| Genomic Edition of Ashbya gossypii Using One-vector CRISPR/Cas9
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Author:
Date:
2020-06-20
[Abstract] The CRISPR/Cas9 system is a novel genetic tool which allows the precise manipulation of virtually any genomic sequence. In this protocol, we use a specific CRISPR/Cas9 system for the manipulation of Ashbya gossypii. The filamentous fungus A. gossypii is currently used for the industrial production of riboflavin (vitamina B2). In addition, A. gossypii produces other high-value compounds such as folic acid, nucleosides and biolipids. A large molecular toolbox is available for the genomic manipulation of this fungus including gene targeting methods, rapid assembly of heterologous expression modules and, recently, a one-vector CRISPR/Cas9 editing system adapted for A. gossypii that allows marker-free engineering strategies to be implemented. The CRISPR/Cas9 ...
[摘要] [摘要] CRISPR/Cas9系统是一种新的基因工具，可以精确地操作几乎所有的基因组序列。在这个协议中，我们使用一个特定的CRISPR/Cas9系统来操纵棉蚜。丝状真菌A.gossypii目前用于核黄素（vitamina B2）的工业生产。此外，棉蚜还产生其他高价值化合物，如叶酸、核苷和生物脂类。一个大型的分子工具箱可用于该真菌的基因组操作，包括基因靶向方法、异源表达模块的快速组装，以及最近一个适用于棉铃虫的单载体CRISPR/Cas9编辑系统，该系统允许实施无标记工程策略。CRISPR/Cas9系统包括RNA引导的DNA内切酶（Cas9）和与基因组靶区互补的引导RNA（gRNA）。Cas9核酸酶需要5′-NGG-3′三核苷酸，称为原间隔基序（PAM），在基因组靶区产生一个双链断裂（DSB），该断裂可以通过同源重组（HR）由合成的致突变供体DNA（dDNA）修复，从而引入一个特定的设计突变。适用于棉铃虫的CRISPR/Cas9系统极大地促进了这种工业真菌的基因组编辑。
[背景] ...
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| Preparation of Sequencing RNA Libraries through Chemical Cross-linking Coupled to Affinity Purification (cCLAP) in Saccharomyces cerevisiae
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Author:
Date:
2018-10-05
[Abstract] Ribonucleoprotein particles (mRNPs) are complexes consisting of mRNAs and RNA-binding proteins (RBPs) which control mRNA transcription localization, turnover, and translation. Some mRNAs within the mRNPs have been shown to undergo degradation or storage. Those transcripts can lack general mRNA elements, like the poly(A) tail or 5’ cap structure, which prevent their identification through the application of widely-used approaches like oligo(dT) purification. Here, we describe a modified cross-linking affinity purification protocol (cCLAP) based on existing cross-linking and immunoprecipitation (CLIP) methods to isolate mRNAs which could be deadenylated, decapped and/or partially degraded in mRNPs, opening the possibility to detect different types of non-coding RNAs (ncRNAs). Once isolated, ...
[摘要] 核糖核蛋白颗粒（mRNP）是由mRNA和RNA结合蛋白（RBP）组成的复合物，其控制mRNA转录定位，转换和翻译。已显示mRNP内的一些mRNA经历降解或储存。那些转录物可能缺乏一般的mRNA元件，如poly（A）尾或5'帽结构，这通过应用广泛使用的方法如oligo（dT）纯化来阻止它们的鉴定。在这里，我们描述了基于现有的交联和免疫沉淀（CLIP）方法的修饰的交联亲和纯化方案（cCLAP），以分离mRNP中可被去腺苷酸化，去除和/或部分降解的mRNA，从而开启了检测不同的可能性。非编码RNA（ncRNA）的类型。分离后，将RNA进行衔接子连接，然后进行下一代测序（NGS）。由于快速有效的交联和淬灭步骤，该方案也适用于瞬时诱导的mRNP颗粒。实例包括由外在应激物触发的处理体（PB）或应力颗粒（SG）。其重现性和广泛应用使该方案成为研究特定RNP的RNA组成的有用且有力的工具。
【背景】mRNP内转录物的表征对于理解细胞转录和转录后过程至关重要。通过交联和免疫沉淀，然后通过RNA-Seq从mRNP颗粒中分离RNA已经成为鉴定mRNA靶标的常用方法（Tagwerker et al。，2006; Hafner et al。，2010; Kishore et al。，2011）。 ...
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