Endogenous C-terminal Tagging by CRISPR/Cas9 in Trypanosoma cruzi
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Author:
Date:
2017-05-20
[Abstract] To achieve the C-terminal tagging of endogenous proteins in T. cruzi we use the Cas9/pTREX-n vector (Lander et al., 2015) to insert a specific tag sequence (3xHA or 3xc-Myc) at the 3’ end of a specific gene of interest (GOI). Chimeric sgRNA targeting the 3’ end of the GOI is PCR-amplified and cloned into Cas9/pTREX-n vector. Then a DNA donor molecule to induce DNA repair by homologous recombination is amplified. This donor sequence contains the tag sequence and a marker for antibiotic resistance, plus 100 bp homology arms corresponding to regions located right upstream of ...
[摘要] 为了实现克氏锥虫内源蛋白的C末端标记,我们使用Cas9 / pTREX-n载体(Lander等,2015)在3'末端插入特异性标签序列(3xHA或3xc-Myc)特定的感兴趣基因(GOI)。将靶向GOI 3'末端的嵌合sgRNA进行PCR扩增,并克隆到Cas9 / pTREX-n载体中。然后扩增通过同源重组诱导DNA修复的DNA供体分子。该供体序列包含标签序列和抗生素抗性标记,加上对应于位于终止密码子右上游的区域和在GOI基因座的Cas9靶位点下游的100bp同源臂。载体pMOTag23M(Oberholzer等,2006)或pMOHX1Tag4H(Lander等,2016b)用作DNA供体扩增的PCR模板。用sgRNA / Cas9 / pTREX-n构建体和DNA供体盒共转染的Epimastigotes然后用抗生素培养5周以选择双重抗性寄生虫。最终通过PCR和Western印迹分析验证了内源基因标记。 【背景】自从CRISPR / ...
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Mating Based Split-ubiquitin Assay for Detection of Protein Interactions
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Author:
Date:
2017-05-05
[Abstract] The mating based split-ubiquitin (mbSUS) assay is an alternative method to the classical yeast two-hybrid system with a number of advantages. The mbSUS assay relies on the ubiquitin-degradation pathway as a sensor for protein-protein interactions, and it is suitable for the determination of interactions between full-length proteins that are cytosolic or membrane-bound. Here we describe the mbSUS assay protocol which has been used for detecting the interaction between K+ channel and SNARE proteins (Grefen et al., 2010 and 2015; Zhang et al., 2015 and 2016)
[摘要] 基于交配的分ubiquitin(mbSUS)测定是具有许多优点的经典酵母双杂交系统的替代方法。 mbSUS测定依赖于泛素降解途径作为蛋白质 - 蛋白质相互作用的传感器,并且它适用于测定细胞溶质或膜结合的全长蛋白质之间的相互作用。在这里,我们描述了已经用于检测K + 通道和SNARE蛋白之间的相互作用的mbSUS测定方案(Grefen等人,2010和2015; Zhang 等等,2015和2016)
背景 图1是mbSUS测定的概况。泛素部分被分成两半,N末端半突变(NubG)以避免重组。泛素部分(Cub)的C末端一半与转录报告基因复合物PLV(Protein A-LexA-VP16)连接。两种蛋白质(X和Y)分别与NubG和CubPLV融合产生蛋白质 - 蛋白质相互作用分析系统。转化后,酵母菌株THY.AP5含有NubG-X融合蛋白,而酵母菌株THY.AP4含有Y-CubPLV融合蛋白。在交配后,在二倍体酵母中,如果蛋白质X和Y彼此相互作用,则将重新组装功能性泛素,这导致PLV的切割。释放的转录蛋白复合物PLV可以开启报告基因(ADE2,HIS3),并允许酵母生长在选择性培养基上。
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Robust Generation of Knock-in Cell Lines Using CRISPR-Cas9 and rAAV-assisted Repair Template Delivery
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Author:
Date:
2017-04-05
[Abstract] The programmable Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated nuclease 9 (Cas9) technology revolutionized genome editing by providing an efficient way to cut the genome at a desired location (Ledford, 2015). In mammalian cells, DNA lesions trigger the error-prone non-homologous end joining (NHEJ) DNA repair mechanism. However, in presence of a DNA repair template, Homology-Directed Repair (HDR) can occur leading to precise repair of the lesion site. This last process can be exploited to enable precise knock-in changes by introducing the desired genomic ...
[摘要] 可编程集群定期间隔短回归度(CRISPR)相关核酸酶9(Cas9)技术通过提供在所需位置切割基因组的有效方式,彻底改变了基因组编辑(Ledford,2015)。 在哺乳动物细胞中,DNA损伤触发易发生非同源末端连接(NHEJ)DNA修复机制。 然而,在DNA修复模板的存在下,可以发生同源性定向修复(HDR),导致病变部位的精确修复。 可以利用最后的方法,通过在修复模板上引入所需的基因组改变来实现精确的敲入变化。 在本协议中,我们描述了使用重组腺相关病毒(rAAV)在人细胞系中进行基于CRISPR-Cas9的C-末端标签序列敲入的长修复模板(> 200个核苷酸)的递送。
尽管有关CRISPR-Cas9产生的敲门模型系统的大量报告,敲门砖报告仍然落后。由于许多应用,产生敲入细胞系仍然是基因组编辑的明显目标。敲入改变的引入通常依赖于修复模板DNA的存在,并且在位点特异性双链(ds)DNA断裂被引入接近改变位点的基因组中后,HDR修复机制的激活。不同的模板可以传送到修复机器,范围从含有广泛同源区域和可选选择盒的经典线性化载体到约200个核苷酸的单链(ss)DNA寡核苷酸(Chen等人, ...
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