| 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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| Complex in vivo Ligation Using Homologous Recombination and High-efficiency Plasmid Rescue from Saccharomyces cerevisiae
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Author:
Date:
2015-07-05
[Abstract] The protocols presented here allow for the facile generation of a wide variety of complex multipart DNA constructs (tagged gene products, gene fusions, chimeric proteins, and other variants) using homologous recombination and in vivo ligation in budding yeast (Saccharomyces cerevisiae). This method is straightforward, efficient and cost-effective, and can be used both for vector creation and for subsequent one-step, high frequency integration into a chromosomal locus in yeast. The procedure utilizes PCR with extended oligonucleotide “tails” of homology between multiple fragments to allow for reassembly in yeast in a single transformation followed by a method for highly efficient plasmid extraction from yeast (for transformation into bacteria). The latter is an ...
[摘要] 这里提出的方案允许使用发芽酵母（酿酒酵母）中的同源重组和体内连接，容易地生成各种复杂的多部分DNA构建体（标记的基因产物，基因融合体，嵌合蛋白和其他变体）。该方法是直接，有效和成本有效的，并且可以用于载体创建和用于后续的一步，高频整合到酵母中的染色体位点。该方法利用PCR扩增多个片段之间的同源性的寡核苷酸“尾”，以允许在单次转化中重组酵母，随后用酵母高效质粒提取（用于转化为细菌）的方法。后者是对现有的酵母质粒提取方法的改进，其历史上已经是恢复所需构建体的限制步骤。我们描述了我们的技术的实用性和便利性，并提供了几个例子。
【背景】酿酒酵母中的同源重组（HR）早已被公认为在体内组装DNA片段的非常方便的方法（Szostak等，1983; Ma等，1987; Oldenburg等，1997）。鉴于酵母中人力资源的效率，它已经被利用了增加其效用，增强其多功能性并允许其应用于广泛的实验目标的方式。这种一般方法的改进包括使用体内连接作为定向诱变的平台（Muhlrad等人，1992），引入反选择以帮助质粒产生（Gunyuzlu等人，2001; Anderson和Haj-Ahmad，2003）和使体内组装适应于不能在酵母中繁殖的载体（Iizasa和Nagano，2006; ...
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| Chromatin Fractionation Assay in Fission Yeast
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Author:
Date:
2014-07-20
[Abstract] The protein recruitment onto chromatin is a critical process for DNA metabolism, including DNA replication, DNA repair and DNA recombination. Especially DNA modification enzymes and checkpoint proteins are loaded onto DNA damage sites in a context-dependent manner. In our recent study (Kunoh and Habu, 2014), the chromatin association of Pcf1, a large subunit of Chromatin Assembly Factor-1 (CAF-1), was monitored after exposure of cells to hydroxyurea which slowed down the DNA replication. Results of the chromatin fractionation assay provided evidence that Pcf1 was recruited to chromatin upon DNA replication stress. A similar procedure enabled to reveal the chromatin association of Orp1, Mcm proteins, and Swi6 (Sadaie et al., 2008; Ogawa et al., 1999). This assay allows us ...
[摘要] 染色质上的蛋白质募集是DNA代谢的关键过程，包括DNA复制，DNA修复和DNA重组。特别是DNA修饰酶和检查点蛋白以上下文依赖性方式装载到DNA损伤位点。在我们最近的研究中（Kunoh和Habu，2014），在将细胞暴露于羟基脲（其减慢DNA复制）后，监测Pcf1的染色质缔合，其为染色质装配因子-1（CAF-1）的大亚基。染色质分馏测定的结果提供了Pcf1在DNA复制应激时募集到染色质的证据。类似的程序能够揭示Orp1，Mcm蛋白和Swi6的染色质缔合（Sadaie等人，2008; Ogawa等人，1999）。该测定允许我们从活细胞分离染色质结合和非结合蛋白。以下各部分的免疫印迹提供了关于我们的靶蛋白的染色质结合状态的信息。
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