺失产生功能缺失等位基因,这为基因组基因座的功能解剖提供了一个直接的起点。我们提出了一个简单易用的策略,包括一个快速克隆双sgRNA载体,有效分离可遗传的Cas9游离基因组缺失,以快速且经济有效地产生靶向遗传基因组缺失。该方法包括拟南芥的gRNA设计、克隆策略和突变检测,可适用于其他植物。 [背景] ...
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Easy and Efficient Permeabilization of Cyanobacteria for in vivo Enzyme Assays Using B-PER
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Author:
Date:
2018-01-05
[Abstract] Cyanobacteria are photosynthetic bacteria that thrive in diverse ecosystems and play major roles in the global carbon cycle. The abilities of cyanobacteria to fix atmospheric CO2 and to allocate the fixed carbons to chemicals and biofuels have attracted growing attentions as sustainable microbial cell factories. A better understanding of activities of enzymes involved in the central carbon metabolism might lead to increased product yields. Currently, cell-free lysates are widely used for the determination of intracellular enzyme activities. However, due to thick cell walls in cyanobacteria, lysis of cyanobacterial cells is inefficient and often laborious. The present protocol describes an easy and efficient method to permeabilize cyanobacterial cells, without lysing them, and ...
[摘要] 蓝细菌是光合细菌,在不同的生态系统中繁衍,在全球碳循环中发挥重要作用。 蓝藻固定大气CO 2和将固定碳分配到化学品和生物燃料的能力作为可持续的微生物细胞工厂已经引起越来越多的关注。 更好地了解参与中央碳代谢的酶的活性可能导致产物产量增加。 目前,无细胞裂解物被广泛用于细胞内酶活性的测定。 然而,由于蓝细菌细胞壁较厚,蓝细菌细胞的裂解效率低下且费力。 目前的方案描述了一种简单而有效的方法来渗透蓝藻细胞,而不溶解它们,并直接使用透化细胞来测定体内代谢酶活性。
【背景】我们之前已经报道了使用B-PER TM试剂(Thermo Fisher Science)(Rasmussen等人,2016)简单,有效且可扩展的蓝细菌的透化。 B-PER TM TM试剂含有溶解在Tris-HCl缓冲液中的未公开的温和洗涤剂,通常用于裂解细菌细胞如大肠杆菌(Escherichia coli)。偶然地,我们发现B-PER TM TM试剂渗透蓝细菌细胞而不是溶解它们,可能是因为厚的蓝细菌细胞壁(Hoiczyk和Hansel,2000)赋予了试剂中使用的去污剂的抗性。在生物技术感兴趣的蓝细菌中进行通透化。聚球蓝细菌PCC 7002(以下简称“Synechococcus”7002)和“Synechocystis”sp。 PCC ...
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Fluorescently Labelled Aerolysin (FLAER) Labelling of Candida albicans Cells
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Author:
Date:
2017-06-05
[Abstract] In this protocol we describe a nonradiolabelled labelling of GPI anchor in Candida albicans. The method uses a fluorescent probe to bind specifically to GPI anchors so that the level of GPI-anchored proteins at the cell surface can be measured. The labelling does not need permeabilization of cells and can be carried out in vivo.
[摘要] 在本协议中,我们描述了白色念珠菌中GPI锚的非放射性标记标记。该方法使用荧光探针特异性结合GPI锚点,从而可以测量细胞表面的GPI锚定蛋白的水平。标记不需要细胞的透化,并且可以在体内进行进行。
背景 GPI(糖基磷脂酰肌醇)锚是在内质网(ER)中发生的翻译后修饰。预先形成的GPI锚定体连接到携带GPI锚定附着信号序列的特异性蛋白质的C末端的ER的内腔中。这些蛋白质随后通过分泌途径转移(并进一步修饰)到细胞表面,其中蛋白质锚定在质膜的细胞外小叶上或共价连接到具有壁的生物中的细胞壁。各种蛋白质在真核生物中获得GPI锚定蛋白,例如水解酶,细胞表面粘附分子或受体蛋白(Orlean和Menon,2007)。在真菌病原体中,例如白色念珠菌,参与宿主识别和粘附的许多粘附素以及几种发病机理和毒力因子是GPI锚定蛋白;此外,白色念珠菌中的几种GPI锚定蛋白具有蛋白水解活性(Richard和Plaine,2007; Nobile等人,2008)。该途径在白色念珠菌中是必不可少的,并且其下调或靶向它可能是打击念珠菌感染的有用策略。本文详细阐述了用于评估白色念珠菌中GPI锚定水平的方案。  荧光标记的Aerolysin(FLAER)是荧光素标记的非溶性细胞溶解素的无活性衍生物,革兰氏阴性细菌毒素,其与真核宿主的细胞膜中的GPI锚定的蛋白质结合并形成孔(Howard等, ...
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