| Defined Mutant Library Sequencing (DML-Seq) for Identification of Conditional Essential Genes
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Author:
Date:
2021-03-05
[Abstract] Transposon insertion sequencing (TIS) is an emerging technique which utilizes a massive transposon mutant library to screen specific phenotype and determine the conditional essential genetic requirements for bacterial fitness under distinct conditions combined with high-throughput parallel sequencing technology. Compared with a massive mutant library in traditional TIS, the defined mutant library sequencing (DML-Seq) has advantages as: 1) efficient mutagenesis; 2) low bottleneck effects; 3) avoid hotpots caused by screening; 4) can be directly used in the following experiments. Here, we described an optimized procedure of DML-Seq for fitness screen to supply classical TIS using the marine pathogenic bacterium Edwardsiella piscicida as an example.
[摘要] [摘要]转座子插入测序（TIS）是一项新兴技术，它利用大量的转座子突变体文库筛选特定表型，并结合高通量并行测序技术，在不同条件下确定细菌适应性的条件性基本遗传要求。与传统TIS中的大规模突变文库相比，已定义的突变文库测序（DML-Seq）具有以下优势：1）高效诱变；2）瓶颈效应低；3）避免因筛选引起的火锅；4）可直接用于以下实验。在这里，我们描述DML-SEQ的优化过程进行健身屏幕使用海洋致病菌提供古典TIS爱德华piscicida作为一个例子。
[背景]转座子插入诱变与下一代测序（NGS）结合已被证明是在多种条件下研究基因功能的有效方法（Chao等，2016； Price等，2018）。通常，TIS分析由转座子插入位点的大规模平行测序和大量插入事件的统计分析组成。
基于TIS的筛选可以在多种条件下基于高度饱和的转座子突变体文库的细菌适应性，提供单个基因座和域的适应性贡献的高分辨率图（Chao等，2016）。每个位点的插入频率或相应突变体的相对丰度通常与施加选择性压力（例如宿主和抗生素施加的压力）后与基因座对适应性的贡献成反比（Chao等人，2016）。这种方法的原理是多种相关方法的基础，包括TIS，转座子测序（TnSeq ），插入测序（INSeq ），转座子定向插入位点测序（TraDIS ...
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| 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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