Evaluation of the Efficiency of Genome Editing Tools by a Frameshift Fluorescence Protein Reporter
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Author:
Date:
2020-05-20
[Abstract] In the last decade, genome editing has been the center of attention as a novel tool for mechanistic investigations and for potential clinical applications. Various genome editing tools like meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALEN), and the clustered regularly interspaced short palindromic repeats (CRISPR)-associated genes (Cas), have been developed in recent years. For the optimal use as well as continued developments of these genome editing tools, the evaluation of their efficiencies and accuracies is vital. Here, we present a protocol for a reporter based on frameshift fluorescence protein which we recently developed to evaluate the efficiency and accuracy of genome editing tools. In this method, a ~20 bp target sequence ...
[摘要] [摘要] 在过去的十年中,基因组编辑作为一种机制研究和潜在临床应用的新工具已成为关注的焦点。近年来,已开发出各种基因组编辑工具,例如大范围核酸酶,锌指核酸酶(ZFN),转录激活子样基于效应子的核酸酶(TALEN)以及成簇的规则间隔的短回文重复序列(CRISPR)相关基因(Cas)。 。对于这些基因组编辑工具的最佳使用和持续发展,评估其效率和准确性至关重要。在这里,我们介绍了一种基于移码荧光蛋白的报告子方案,我们最近开发了该方案以评估效率和 基因组编辑工具的实用性。在这种方法中,在天蓝色荧光蛋白(CFP)的起始密码子后插入一个约20 bp的包含移码的靶序列,以使其荧光失活,并且只有新的插入/缺失事件会重新激活CFP 荧光。 。为了增加可追溯性,将内部核糖体进入位点和红色荧光蛋白mCherryFP 放置在报告子的下游。由in / del介导的荧光恢复产生的CFP阳性细胞的百分比可以通过荧光测量装置定量,作为基因组编辑频率的读数。作为演示,我们在这里介绍CRISPR-Cas9技术的使用以及流式细胞仪作为荧光变化的读数。
[背景] 基因组编辑工具对于生物学机制的研究以及遗传疾病的预防和/或治疗非常重要(Maeder和Gersbach,2016)。在最近的几十年中,引入了几种基因组编辑工具,包括大范围核酸酶(Epinat 等,2003),锌指核酸酶(ZFN)(Kim ...
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Rapid Generation of Human Neuronal Cell Models Enabling Inducible Expression of Proteins-of-interest for Functional Studies
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Author:
Date:
2020-05-05
[Abstract] CRISPR-Cas9 technology has transformed the ability to edit genomic sequences and control gene expression with unprecedented ease and scale. However, precise genomic insertions of coding sequences using this technology remain time-consuming and inefficient because they require introducing adjacent single-strand cuts through Cas9 nickase action and invoking the host-encoded homology-directed repair program through the concomitant introduction of large repair templates. Here, we present a system for the rapid study of any protein-of-interest in two neuronal cell models following its inducible expression from the human AAVS1 safe harbor locus. With lox-flanked foundation cassettes in the AAVS1 site and a tailor-made plasmid for accepting coding sequences-of-interest in ...
[摘要] [摘要] CRISPR-Cas9技术以前所未有的简便性和规模改变了编辑基因组序列和控制基因表达的能力。但是,由于需要引入相邻的单链,因此使用该技术进行精确的基因组编码插入仍然很耗时且效率低下。通过减少Cas9切口酶的作用并通过同时引入大型修复模板来调用宿主编码的同源性指导的修复程序。在此,我们提出了一种系统,用于在其诱导后的两个神经元细胞模型中快速研究任何目的蛋白该系统可从人类AAVS1 安全港基因座表达,在AAVS1 位点具有lox侧翼的基础盒和定制的质粒以接受感兴趣的编码序列,该系统使研究人员能够为诱导型产生自己的神经元细胞模型任何编码表达序列不到一个月的时间。由于可用性Preinserted 增强型绿色的Fluo 可以与目标蛋白质融合的最新蛋白质(EGFP)编码序列,该系统可帮助功能研究通过活细胞显微镜以及利用非常有效的可用性进行的相互作用组分析来跟踪目标蛋白质EGFP捕获矩阵。
[背景] ...
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Dual Fluorescence Reporter Based Analytical Flow Cytometry for miRNA Induced Regulation in Mammalian Cells
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Author:
Date:
2018-09-05
[Abstract] MicroRNA-induced gene regulation is a growing field in basic and translational research. Examining this regulation directly in cells is necessary to validate high-throughput data originated from RNA sequencing technologies. For this several studies employ luciferase-based reporters that usually measure the whole cell population, which comes with low resolution for the complexity of the miRNA-induced regulation. Here, we provide a protocol using a dual-fluorescence reporter and flow cytometry reaching single cell resolution; the protocol contains a simplified workflow that includes: vector generation, data acquisition, processing, and analysis using the R environment. Our protocol enables high-resolution measurements of miRNA induced post-transcriptional gene regulation and combined with ...
[摘要] MicroRNA诱导的基因调控是基础和转化研究中不断增长的领域。 直接在细胞中检查该调节对于验证源自RNA测序技术的高通量数据是必要的。 对于这一研究,一些研究采用基于荧光素酶的报告基因,通常测量全细胞群,其具有低分辨率的miRNA诱导调节的复杂性。 在这里,我们提供使用双荧光报告基因和流式细胞仪达到单细胞分辨率的方案; 该协议包含一个简化的工作流程,包括:使用R环境进行矢量生成,数据采集,处理和分析。 我们的协议可实现miRNA诱导的转录后基因调控的高分辨率测量,并结合系统生物学,可用于估计miRNA的熟练程度。
【背景】MicroRNAs(miRNA)是高度保守的小型非蛋白质编码RNA(21-22nt),可调节转录后基因表达并调节基因生物学过程,如发育和细胞稳态(Lagos-Quintana et al。,2001; Fabian et al。,2010; Bartel,2018),包括miRNA表达与肿瘤进展和侵袭性相关的几种病理(Lu et al。, 2005; Di Leva和Croce,2013; Krishnan et al。,2015; Bertoli et ...
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