| Production of Guide RNAs in vitro and in vivo for CRISPR Using Ribozymes and RNA Polymerase II Promoters
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Author:
Date:
2017-02-20
[Abstract] CRISPR/Cas9-mediated genome editing relies on a guide RNA (gRNA) molecule to generate sequence-specific DNA cleavage, which is a prerequisite for gene editing. Here we establish a method that enables production of gRNAs from any promoters, in any organisms, and in vitro (Gao and Zhao, 2014). This method also makes it feasible to conduct tissue/cell specific gene editing.
[摘要] CRISPR / Cas9介导的基因组编辑依赖于指导性RNA（gRNA）分子来产生序列特异性DNA切割，这是基因编辑的前提条件。在这里，我们建立了一种能够从任何启动子，任何生物体内和体外生产gRNA的方法（Gao和Zhao，2014）。该方法也可以进行组织/细胞特异性基因编辑。
背景 几乎所有报道的CRISPR介导的基因编辑病例都使用小核RNA如U6和U3 snRNA启动子的启动子来驱动体内生产gRNAs（Cong等人，2013; Mali等人，2013）。然而，U6和U3启动子有几个主要的局限性：1）它们是组成型活性的，不可调谐的; 2）它们缺乏细胞/组织特异性; 3）它们在许多生物体中没有明确定义; 4）U6需要G和U3需要A用于转录起始，从而限制目标选择; 5）由于缺乏商业RNA聚合酶III，它们不适合体外转录。不幸的是，构成大部分特征启动子的RNA聚合酶II启动子不能直接用于体内的gRNA生产，原因如下：1）RNA聚合酶II启动子的主要转录物经历广泛的加工如5'-端封端，3'末端聚腺苷酸化和拼接出内含子。一些修饰可能使设计的gRNA无功能。 2）将成熟的RNA分子转运到细胞质中;因此它们与位于细胞核中的预期目标物理分离。这就是为什么使用U6和U3 ...
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| Enzymatic Reactions and Detection of C3 Cleavage Fragments
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Author:
Date:
2014-08-20
[Abstract] The complement component C3 is the major effector molecule of the complement system. C3 circulates in the blood and interstitial fluids as pro-enzyme and is activated by enzymatic cleavage into a C3a portion, a classic anaphylatoxin that functions as chemoattractant and immune cell activator, and the C3b portion, the body’s most potent opsonin. C3 cleavage is in most cases mediated by an enzyme complex called the C3 convertase. However, it is now becoming increasingly clear that the cleavage of C3 by a range of ‘single’ proteases into bioactive C3a and C3b fragments is of high physiological significance. Here, we describe a protocol for the enzymatic cleavage of human C3 by the serine protease cathepsin L and the detection of the cleavage products C3a and C3b by western blotting as an ...
[摘要] 补体组分C3是补体系统的主要效应分子。 C3作为前酶在血液和间质液中循环，并通过酶裂解活化为C3a部分，用作化学引诱物和免疫细胞活化剂的经典过敏毒素和作为机体最有效调理素的C3b部分。 C3切割在大多数情况下由称为C3转化酶的酶复合物介导。 然而，现在越来越清楚的是，通过一系列"单一"蛋白酶将C3切割成生物活性C3a和C3b片段具有高生理学意义。 在这里，我们描述了通过丝氨酸蛋白酶组织蛋白酶L酶切割人C3的方案和通过蛋白质印迹检测裂解产物C3a和C3b作为这种酶反应的实例。
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