| A Novel Method to Construct Binary CRISPR Vectors for Plant Transformation by Single Round of PCR Amplification
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Author:
Date:
2021-04-05
[Abstract] CRISPR/Cas9 is an established and flexible tool for genome editing. However, most methods used to generate expression clones for the CRISPR/Cas9 are time-consuming. Hence, we have developed a one-step protocol to introduce sgRNA expression cassette(s) directly into binary vectors (Liu et al., 2020). In this approach, we have optimized the multiplex PCR to produce an overlapping PCR product in a single reaction to generate the sgRNA expression cassette. We also amplified two sgRNA expression cassettes through a single round of PCR. Then, the sgRNA expression cassette(s) is cloned into the binary vectors in a Gateway LR or Golden gate reaction. The system reported here provides a much more efficient and simpler procedure to construct expression clones for CRISPR/Cas9-mediated genome ...
[摘要] [摘要] CRISPR / Cas9是一种成熟且灵活的基因组编辑工具。但是，大多数用于生成CRISPR / Cas9表达克隆的方法都很耗时。因此，我们开发了一种将sgRNA表达盒直接引入二元载体的一步协议（Liu等人，2020年）。在这种方法中，我们优化了多重PCR，以在单个反应中产生重叠的PCR产物，从而生成sgRNA表达盒。我们还通过单轮PCR扩增了两个sgRNA表达盒。然后，在Gateway LR或Golden gate反应中将sgRNA表达盒克隆到二元载体中。本文报道的系统为构建用于CRISPR / Cas9介导的基因组编辑的表达克隆提供了更有效，更简单的程序。在此协议中，我们描述了使用此系统的详细分步说明。
[背景]乙acteria保卫针对病毒通过蛋白系统，由群集规则间隔开的短回文重复序列（CRISPR）中，CRISPR相关（CAS）蛋白质，CRISPR的RNA（crRNAs）和反式编码crRNA（tracrRNA）。现在，研究人员已经将其系统开发为用于靶向基因组编辑的关键工具。CRISPR –二元载体表达两个元素–具有靶序列的sgRNA（target-sgRNA）和Cas9蛋白–切割靶基因组区域。冯等人。（2013年）已经构建了网关载体，通过农杆菌介导的转化在植物中共表达Cas9和sgRNA ...
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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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| A SsrA/NIa-based Strategy for Post-Translational Regulation of Protein Levels in Gram-negative Bacteria
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Author:
Date:
2020-07-20
[Abstract] Strategies to control the levels of key enzymes of bacterial metabolism are commonly based on the manipulation of gene of interest within the target pathway. The development of new protocols towards the manipulation of biochemical processes is still a major challenge in the field of metabolic engineering. On this background, the FENIX (functional engineering of SsrA/NIa-based flux control) system allows for the post-translational regulation of protein levels, providing both independent control of the steady-state protein amounts and inducible accumulation of target proteins. This strategy enables an extra layer of control over metabolic fluxes in bacterial cell factories (see Graphical abstract below). The protocol detailed here describes the steps needed to design FENIX-tagged ...
[摘要] [摘要 ] 控制细菌代谢关键酶水平的策略通常基于目标途径内目标基因的操纵。朝着生化过程的操纵发展新协议仍然是代谢工程领域的主要挑战。在此背景下，FENIX（基于SsrA / NIa的流量控制功能工程）系统可进行蛋白质水平的翻译后调节，既提供对稳态蛋白质量的独立控制，又可诱导焦油获得蛋白质的积累。这一战略ENABL 上课了代谢流和细菌细胞工厂控制的一个额外层（见图形抽象下文）。此处详细介绍的协议描述了设计FENIX标签的蛋白质并使系统适应几乎所有代谢通量微调途径的步骤。
D：\重新格式化\ 2020-5-6 \ 1903046--1448贡萨洛·杜兰特714707 \图jpg \图1.jpg
图形概要
[背景 ] 控制蛋白质生产已成为已被一个具有挑战性的问题主要是解决由操纵它的生产的不同层次的调节，如所产生的DNA水平或蛋白质的（一个或多个）的量（吴等人，2016 ; Avcilar- Kucukgoze 等人，2017）。在DNA方面，已经在几种微生物中广泛研究了转录和翻译，从而能够设计和开发大量合成电路以改善生物生产过程（Guzmán 等，1995 ； Lutz 和Bujard ，1997）。相比之下，对蛋白质水平的研究较少，主要基于RNA干扰，核糖调节剂和特定的转录调节子（Isaacs ...
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