Screening Method for CRISPR/Cas9 Inhibition of a Human DNA Virus: Herpes Simplex Virus
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Author:
Date:
2020-09-05
[Abstract] The efficiency of cleavage of individual CRISPR/Cas9-sgRNAs remains difficult to predict based on the CRISPR target sequence alone. Different intracellular environments (dependent on cell type or cell cycle state for example) may affect sgRNA efficiency by altering accessibility of genomic DNA through DNA modifications such as epigenetic marks and DNA-binding proteins (e.g., histones) as well as alteration of the chromatin state of genomic DNA within the nucleus.
We recently reported a multi-step screening method for the identification of efficient sgRNAs targeting the Herpes simplex virus (HSV-1) genome and reported a differential mechanism for viral inhibition by CRISPR-Cas9 in the latent versus lytic phase. The screening platform detailed in this protocol allows ...
[摘要] [摘要 ] 单独基于CRISPR靶序列仍难以预测单个CRISPR / Cas9-sgRNA的切割效率。不同的细胞内环境(例如,取决于细胞类型或细胞周期状态)可能会通过DNA修饰(例如表观遗传标记和DNA结合蛋白(例如组蛋白))和染色质状态的改变来改变基因组DNA的可及性,从而影响sgRNA效率。核内基因组DNA的标记。
我们recen TLY 报道的多步骤方法筛选用于有效sgRNAs靶向的识别的单纯疱疹病毒(HSV-1)的基因组,并报告为在潜对裂解病毒相抑制CRISPR-Cas9的差动机构。该协议中详述的筛选平台允许在无细胞系统中以及在病毒靶细胞(例如人包皮成纤维细胞)的背景下逐步测试切割效率,然后对CRISPR / sgRNA的作用进行功能测试病毒蛋白的表达,复制,和再活化。该策略可以容易地应用于其他靶细胞,例如多能干细胞衍生的人感觉神经元或其他人DNA病毒。
背景 ] 单纯疱疹病毒(HSV)是的一种嗜神经性DNA病毒疱疹病毒科家族引起终身和无法治愈感染在大多数人群的,并可能导致显著发病率和死亡率(Liesegang环等人,1989; Roizman 等人。,2013) ...
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Deoxycholate Fractionation of Fibronectin (FN) and Biotinylation Assay to Measure Recycled FN Fibrils in Epithelial Cells
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Author:
Date:
2018-08-20
[Abstract] Fibronectin (FN) is an extracellular matrix protein that is secreted by many cell types and binds predominantly to the cell surface receptor Integrin α5β1. Integrin α5β1 binding initiates the step-wise assembly of FN into fibrils, a process called fibrillogenesis. We and several others have demonstrated critical effects of fibrillogenesis on cell migration and metastasis. While immunostaining and microscopy methods help visualize FN incorporation into fibrils, with each fibril being at least 3 μm in length, the first study that developed a method to biochemically fractionate FN to quantify fibril incorporated FN was published by Jean Schwarzbauer’s group in 1996. Our protocol was adapted from the original publication, and has been tested on multiple cell types including as shown here in ...
[摘要] 纤连蛋白(FN)是一种细胞外基质蛋白,由许多细胞类型分泌,主要与细胞表面受体整合素α5β1结合。整合素α5β1结合启动FN逐步组装成原纤维,这一过程称为原纤维形成。我们和其他几个人已经证明了原纤维形成对细胞迁移和转移的关键作用。虽然免疫染色和显微镜方法有助于可视化FN掺入原纤维,每个原纤维的长度至少为3μm,但是第一项研究开发了一种生物化学分离FN以量化原纤维并入FN的方法,由Jean Schwarzbauer小组于1996年出版。我们的方案改编自原始出版物,并已在多种细胞类型上进行测试,包括如此处所示的MCF10A乳腺上皮细胞和Caki-1肾癌上皮细胞。使用两种洗涤剂提取物,将细胞FN分离成不溶于洗涤剂或掺入原纤维的FN和可溶性FN或未掺入的级分。为了确定原纤维形成是否利用FN的再循环池,我们使用了生物素标记的FN(FN-生物素)再循环测定,其已经从先前的研究中修改。使用再循环测定和脱氧胆酸盐分离方法的组合,可以定量地证明在不同实验条件下细胞中原纤维形成的程度,并确定原纤维形成的FN来源
【背景】 纤连蛋白(FN)是普遍产生的细胞外基质(ECM)组分(Uitto et al。,1989; Mao和Schwarzbauer,2005)。纤连蛋白库是转录产生的,可以通过几种生长因子如TGF-β1增加(Yokoi et al。,2002; Mimura ...
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CRISPR/Cas Gene Editing of a Large DNA Virus: African Swine Fever Virus
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Author:
Date:
2018-08-20
[Abstract] Gene editing of large DNA viruses, such as African swine fever virus (ASFV), has traditionally relied on homologous recombination of a donor plasmid consisting of a reporter cassette with surrounding homologous viral DNA. However, this homologous recombination resulting in the desired modified virus is a rare event. We recently reported the use of CRISPR/Cas9 to edit ASFV. The use of CRISPR/Cas9 to modify the African swine fever virus genome resulted in a fast and relatively easy way to introduce genetic changes. To accomplish this goal we first infect primary swine macrophages with a field isolate, ASFV-G, and transfect with the CRISPR/Cas9 donor plasmid along with a plasmid that will express a specific gRNA that targets our gene to be deleted. By inserting a reporter cassette, we are ...
[摘要] 大型DNA病毒(例如非洲猪瘟病毒(ASFV))的基因编辑传统上依赖于由报道盒组成的供体质粒与周围同源病毒DNA的同源重组。然而,这种导致所需修饰病毒的同源重组是罕见的事件。我们最近报道了使用CRISPR / Cas9编辑ASFV。使用CRISPR / Cas9修饰非洲猪瘟病毒基因组导致了引入遗传变化的快速且相对简单的方法。为了实现这一目标,我们首先用田间分离株ASFV-G感染原代猪巨噬细胞,并用CRISPR / Cas9供体质粒转染质粒,该质粒将表达靶向我们基因的特异性gRNA被删除。通过插入报告盒,我们能够通过有限稀释和噬菌斑纯化从亲本中纯化我们的重组病毒。我们以前曾报道将传统的同源重组方法与CRISPR / Cas9进行比较,结果导致重组增加超过4个对数。
【背景】 非洲猪瘟(ASF)是一种由ASF病毒(ASFV)引起的高度致命的猪传染性病毒性疾病。 ASFV的基因组由大约180-190千碱基对的双链DNA基因组组成。 ASFV引起一系列疾病,从高度致命到亚临床,取决于宿主特征和病毒株(Tulman et al。,2009)。 ASFV没有商业疫苗;实验上,2007年格鲁吉亚爆发的唯一能够抵御目前流行病毒株的疫苗(ASFV-G)是含有一个或多个病毒基因组缺失的减毒活疫苗,例如:( O'Donnell et ...
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