Rapid Genome Engineering of Pseudomonas Assisted by Fluorescent Markers and Tractable Curing of Plasmids
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Author:
Date:
2021-02-20
[Abstract] Precise genome engineering has become a commonplace technique for metabolic engineering. Also, insertion, deletion and alteration of genes and other functional DNA sequences are essential for understanding and engineering cells. Several techniques have been developed to this end (e.g., CRISPR/Cas-assisted methods, homologous recombination, or λ Red recombineering), yet most of them rely on the use of auxiliary plasmids, which have to be cured after the editing procedure. Temperature-sensitive replicons, counter-selectable markers or repeated passaging of plasmid-bearing cells have been traditionally employed to circumvent this hurdle. While these protocols work reasonably well in some bacteria, they are not applicable for other species or are time consuming and laborious. Here, we present ...
[摘要] [摘要]精确的基因组工程已成为代谢工程的一种普遍技术。同样,基因和其他功能性DNA序列的插入,缺失和改变对于理解和改造细胞也是必不可少的。几种技术已经发展到该端部(例如,CRISPR / CAS-辅助方法,同源重组,或 λ 红色重组),但其中大多数依赖于辅助质粒的使用,必须在编辑程序后将其固化。传统上已采用对温度敏感的复制子,反向选择标记或带有质粒的细胞的重复传代来规避这一障碍。尽管这些协议在某些细菌中可以很好地发挥作用,但它们不适用于其他物种,或者既费时又费力。在这里,我们提出了快速和通用的荧光假单胞菌荧光标记辅助基因组编辑协议,然后通过用户控制的质粒复制干净固化辅助质粒。一种荧光标记有助于鉴定基因组编辑的菌落,而第二种报道分子能够检测无质粒的细菌克隆。该协议不仅是用于假单胞菌物种的最快方法,而且可以轻松地适应任何类型的基因组修饰,包括序列删除,插入和替换。
图形概要:
带有可治愈质粒的假单胞菌的快速基因组工程
[背景]靶向,精确的基因组操纵技术已经大大推进了微生物工程领域。这样的方法不仅允许评估基因型与表型的关系,而且使微生物细胞工厂的复杂工程化成为可能。近年来,CRISPR / Cas9方法为真核生物的精确基因组工程铺平了道路。在细菌中,CRISPR / ...
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An Affinity-directed Protein Missile (AdPROM) System for Targeted Destruction of Endogenous Proteins
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Author:
Date:
2017-11-20
[Abstract] We recently reported an Affinity-directed PROtein Missile (AdPROM) system for the targeted proteolysis of endogenous proteins of interest (POI) (Fulcher et al., 2016 and 2017). AdPROM consists of the Von Hippel Lindau (VHL) protein, a Cullin 2 E3 ligase substrate receptor (Bosu and Kipreos, 2008), conjugated to a high affinity polypeptide binder (such as a camelid nanobody) that recognises the target protein in cells. When introduced in cells, the target protein is recruited to the CUL2 E3 ubiquitin ligase complex for ubiquitin-mediated proteasomal degradation. For target protein recruitment, we have utilised both camelid-derived VHH domain nanobodies as well as synthetic polypeptide monobodies based on the human type III fibronectin domain (Sha et al., 2013; Fridy et ...
[摘要] 我们最近报道了一种针对内源性感兴趣蛋白(POI)的靶向蛋白水解的亲和指导PROtein导弹(AdPROM)系统(Fulcher等人,2016和2017)。 AdPROM由Von Hippel Lindau(VHL)蛋白组成,Cullin 2 E3连接酶底物受体(Bosu and Kipreos,2008),与识别细胞中靶蛋白的高亲和力多肽结合剂(如骆驼科纳米抗体)缀合。当在细胞中引入时,靶蛋白质被招募到CUL2 E3泛素连接酶复合体用于泛素介导的蛋白酶体降解。对于靶蛋白的募集,我们使用了基于人类III型纤连蛋白结构域的骆驼科动物来源的VHH结构域纳米抗体以及合成多肽单体(Sharm等人,2013; Fridy等人。,2014; Schmidt et al。,2016)。在此协议中,我们描述了生成AdPROM构建体及其在人细胞系中用于靶蛋白质破坏的详细方法。 AdPROM允许对POI进行功能表征,并且其目标蛋白质破坏的效率克服了RNA干扰方法的许多局限性,这些方法需要长时间的治疗并与脱靶效应相关联,而CRISPR / Cas9基因编辑并不总是可行的。 【背景】该协议使人们能够在哺乳动物细胞系中设计,构建和表达AdPROM VHL-nano ...
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Knock-in Blunt Ligation Utilizing CRISPR/Cas9
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Author:
Date:
2017-03-05
[Abstract] The incorporation of the CRISPR/Cas9 bacterial immune system into the genetic engineering toolbox has led to the development of several new methods for genome manipulation (Auer et al., 2014; Byrne et al., 2015). We took advantage of the ability of Cas9 to generate blunt-ended double-strand breaks (Jinek et al., 2012) to introduce exogenous DNA in a highly precise manner through the exploitation of non-homologous end-joining DNA repair machinery (Geisinger et al., 2016). This protocol has been successfully applied to traditional immortalized cell lines and human induced pluripotent stem cells. Here we present a generalized protocol for knock-in blunt ligation, using HEK293 cells as an example.
[摘要] 将CRISPR / Cas9细菌免疫系统并入基因工程工具箱已经导致了几种用于基因组操作的新方法的开发(Auer等人,2014; Byrne等人,2015)。我们利用Cas9产生平端双链断裂的能力(Jinek等人,2012),以高度精确的方式通过开发非同源末端引物来引入外源DNA,加入DNA修复机械(Geisinger等人,2016)。该方案已成功应用于传统的永生化细胞系和人诱导多能干细胞。在这里,我们提出了使用HEK293细胞作为例子的敲入钝性连接的一般化方案。
背景 当我们概念化敲门钝性结扎(Geisinger等人,2016)时,开发用于CRISPR / Cas9的绝大多数方法都集中在提高同源重组的效率。然而,有一个例外:在斑马鱼中开发的同源性独立的基于质粒的敲入方法(Auer等人,2014)。这种方法,如敲入钝性连接,依赖于典型的非同源末端连接的机制,以线性化的,平端的双链DNA片段以高精度插入到基因组双链断裂中,核苷酸损失最小。这两种方法类似于为锌指核酸酶和被称为专性连接门控重组的TALEN开发的方法(ObLiGaRe; Maresca等人,2013),其依赖于产生相容的突出端以促进将靶DNA插入基因组。 ...
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