p426-SNR52p-gRNA.CAN1.Y-SUP4t

公司名称: Addgene

产品编号: 43803

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Markerless Gene Editing in the Hyperthermophilic Archaeon Thermococcus kodakarensis

Alexandra M. Gehring, Travis J. Sanders and Thomas J. Santangelo

Published online: 2017-11-20

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Method for Multiplexing CRISPR/Cas9 in Saccharomyces cerevisiae Using Artificial Target DNA Sequences

Author:

Rachael M. Giersch and Gregory C. Finnigan,

Date:

2017-09-20

[Abstract] Genome manipulation has become more accessible given the advent of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) editing technology. The Cas9 endonuclease binds a single stranded (single guide) RNA (sgRNA) fragment that recruits the complex to a corresponding genomic target sequence where it induces a double stranded break. Eukaryotic repair systems allow for the introduction of exogenous DNA, repair of existing mutations, or deletion of endogenous gene products. Targeting of Cas9 to multiple genomic positions (termed ‘multiplexing’) is achieved by the expression of ... [摘要] 鉴于CRISPR（集群定期间隔短回归重复）编辑技术的出现，基因组操纵变得更加易于使用。 Cas9核酸内切酶将募集复合物的单链（单向导）RNA（sgRNA）片段结合到相应的基因组靶序列，引发双链断裂。真核修复系统允许引入外源DNA，修复现有突变或内源基因产物的缺失。通过在同一核内表达多个sgRNA来实现Cas9对多个基因组位置的定位（称为“多重”）。然而，CRISPR领域的持续关注是将Cas9意外地定位到基因组内的替代（“脱靶”）DNA位置。我们将安装的人造Cas9靶序列的使用（称为人造基因座上的Cas9复制）描述为允许（i）与单个sgRNA复用的酵母基因组中的用途; （ii）减少/消除可能的脱靶效应，以及（iii）精确控制预定目标序列的放置。
【背景】CRISPR（集群定期间隔回归重复）机制已经在原核生物中演变为具有很高精度编辑任何基因组的能力的原始适应性免疫系统（Jinek等，2012; Sorek等，2013）。这种生物技术需要使用来自化脓性链球菌（或othologous物种）的内切核酸酶（Cas9），单个RNA'引导'序列和外源供体DNA（如果需要）。仅在短短几年内，CRISPR / ...

CRISPR-PCS Protocol for Chromosome Splitting and Splitting Event Detection in Saccharomyces cerevisiae

Author:

Yu Sasano and Satoshi Harashima,

Date:

2017-05-20

[Abstract] Chromosome engineering is an important technology with applications in basic biology and biotechnology. Chromosome splitting technology called PCS (PCR-mediated Chromosome Splitting) has already been developed as a fundamental chromosome engineering technology in the budding yeast. However, the splitting efficiency of PCS technology is not high enough to achieve multiple splitting at a time. This protocol describes a procedure for achieving simultaneous and multiple chromosome splits in the budding yeast Saccharomyces cerevisiae by a new technology called CRISPR-PCS. At least four ... [摘要] 染色体工程是应用于基础生物学和生物技术的重要技术。染色体分裂技术称为PCS（PCR介导的染色体分裂）已经被开发为发芽酵母中的基本染色体工程技术。然而，PCS技术的分割效率不够高，不能一次实现多次分割。该协议描述了通过称为CRISPR-PCS的新技术在芽状酵母酿酒酵母中实现同时和多个染色体分裂的过程。基因组中至少四个独立的位点可以通过一次转化来分裂。与常规PCS技术相比，获得多重分裂酵母菌株的总时间和劳动力大大降低。

背景能够快速有效地操纵多个遗传基因座或染色体区域的染色体工程技术变得越来越重要。这些技术为阐明染色体和基因组功能提供了有力的手段。此外，它可以用于通过创建广泛的遗传变体来繁殖有用的菌株。在芽殖酵母酿酒酵母（Saccharomyces ...

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