| A Method to Convert mRNA into a Guide RNA (gRNA) Library without Requiring Previous Bioinformatics Knowledge of the Organism
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Author:
Date:
2017-05-20
[Abstract] While the diversity of species represents a diversity of special biological abilities, many of the genes that encode those special abilities in a variety of species are untouched, leaving an untapped gold mine of genetic information; however, despite current advances in genome bioinformatics, annotation of that genetic information is incomplete in most species, except for well-established model organisms, such as human, mouse, or yeast. A guide RNA (gRNA) library using the clustered regularly interspersed palindromic repeats (CRISPR)/Cas9 (CRISPR-associated protein 9) system can be used for the phenotypic screening of uncharacterized genes by forward genetics. The construction of a gRNA library usually requires an abundance of chemically synthesized oligos designed from annotated genes; ...
[摘要] 虽然物种的多样性代表着特殊的生物学能力的多样性，但许多编码这些特殊能力的基因却是不变的，留下了未开发的遗传信息金矿;然而，尽管基因组生物信息学方面取得了进展，但除了成熟的模型生物体，如人，小鼠或酵母，遗传信息的注释在大多数物种中是不完全的。使用聚簇常规散布的回文重复序列（CRISPR）/ Cas9（CRISPR相关蛋白9）系统的引导RNA（gRNA）文库可用于通过正向遗传学对非特异性基因的表型筛选。 gRNA文库的构建通常需要从注释基因设计的大量化学合成寡核苷酸;如果想在没有目标DNA序列的先验知识的情况下将mRNA转换成gRNA，那么主要的挑战就是发现原始邻近基序（PAM）侧翼的序列并切出20-bp片段。最近，我开发了基于分子生物学技术将mRNA转化为gRNA文库（Arakawa，2016）（图1）。我在这里描述了如何从mRNA构建gRNA文库的详细协议。
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图1.将mRNA转化为gRNA文库构建的方法（Sanjana等人，2014）。总结了该方法的方案。在步骤中详细描述了D-O的每个步骤。 ...
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| CytoTrap Two-Hybrid Screening Assay
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Author:
Date:
2014-12-05
[Abstract] CytoTrap two-hybrid system provides an alternate strategy to detect protein-protein interactions in yeast. In this system, bait protein is fused with human son of sevenless (hSos) protein (Li et al., 1993), and a cDNA library or prey protein is expressed by fusion with myristoylation signal which anchors the prey fusion protein to yeast cell membrane. Protein interaction between bait and prey proteins recruits the hSos protein to the cell membrane, where hSos activates the Ras signaling pathway, leading to the survival of temperature-sensitive Saccharomyces cerevisiae (S. cerevisiae) strain cdc25H at 36 °C. In the CytoTrap two-hybrid system, detection of protein interaction occurs in the cytoplasm near cell membrane and is not dependent on transcription ...
[摘要] CytoTrap双杂交系统提供了检测酵母中蛋白质 - 蛋白质相互作用的替代策略。在该系统中，诱饵蛋白与七（hSos）蛋白质的人子融合（Li等，1993），cDNA文库或猎物蛋白质与肉豆蔻酰化信号的融合表达，将猎物融合蛋白锚定到酵母细胞膜。诱饵和猎物蛋白之间的蛋白质相互作用将hSos蛋白质募集到细胞膜上，其中hSos激活Ras信号通路，导致温度敏感的酿酒酵母（S.cerevisiae）菌株cdc25H在36℃下的存活。在CytoTrap双杂交系统中，蛋白质相互作用的检测发生在细胞膜附近的细胞质中，不依赖于报告基因的转录激活。因此，该系统对于识别需要在细胞质中进行翻译后修饰的转录因子和蛋白质的相互作用伴侣特别有用，其在常规基于反式激活的酵母双杂交系统中不能用作诱饵蛋白。在这里，我们描述了来自模拟植物拟南芥的cDNA文库的构建以及用于筛选来自该文库的AtSR1 / CAMTA3，Ca2 + / CaM调节的转录因子的相互作用蛋白的程序。该过程可以适应于识别来自其他生物体的感兴趣的蛋白质的相互作用的伴侣。
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