| Dual sgRNA-based Targeted Deletion of Large Genomic Regions and Isolation of Heritable Cas9-free Mutants in Arabidopsis
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Author:
Date:
2020-10-20
[Abstract] CRISPR/Cas9 system directed by a gene-specific single guide RNA (sgRNA) is an effective tool for genome editing such as deletions of few bases in coding genes. However, targeted deletion of larger regions generate loss-of-function alleles that offer a straightforward starting point for functional dissections of genomic loci. We present an easy-to-use strategy including a fast cloning dual-sgRNA vector linked to efficient isolation of heritable Cas9-free genomic deletions to rapidly and cost-effectively generate a targeted heritable genome deletion. This step-by-step protocol includes gRNA design, cloning strategy and mutation detection for Arabidopsis and may be adapted for other plant species.
[摘要] [摘要] CRISPR/Cas9由基因特异性单导RNA（sgRNA）引导的系统是一种有效的基因组编辑工具，如编码基因中少部分碱基的删除。然而，大区域的靶向缺失产生功能缺失等位基因，这为基因组基因座的功能解剖提供了一个直接的起点。我们提出了一个简单易用的策略，包括一个快速克隆双sgRNA载体，有效分离可遗传的Cas9游离基因组缺失，以快速且经济有效地产生靶向遗传基因组缺失。该方法包括拟南芥的gRNA设计、克隆策略和突变检测，可适用于其他植物。
[背景] ...
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| Assembly of Genetic Circuits with the Mammalian ToolKit
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Author:
Date:
2020-03-05
[Abstract] The ability to rapidly assemble and prototype cellular circuits is vital for biological research and its applications in biotechnology and medicine. The Mammalian ToolKit (MTK) is a Golden Gate-based cloning toolkit for fast, reproducible and versatile assembly of large DNA vectors and their implementation in mammalian models. The MTK consists of a curated library of characterized, modular parts that can be assembled into transcriptional units and further weaved into complex circuits. These circuits are easily repurposed and introduced in mammalian cells by different methods.
[摘要] [摘要 ] 快速组装和原型细胞电路的能力对于生物学研究及其在生物技术和医学中的应用至关重要。哺乳动物工具箱（MTK）是基于金门大桥的克隆工具箱，用于快速，可复制和通用的大型DNA载体组装及其在哺乳动物模型中的实现。MTK由精选的，模块化的零件组成的精选库组成，这些零件可以组装成转录单位，并进一步编织成复杂的电路。这些电路很容易重新利用，并通过不同的方法引入哺乳动物细胞。
[背景 ] 分子克隆是现代生物技术与重新利用重组DNA导入多种基因电路可以表示目的的频谱的能力的标志。但是，探索遗传电路构建中可能存在的排列的主要局限性在于能否对电路设计进行快速原型设计，测试和实施改进。为了实现这一目标，需要从常规的克隆方法（如Gibson克隆（Akama-Garren 等人，2016）或限制性酶切消化）中加快从设计遗传回路到将其递送至细胞的时间。我们设计了一个框架，在该框架中，传统的基因电路被分解成其组成部分，以便人们可以轻松地交换这些组成部分，以快速组装出巨大的组合，从而评估每次迭代如何影响功能。受早期克隆工具包迭代的启发（Weber 等人，2011 a和2011b ; Duportet 等人，2014; Lee 等人，2015; Martella 等人，2017;Pérez-González 等人，2017; Halleran 等人，2017）等人，2018年; ...
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