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QIAquick Gel Extraction Kit

QIAquick凝胶提取试剂盒

Company: QIAGEN
Catalog#: 28704
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Protocol for Construction of a Tunable CRISPR Interference (tCRISPRi) Strain for Escherichia coli
Author:
Date:
2017-10-05
[Abstract]  We present a protocol for construction of tunable CRISPR interference (tCRISPRi) strains for Escherichia coli. The tCRISPRi system alleviates most of the known problems of plasmid-based expression methods, and can be immediately used to construct libraries of sgRNAs that can complement the Keio collection by targeting both essential and nonessential genes. Most importantly from a practical perspective, construction of tCRISPRi to target a new gene requires only one-step oligo recombineering. Additional advantages of tCRISPRi over other existing CRISPRi methods include: (1) tCRISPRi shows significantly less than 10% leaky repression; (2) tCRISPRi uses a tunable arabinose operon promoter and modifications in transporter genes to allow a wide dynamic range with graded control by ... [摘要]  我们提出了构建大肠杆菌可调CRISPR干扰(tCRISPRi)菌株的方案。 tCRISPRi系统缓解了基于质粒的表达方法的大多数已知问题,并且可以立即用于构建可通过靶向必需基因和非必需基因来补充Keio收集物的sgRNA的文库。 最重要的是从实践的角度来看,建立tCRISPRi来靶向一个新的基因只需要一步寡核苷酸重组。 tCRISPRi与其他现有CRISPRI方法的其他优点包括:(1)tCRISPRi显示低于10%的泄漏抑制; (2)tCRISPRi使用可调阿拉伯糖操纵子启动子和转运蛋白基因的修饰,以允许通过阿拉伯糖诱导剂分级控制的宽动态范围; (3)tCRISPRi是无质粒的,整个系统整合到染色体中; (4)tCRISPRi菌株显示出理想的生理特性。
【背景】已经开发了各种CRISPR干扰系统,用于从细菌到真核生物的生物体。对于正在考虑使用CRISPRi细菌的人员,我们提供了关于我们的tCRISPRi系统的以下背景资料(Li等等,2016)及其与其他CRISPRi系统的比较。
Morgan-Kiss 等人。 (2002)开发了基于质粒的剂量诱导型启动子pBAD。它们的系统允许来自pBAD启动子的蛋白质的可调节表达,取决于阿拉伯糖水平。阿拉伯糖转运蛋白基因和araFGH在菌株中是无活性的。他们的菌株也有两个拷贝的lacY ...

Dense sgRNA Library Construction Using a Molecular Chipper Approach
Author:
Date:
2017-06-20
[Abstract]  Genetic screens using single-guide-RNA (sgRNA) libraries and CRISPR technology have been powerful to identify genetic regulators for both coding and noncoding regions of the genome. Interrogating functional elements in noncoding regions requires sgRNA libraries that are densely covering, and ideally inexpensive, easy to implement and flexible for customization. We present a Molecular Chipper protocol for generating dense sgRNA libraries from genomic regions of interest. This approach utilizes a combination of random fragmentation and a Type III restriction enzyme to derive a dense coverage of sgRNA library from input DNA. [摘要]  使用单导向RNA(sgRNA)文库和CRISPR技术的遗传筛选功能强大可以识别基因组编码区和非编码区的遗传调控因子。 在非编码区域中询问功能元件需要密集覆盖的sgRNA文库,理想的便宜,易于实现和灵活定制。 我们提出了一个分子切片方案从感兴趣的基因组区域产生密集的sgRNA文库。 该方法利用随机断裂和III型限制酶的组合从输入DNA导出sgRNA文库的致密覆盖。
【背景】使用化脓性链球菌(sp)的基因组编辑Cas9和sgRNA文库是通过产生双重缺失功能序列改变来筛选哺乳动物细胞功能性遗传调节因子的有力工具(Wiedenheft et al。,2012; Mali et al。,2013; Koike-Yusa等,2014; Shalem等,2014; Wang等,2014; Zhou等,2014)。 Cas9结合sgRNA,其可被设计为将Cas9靶向基因组中定义的基因座。 Cas9的核酸酶活性切割靶DNA位点,导致双链DNA断裂,在通过非同源末端连接途径进行DNA修复时,经常导致感兴趣的基因座短缺失。
CRISPR-Cas9系统强大的基因组编辑能力导致使用sgRNA文库来询问蛋白质编码基因以及非编码区域。通过sgRNA富集功能筛选,报告了几种用于蛋白质编码基因和/或有限数量的非编码基因的sgRNA文库,以鉴定调控特定细胞功能的基因和网络(Koike-Yusa等,2014; ...

Targeted Nucleotide Substitution in Mammalian Cell by Target-AID
Author:
Date:
2017-06-05
[Abstract]  Programmable RNA-guided nucleases based on CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated protein) systems have been applied to various type of cells as powerful genome editing tools. By using activation-induced cytidine deaminase (AID) in place of the nuclease activity of the CRISPR/Cas9 system, we have developed a genome editing tool for targeted nucleotide substitution (C to T or G to A) without donor DNA template (Figure 1; Nishida et al., 2016). Here we describe the detailed method for Target-AID to perform programmable point mutagenesis in the genome of mammalian cells. A specific method for targeting the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene in Chinese Hamster Ovary (CHO) cell was described here as an ... [摘要]  基于CRISPR的可编程RNA引导核酸酶(集群定期交织的短回文重复)-Cas(CRISPR相关蛋白)系统已被应用于各种类型的细胞作为强大的基因组编辑工具。通过使用激活诱导的胞苷脱氨酶(AID)代替CRISPR / Cas9系统的核酸酶活性,我们开发了一种用于靶向核苷酸替代(C至T或G至A)的基因组编辑工具,无供体DNA模板(图1 ; Nishida等人,2016)。这里我们描述Target-AID在哺乳动物细胞基因组中进行可编程点突变的详细方法。在这里描述了用于靶向中国仓鼠卵巢(CHO)细胞中的次黄嘌呤 - 鸟嘌呤磷酸核糖基转移酶(HPRT)基因的具体方法作为实例,而该方法主要应适用于任何感兴趣的基因广泛的细胞类型。


图1. Target-AID及其可靶向位点的示意图。在指导RNA(gRNA)依赖性方式中,通过接头与nCas9(D10A)融合的PmCDA1在-21周围进行可编程胞苷突变至相对于哺乳动物细胞中非互补链上的PAM序列的-16位。可目标地点是根据以前的工作中观察到的有效的基础替代(> 20%)来确定的。
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