| 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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| Agrobacterium tumefaciens-mediated Transformation of Walnut (Juglans regia)
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Author:
Date:
2014-10-05
[Abstract] Like many woody plant species, walnut (Juglans regia) can be difficult to genetically transform and regenerate. However, somatic embryos have been used successfully for over two decades as a target tissue for transformation and regeneration of transgenic walnut plants. Walnut somatic embryos, initiated originally from developing zygotic embryos or anther tissue, will proliferate numerous secondary embryos from single cells in the epidermal layer. These single cells in intact somatic embryos can be efficiently transformed by Agrobacterium tumefaciens (A. tumefaciens). This gene transfer system is most efficient when Agrobacterium binary vector plasmids contain a scorable maker gene (e.g. uidA) and a selectable marker gene (e.g. nptII). This ...
[摘要] 像许多木本植物物种一样，核桃（ Juglans regia ）可能难以进行遗传转化和再生。然而，体细胞胚已经成功使用超过二十年作为转基因核桃植物的转化和再生的靶组织。核桃体细胞胚，最初从发育的合子胚或花药组织开始，将从表皮层中的单个细胞增殖许多次级胚。在完整的体细胞胚中的这些单细胞可以通过根癌土壤杆菌（根瘤土壤杆菌）有效地转化（ tumefaciens ）。当农杆菌二元载体质粒含有可打分的基因（例如uidA）和选择性标记基因（例如nptII）时，该基因转移系统是最有效的。该系统应适用于从单个农杆菌感受细胞经历重复胚胎发生的任何作物。在这里我们详细描述转化体细胞胚胎的方法，使这种技术可以应用于核桃和其他木本植物物种。
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