A CRISPR Competition Assay to Identify Cancer Genetic Dependencies
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Author:
Date:
2020-07-20
[Abstract] The CRISPR/Cas9 system is a powerful tool for genome editing, wherein the RNA-guided nuclease Cas9 can be directed to introduce double-stranded breaks (DSBs) at a targeted locus. In mammalian cells, these DSBs are typically repaired through error-prone processes, resulting in insertions or deletions (indels) at the targeted locus. Researchers can use these Cas9-mediated lesions to probe the consequences of loss-of-function perturbations in genes of interest. Here, we describe an optimized protocol to identify specific genes required for cancer cell fitness through a CRISPR-mediated cellular competition assay. Identifying these genetic dependencies is of utmost importance, as they provide potential targets for anti-cancer drug development. This protocol provides researchers with a robust ...
[摘要] [摘要] CRISPR / Cas9系统是用于基因组编辑的强大工具,其中RNA引导的核酸酶Cas9可以直接在目标基因座处引入双链断裂(DSB)。在哺乳动物细胞中,这些DSB通常通过容易出错的过程进行修复,从而导致在目标基因座处插入或缺失(indel)。研究人员可以使用这些Cas9介导的病变来探究结果目标基因的功能丧失扰动。在这里,我们描述了一种优化的协议,可通过CRISPR介导的细胞竞争测定法来鉴定癌细胞适应性所需的特定基因。鉴定这些遗传依赖性至关重要,因为它们为抗癌药物的开发提供了潜在的靶标。该协议为研究人员提供了一种强大且可扩展的方法,以研究多种细胞系和癌症类型中的基因依赖性,并验证高通量或全基因组筛选的结果。
[背景] CRISPR / Cas9系统被认为已发展成为一种适应性的原核病毒防御系统(Mojica 等,2005; Makarova 等,2006)。它被发现后不久,就被研究人员选中,并进行了基因组编辑以供实验室使用(Doudna和Charpentier,2014年; Hsu 等人,2014年)。通过转基因表达Cas9核酸酶以及与靶序列互补的短链RNA(sgRNA),可以将双链断裂(DSB)引入各种细胞和生物体的目标位点(Cong 等,2013)。 ...
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In vitro RNA-dependent RNA Polymerase Assay Using Arabidopsis RDR6
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Author:
Date:
2018-01-05
[Abstract] RNA-dependent RNA polymerases (RdRPs) in eukaryotes convert single-stranded RNAs into double-stranded RNAs, thereby amplifying small interfering RNAs that play crucial roles in the regulation of development, maintenance of genome integrity and antiviral immunity. Here, we describe a method of in vitro RdRP assay using recombinant Arabidopsis RDR6 prepared by an insect expression system. By using this classical biochemical assay, we revealed that RDR6 has a strong template preference for RNAs lacking a poly(A) tail. This simple method will be applicable to other RdRPs in Arabidopsis and different organisms.
[摘要] 真核生物中的RNA依赖性RNA聚合酶(RdRP)将单链RNA转化为双链RNA,从而扩增在调节发育,维持基因组完整性和抗病毒免疫方面起关键作用的小干扰RNA。 在此,我们描述了使用通过昆虫表达系统制备的重组拟南芥RDR6的体外RdRP测定的方法。 通过使用这种经典的生物化学分析,我们发现RDR6有一个强大的模板偏好RNAs缺乏poly(A)尾巴。 这个简单的方法将适用于拟南芥属和其他生物体中的其他RdRPs。
【背景】已经在所有真核生物王国 - 植物,真菌,原生动物和动物中发现RNA依赖性RNA聚合酶(RdRP)基因(Zong等人,2009)。它们将单链RNA(ssRNA)转化为双链RNA(dsRNA),从而扩增在各种生物过程中发挥关键作用的小干扰RNA(siRNA),包括调节发育(Peragine等人, ,2004; Li等人,2005),维持基因组完整性(Volpe等人,2002; Xie等人,2004年, )和抗病毒免疫性(Mourrain等人,2000; Yu等人,2003; Garcia-Ruiz等人,2010; Wang ,2010)。除了这种RdRP活性之外,RdRP还具有称为末端核苷酸转移酶(TNTase)活性的另一种酶活性(Curaba和Chen,2008; ...
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In vitro Engineered DNA-binding Molecule-mediated Chromatin Immunoprecipitation (in vitro enChIP) Using CRISPR Ribonucleoproteins in Combination with Next-generation Sequencing (in vitro enChIP-Seq) for the Identification of Chromosomal Interactions
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Author:
Date:
2017-11-20
[Abstract] We have developed locus-specific chromatin immunoprecipitation (locus-specific ChIP) technologies consisting of insertional ChIP (iChIP) and engineered DNA-binding molecule-mediated ChIP (enChIP). Locus-specific ChIP is a method to isolate a genomic region of interest from cells while it also identifies what binds to this region using mass spectrometry (for protein) or next generation sequencing (for RNA or DNA) as described in Fujita et al. (2016a). Recently, we identified genomic regions that physically interact with a locus using an updated form of enChIP, in vitro enChIP, in combination with NGS (in vitro enChIP-Seq) (Fujita et al., 2017a). Here, we describe a protocol on in vitro enChIP to isolate a target locus for identification of ...
[摘要] 我们开发了基因座特异性染色质免疫沉淀(基因座特异性芯片)技术,包括插入ChIP(iChIP)和工程DNA-结合分子介导ChIP(enChIP)。 基因座特异性ChIP是一种从细胞中分离感兴趣的基因组区域的方法,同时它还使用质谱(用于蛋白质)或下一代测序(用于RNA或DNA)鉴定什么与该区域结合,如Fujita等人 (2016a)。 最近,我们使用更新后的enChIP形式,结合NGS( in vitro enChIP-Seq),鉴定了与基因座物理相互作用的基因组区域(Fujimita et al。,2017a)。 在这里,我们描述了一个体外试验的方法,用于分离靶基因座以鉴定与基因座物理相互作用的基因组区域。 【背景】阐明基因组功能强调的分子机制需要鉴定与感兴趣的基因组区域相互作用的分子。为此,我们开发了由插入ChIP(iChIP)和工程化DNA结合分子介导的ChIP(enChIP)组成的基因座特异性染色质免疫沉淀技术(基因座特异性ChIP)技术(Fujita等人 ...
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