Multiplexed GuideRNA-expression to Efficiently Mutagenize Multiple Loci in Arabidopsis by CRISPR-Cas9
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Author:
Date:
2017-03-05
[Abstract] Since the discovery of the CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein (Cas) as an efficient tool for genome editing in plants (Li et al., 2013; Shan et al., 2013; Nekrasov et al., 2013), a large variety of applications, such as gene knock-out, knock-in or transcriptional regulation, has been published. So far, the generation of multiple mutants in plants involved tedious crossing or mutagenesis followed by time-consuming screening of huge populations and the use of the Cas9-system appeared a promising method to overcome these issues. We designed a binary vector that combines both the coding sequence of the codon optimized Streptococcus pyogenes Cas9 nuclease under the control of the Arabidopsis thaliana ...
[摘要] 自从发现CRISPR(聚集的定期交织的短回文重复) - 相关蛋白(Cas)作为植物基因组编辑的有效工具(Li等人,2013; Shan等人已经出版了诸如基因敲除,敲入或转录调控等各种各样的应用,例如,2013; Nekrasov等人,2013)。到目前为止,植物中多种突变体的产生涉及繁琐的杂交或诱变,随后大量人群的耗时筛选,Cas9系统的使用似乎是有希望的方法来克服这些问题。我们设计了一种二元载体,其结合了在拟南芥UBIQUITIN10(UBQ10)启动子和引导RNA(gRNA)控制下的优化的化脓性链球菌(Caspase)密码子的编码序列)由 A驱动的表现盒。拟南芥U6 - 启动子,用于在拟南芥中进行有效的多重编辑(阎等人,2016年)。在这里,我们描述了一个逐步的方案,以经济有效的方式生成含有多个gRNA的二元载体和基于经典克隆方法的Cas9核酸酶。背景 RNA引导的Cas9系统源于针对外源DNA的细菌防御系统(Sorek等人,2013)。由于其高效率,易于处理和多重编辑的可能性,已经被认为是基因组编辑的选择方法。通常,Cas9基因编辑系统涉及单个合成RNA分子,其指导Cas9蛋白质靶向所需DNA位点以进行基因组修饰或转录控制的gRNA。 gRNA-Cas9复合物通过gRNA-DNA配对识别靶向的DNA,并需要存在原始相邻基序(PAM)。 ...
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siRNA Screening for Genes Involved in HSV-1 Replication
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Author:
Date:
2014-08-20
[Abstract] Small interfering RNAs (siRNAs) are small (typically 18-24 nucleotides) RNA molecules capable of silencing gene expression post-transcriptionally and as such, they provide a simple method by which the role of individual genes in complex cellular systems can be easily assessed. As siRNAs are easy to use experimentally, and can be designed to target any gene (including pathogens), their use is perfectly suited to and easily adapted to high-throughput genome-wide screening methodologies and a range of phenotypic assays. Here we describe the use of a large siRNA library (>8,000 genes targeted individually) to screen for and identify host factors functionally involved in the replication of a human herpesvirus (Herpes simplex virus type 1; HSV-1) (Griffiths et al., 2013; Griffiths, ...
[摘要] 小干扰RNA(siRNA)是能够沉默转录后基因表达的小(通常为18-24个核苷酸)RNA分子,因此,它们提供了一种简单的方法,通过其可以容易地评估单个基因在复杂细胞系统中的作用。 由于siRNA易于实验使用,并且可以设计为靶向任何基因(包括病原体),它们的使用完全适合于并且容易地适应于高通量全基因组筛选方法学和一系列表型测定。 这里我们描述了使用大的siRNA文库(> 8,000个基因单独靶向)来筛选和鉴定功能上涉及人疱疹病毒(1型单纯疱疹病毒; HSV-1)(Griffiths)的复制的宿主因子, et al。,2013; Griffiths,2013)。
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