| 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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| 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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