| 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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| Endogenous C-terminal Tagging by CRISPR/Cas9 in Trypanosoma cruzi
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Author:
Date:
2017-05-20
[Abstract] To achieve the C-terminal tagging of endogenous proteins in T. cruzi we use the Cas9/pTREX-n vector (Lander et al., 2015) to insert a specific tag sequence (3xHA or 3xc-Myc) at the 3’ end of a specific gene of interest (GOI). Chimeric sgRNA targeting the 3’ end of the GOI is PCR-amplified and cloned into Cas9/pTREX-n vector. Then a DNA donor molecule to induce DNA repair by homologous recombination is amplified. This donor sequence contains the tag sequence and a marker for antibiotic resistance, plus 100 bp homology arms corresponding to regions located right upstream of the stop codon and downstream of the Cas9 target site at the GOI locus. Vectors pMOTag23M (Oberholzer et al., 2006) or pMOHX1Tag4H (Lander et al., 2016b) are used as PCR templates for ...
[摘要] 为了实现克氏锥虫内源蛋白的C末端标记，我们使用Cas9 / pTREX-n载体（Lander等，2015）在3'末端插入特异性标签序列（3xHA或3xc-Myc）特定的感兴趣基因（GOI）。将靶向GOI 3'末端的嵌合sgRNA进行PCR扩增，并克隆到Cas9 / pTREX-n载体中。然后扩增通过同源重组诱导DNA修复的DNA供体分子。该供体序列包含标签序列和抗生素抗性标记，加上对应于位于终止密码子右上游的区域和在GOI基因座的Cas9靶位点下游的100bp同源臂。载体pMOTag23M（Oberholzer等，2006）或pMOHX1Tag4H（Lander等，2016b）用作DNA供体扩增的PCR模板。用sgRNA / Cas9 / pTREX-n构建体和DNA供体盒共转染的Epimastigotes然后用抗生素培养5周以选择双重抗性寄生虫。最终通过PCR和Western印迹分析验证了内源基因标记。
【背景】自从CRISPR / ...
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| Efficient Generation of Multi-gene Knockout Cell Lines and Patient-derived Xenografts Using Multi-colored Lenti-CRISPR-Cas9
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Author:
Date:
2017-04-05
[Abstract] CRISPR-Cas9 based knockout strategies are increasingly used to analyze gene function. However, redundancies and overlapping functions in biological signaling pathways can call for generating multi-gene knockout cells, which remains a relatively laborious process. Here we detail the application of multi-color LentiCRISPR vectors to simultaneously generate single and multiple knockouts in human cells. We provide a complete protocol, including guide RNA design, LentiCRISPR cloning, viral production and transduction, as well as strategies for sorting and screening knockout cells. The validity of the process is demonstrated by the simultaneous deletion of up to four programmed cell death mediators in leukemic cell lines and patient-derived acute lymphoblastic leukemia xenografts, in which ...
[摘要] 基于CRISPR-Cas9的敲除策略越来越多地用于分析基因功能。然而，生物信号通路中的冗余和重叠功能可能需要产生多基因敲除细胞，这仍然是一个相对费力的过程。在这里，我们详细介绍了多色LentiCRISPR载体在人体细胞中同时产生单次和多次敲除的应用。我们提供了一个完整的方案，包括指导RNA设计，LentiCRISPR克隆，病毒生产和转导，以及排序和筛选敲除细胞的策略。该过程的有效性通过同时删除白血病细胞系中多达四个程序性细胞死亡介质和来自患者来源的急性淋巴细胞白血病异种移植物，其中单细胞克隆是不可行的。该协议允许任何具有基本细胞生物学设备的实验室，生物安全2级设备和荧光激活细胞分选功能，可在一个月内有效产生单基因和多基因敲除细胞系或原代细胞。
从对细菌基因组中被称为聚簇定期交织的短回文重复（CRISPR）的遗传元件的好奇的初步观察开始（Ishino等人，1987; Mojica等人，2000 ）和随后在哺乳动物细胞中的基因编辑（Cong等人，2013; Mali等人，2013），CRISPR-Cas9已经成为廉价和有效的基因编辑。随着从烟草植物细胞到斑马鱼和原代人类细胞（Hsu等人，2014）的细胞系统的成功应用，CRISPR-Cas9可以通过短的20个核苷酸RNA序列的设计来引导在大基因组内的靶向DNA双链断裂（DSB）（Park等人，2016）。 ...
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