| 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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| 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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| Isolation and Culture of Neurospheres for the Study of Pathogenesis of Prion Disease
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Author:
Date:
2014-03-20
[Abstract] Neurosphere contains neural stem cells that are capable of self-renewal and multilineage differentiation including neurons, astrocytes, and oligodendrocytes (Gage, 2000). Cell culture model using differentiated neurosphere cultures are suggested to be a valuable tool for studying the pathogenesis of prion disease at the cellular level (Iwamaru et al., 2013). This protocol describes the procedure for a culture of whole brain-derived neurospheres from newborn mouse brains. Neurosphere formation steadily occurs within a week from the cultures of neonatal whole brains and these cells have stem cell properties.
[摘要] 神经球包含能够自我更新和多向分化的神经干细胞，包括神经元，星形胶质细胞和少突胶质细胞（Gage，2000）。 使用分化的神经球培养物的细胞培养模型被认为是用于研究朊病毒疾病在细胞水平的发病机制的有价值的工具（Iwamaru等人，2013）。 该协议描述了来自新生小鼠脑的全脑衍生的神经球的培养的程序。 神经球形成稳定地发生在新生儿全脑的培养物的一周内，并且这些细胞具有干细胞特性。
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