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Company: New England Biolabs
Catalog#: R0535S
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Single-step Precision Genome Editing in Yeast Using CRISPR-Cas9
Author:
Date:
2018-03-20
[Abstract]  Genome modification in budding yeast has been extremely successful largely due to its highly efficient homology-directed DNA repair machinery. Several methods for modifying the yeast genome have previously been described, many of them involving at least two-steps: insertion of a selectable marker and substitution of that marker for the intended modification. Here, we describe a CRISPR-Cas9 mediated genome editing protocol for modifying any yeast gene of interest (either essential or nonessential) in a single-step transformation without any selectable marker. In this system, the Cas9 nuclease creates a double-stranded break at the locus of choice, which is typically lethal in yeast cells regardless of the essentiality of the targeted locus due to inefficient non-homologous end-joining ... [摘要]  芽殖酵母中的基因组修饰已经非常成功,主要归功于其高度同源性的DNA修复机制。之前已经描述了几种用于修饰酵母基因组的方法,其中许多方法涉及至少两个步骤:插入选择标记并用该标记取代预期的修饰。在这里,我们描述了CRISPR-Cas9介导的基因组编辑方案,用于在没有任何选择标记的情况下在单步转化中修饰任何感兴趣的酵母基因(基本或非必需)。在该系统中,Cas9核酸酶在选择的基因座处产生双链断裂,这在酵母细胞中通常是致死的,而不管由于无效的非同源末端连接修复导致的靶基因座的重要性。该致死性通过使用源自PCR的修复模板的同源重组导致有效的修复。在涉及必需基因的情况下,用功能性等位基因编辑基因组病变的必要性作为额外的选择层。作为一个激励性的例子,我们描述了使用这种策略替代HEM2,一种必需的酵母基因,以及相应的人类直向同源物ALAD。

【背景】酿酒酵母(Baccharomyces cerevisiae,Baker's酵母)作为一种遗传易处理的生物体具有悠久的历史,并且有许多操作酵母基因组的方法。然而,直到最近,有必要应用选择以分离具有所需遗传改变的克隆(Kearse等人,2012; DiCarlo等人,2013; Lee等人,等,2015; ...

Targeted Genome Editing of Virulent Phages Using CRISPR-Cas9
Author:
Date:
2018-01-05
[Abstract]  This protocol describes a straightforward method to generate specific mutations in the genome of strictly lytic phages. Briefly, a targeting CRISPR-Cas9 system and a repair template suited for homologous recombination are provided inside a bacterial host, here the Gram-positive model Lactococcus lactis MG1363. The CRISPR-Cas9 system is programmed to cleave a specific region present on the genome of the invading phage, but absent from the recombination template. The system either triggers the recombination event or exerts the selective pressure required to isolate recombinant phages. With this methodology, we generated multiple gene knockouts, a point mutation and an insertion in the genome of the virulent lactococcal phage p2. Considering the broad host range of the plasmids used ... [摘要]  该协议描述了一个直接的方法来产生严格裂解噬菌体的基因组中的特定突变。 简而言之,在细菌宿主(此处为革兰氏阳性模型乳酸乳球菌MG1363)内提供靶向CRISPR-Cas9系统和适合于同源重组的修复模板。 CRISPR-Cas9系统被编程为切割入侵噬菌体的基因组上存在的特定区域,但是缺少重组模板。 该系统触发重组事件或施加分离重组噬菌体所需的选择性压力。 利用这种方法,我们在毒性乳酸球菌噬菌体p2的基因组中产生了多个基因敲除,点突变和插入。 考虑到本协议中使用的质粒的广泛宿主范围,后者可以外推到其他噬菌体 - 宿主对。

【背景】噬菌体是在每个生态系统中发现丰富的细菌病毒(Suttle,2005; Breitbart and Rohwer,2005),毫不奇怪,它们是牛奶的天然居民。噬菌体p2是乳品工业中发现的强毒乳球菌噬菌体的最普遍组( Sk1virus )的模型(Deveau等人,2006; Mahony等人。,2012),它感染革兰氏阳性细菌乳酸乳球菌MG1363,也是基础研究的模式菌株。尽管p2作为参照噬菌体的地位,但几乎一半的基因编码未表征的蛋白质。同样,由宏基因组学确定的绝大多数噬菌体基因在公共数据库中没有功能分配和同系物(Hurwitz等人,2016; Paez-Espino等人, 2016)。

研究基因的方法之一是通过修饰和随后观察所得到的表型。噬菌体基因组只能在宿主内以其生物活性形式进行修饰。强毒噬菌体严格裂解;因此,它们的基因组从未整合到细菌染色体中。这为DNA的体内修饰增加了一个时间限制,只能在短的感染周期内对其进行操作。 ...

Genome Editing in Diatoms Using CRISPR-Cas to Induce Precise Bi-allelic Deletions
Author:
Date:
2017-12-05
[Abstract]  Genome editing in diatoms has recently been established for the model species Phaeodactylum tricornutum and Thalassiosira pseudonana. The present protocol, although developed for T. pseudonana, can be modified to edit any diatom genome as we utilize the flexible, modular Golden Gate cloning system. The main steps include how to design a construct using Golden Gate cloning for targeting two sites, allowing a precise deletion to be introduced into the target gene. The transformation protocol is explained, as are the methods for screening using band shift assay and/or restriction site loss. [摘要]  最近为三角褐指藻(Phaeodactylum tricornutum)和海绵假丝酵母(Thalassiosira pseudonana)建立了硅藻基因组编辑。 目前的协议,虽然开发的 T。 pseudonana ,可以修改编辑任何硅藻基因组,因为我们利用灵活,模块化的金门克隆系统。 主要步骤包括如何设计构建使用金门克隆靶向两个网站,允许一个精确的删除被引入目标基因。 解释转化方案,以及使用带移位测定和/或限制性位点丢失进行筛选的方法。

【背景】CRISPR-Cas正在迅速成为分子研究的一个关键方法。基于在细菌和古细菌中发现的病毒防御机制,CRISPR-Cas诱导基因组中精确位置的双链断裂(DSBs)。它涉及使用与CRISPR ...

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