| Method for Multiplexing CRISPR/Cas9 in Saccharomyces cerevisiae Using Artificial Target DNA Sequences
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Author:
Date:
2017-09-20
[Abstract] Genome manipulation has become more accessible given the advent of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) editing technology. The Cas9 endonuclease binds a single stranded (single guide) RNA (sgRNA) fragment that recruits the complex to a corresponding genomic target sequence where it induces a double stranded break. Eukaryotic repair systems allow for the introduction of exogenous DNA, repair of existing mutations, or deletion of endogenous gene products. Targeting of Cas9 to multiple genomic positions (termed ‘multiplexing’) is achieved by the expression of multiple sgRNAs within the same nucleus. However, an ongoing concern of the CRISPR field has been the accidental targeting of Cas9 to alternative (‘off-target’) DNA locations within a genome. We ...
[摘要] 鉴于CRISPR（集群定期间隔短回归重复）编辑技术的出现，基因组操纵变得更加易于使用。 Cas9核酸内切酶将募集复合物的单链（单向导）RNA（sgRNA）片段结合到相应的基因组靶序列，引发双链断裂。真核修复系统允许引入外源DNA，修复现有突变或内源基因产物的缺失。通过在同一核内表达多个sgRNA来实现Cas9对多个基因组位置的定位（称为“多重”）。然而，CRISPR领域的持续关注是将Cas9意外地定位到基因组内的替代（“脱靶”）DNA位置。我们将安装的人造Cas9靶序列的使用（称为人造基因座上的Cas9复制）描述为允许（i）与单个sgRNA复用的酵母基因组中的用途; （ii）减少/消除可能的脱靶效应，以及（iii）精确控制预定目标序列的放置。
【背景】CRISPR（集群定期间隔回归重复）机制已经在原核生物中演变为具有很高精度编辑任何基因组的能力的原始适应性免疫系统（Jinek等，2012; Sorek等，2013）。这种生物技术需要使用来自化脓性链球菌（或othologous物种）的内切核酸酶（Cas9），单个RNA'引导'序列和外源供体DNA（如果需要）。仅在短短几年内，CRISPR / ...
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| Dictyostelium Cultivation, Transfection, Microscopy and Fractionation
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Author:
Date:
2015-06-05
[Abstract] The real time visualisation of fluorescently tagged proteins in live cells using ever more sophisticated microscopes has greatly increased our understanding of the dynamics of key proteins during fundamental physiological processes such as cell locomotion, chemotaxis, cell division and membrane trafficking. In addition the fractionation of cells and isolation of organelles or known compartments can often verify any subcellular localisation and the use of tagged proteins as bait for the immunoprecipitation of material from cell fractions can identify specific binding partners and multiprotein complexes thereby helping assign a function to the tagged protein. We have successfully applied these techniques to the Dictyostelium discoideum protein TSPOON that is part of an ancient ...
[摘要] 使用更复杂的显微镜，活细胞中荧光标记的蛋白质的实时可视化大大增加了我们对基本生理过程如细胞运动，趋化性，细胞分裂和膜运输过程中关键蛋白质动力学的了解。此外，细胞的分级和分离细胞器或已知的隔室通常可以验证任何亚细胞定位，并且使用标记的蛋白质作为诱饵用于来自细胞部分的物质的免疫沉淀可以鉴定特异性结合配偶体和多蛋白复合物，从而有助于赋予功能标记蛋白。我们已经成功地将这些技术应用于作为古代异构六聚体膜转运复合物的一部分的盘基网柄菌discoideum蛋白TSPOON（Hirst等，2013）。 ...
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