Spectinomycin

Company: VWR

Catalog#: 101454-196

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Construction of Antisense RNA-mediated Gene Knock-down Strains in the Cyanobacterium Anabaena sp. PCC 7120

Amit Srivastava, Anand Ballal, Karl Forchhammer and Anil Kumar Tripathi

Published online: 2020-02-20

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Dual sgRNA-based Targeted Deletion of Large Genomic Regions and Isolation of Heritable Cas9-free Mutants in Arabidopsis

Author:

Yu Jin and Sebastian Marquardt,

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。

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