Improved Genome Editing in Human Cell Lines Using the CRISPR Method

The Cas9/CRISPR system has become a popular choice for genome editing. In this system, binding of a single guide (sg) RNA to a cognate genomic sequence enables the Cas9 nuclease to induce a double-strand break at that locus. This break is next repaired by an error-prone mechanism, leading to mutation and gene disruption. In this study we describe a range of refinements of the method, including stable cell lines expressing Cas9, and a PCR based protocol for the generation of the sgRNA. We also describe a simple methodology that allows both elimination of Cas9 from cells after gene disruption and re-introduction of the disrupted gene. This advance enables easy assessment of the off target effects associated with gene disruption, as well as phenotype-based structure-function analysis. In our study, we used the Fan1 DNA repair gene as control in these experiments. Cas9/CRISPR-mediated Fan1 disruption occurred at frequencies of around 29%, and resulted in the anticipated spectrum of genotoxin hypersensitivity, which was rescued by re-introduction of Fan1.     

Peculiarities in Creation of Genetic Engineering Constructions for Knock-In Variant of Genome Editing of Arabidopsis thaliana Cell Culture

Russian Journal of Plant Physiology - The peculiarities in the creation of genetic engineering tools for the knock-in variant of genome editing are considered in detail on the example of gfp gene...     

基因组编辑技术研究进展

利用基因组编辑技术可以对生物基因组特定位点进行人工修饰,研究相关基因的功能,进而应用于基础研究和临床治疗方面。序列特异性的DNA结合结构域与非特异性的DNA修饰结构域组合而成的人工酶是基因组编辑工具的重要组成部分。主要介绍了锌指核酸酶(ZFNs)、转录激活样效应因子核酸酶(TALEN)、归巢核酸内切酶(Meganucleases)和成簇间隔短回文重复(CRISPR)4种基因组编辑技术的特点、原理、构建方法及应用,为相关的研究和应用提供参考。     

The Applications of Genome Editing in Xenotransplantation

正Higher living standards and better medical care are increasing the lifespan of people around the world.Aging populations,however,have an increased incidence of loss of function or failure of cell,tissue or organ.This has led to the development of new medical disciplines,such as organ transplantation and more recently regenerative medicine.Organ transplantation using human donors(allotransplantation)has made enormous progress thanks to the     

Genome Editing: Revolutionizing the Crop Improvement

Plant Molecular Biology Reporter - Modern biotechnology is progressed to sequence-specific, site-directed, precise, and safe strategies for genetic manipulation. The genome-editing strategy is...     

Target-Specific Precision of CRISPR-Mediated Genome Editing

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CRISPR/Cas9 and Genome Editing in Drosophila

Recent advances in our ability to design DNA binding factors with specificity for desired sequences have resulted in a revolution in genetic engineering, enabling directed changes to the genome to be made relatively easily. Traditional techniques for generating genetic mutations in most organisms have relied on selection from large pools of randomly induced mutations for those of particular interest, or time-consuming gene targeting by homologous recombination. Drosophila melanogaster has always been at the forefront of genetic analysis, and application of these new genome editing techniques to this organism will revolutionise our approach to performing analysis of gene function in the future. We discuss the recent techniques that apply the CRISPR/Cas9 system to Drosophila, highlight potential uses for this technology and speculate upon the future of genome engineering in this model organism.     

基因组编辑:植物生物技术的机遇与挑战

基于序列特异性核酸酶的基因组编辑技术可以在不同物种中对目标基因进行定点敲除,并可实现特定基因片段置换,基因的定点插入等基因组靶向修饰。基因组编辑是一种精准和高效的基因工程方法,近年来快速发展并得到了广泛的应用,并将改变生物技术的现状。目前,基因组编辑在不同植物,特别是农作物中的技术体系已建立,初步展示了其在植物生物技术领域的巨大潜力。介绍了不同基因组编辑系统的工作原理,并对基因组编辑技术在植物研究中的应用及成功案例进行了综述,最后对基因组编辑在植物生物技术领域所面临的机遇与挑战进行了讨论。     

Development of an Efficient Genome Editing Method by CRISPR/Cas9 in a Fish Cell Line

CRISPR/Cas9 system has been used widely in animals and plants to direct mutagenesis. To date, no such method exists for fish somatic cell lines. We describe an efficient procedure for genome editing in the Chinook salmon Oncorhynchus tshawytscha CHSE. This cell line was genetically modified to firstly overexpress a monomeric form of EGFP (cell line CHSE-E Geneticin resistant) and additionally to overexpress nCas9n, a nuclear version of Cas9 (cell line CHSE-EC, Hygromycin and Geneticin resistant). A pre-validated sgRNA was produced in vitro and used to transfect CHSE-EC cells. The EGFP gene was disrupted in 34.6 % of cells, as estimated by FACS and microscopy. The targeted locus was characterised by PCR amplification, cloning and sequencing of PCR products; inactivation of the EGFP gene by deletions in the expected site was validated in 25 % of clones. This method opens perspectives for functional genomic studies compatible with high-throughput screening.     

A Toolkit of CRISPR-Based Genome Editing Systems in Drosophila

The last couple of years have witnessed an explosion in development of CRISPR-based genome editing technologies in cell lines as well as in model organisms. In this review, we focus on the applications of this popular system in Drosophila. We discuss the effectiveness of the CRISPR/Cas9 systems in terms of delivery, mutagenesis detection, parameters affecting efficiency, and off-target issues, with an emphasis on how to apply this powerful tool to characterize gene functions.     

Stimulation of Cell Elongation by Tetraploidy in Hypocotyls of Dark-Grown Arabidopsis Seedlings

Plant size is largely determined by the size of individual cells. A number of studies showed a link between ploidy and cell size in land plants, but this link remains controversial. In this study, post-germination growth, which occurs entirely by cell elongation, was examined in diploid and autotetraploid hypocotyls of Arabidopsis thaliana (L.) Heynh. Final hypocotyl length was longer in tetraploid plants than in diploid plants, particularly when seedlings were grown in the dark. The longer hypocotyl in the tetraploid seedlings developed as a result of enhanced cell elongation rather than by an increase in cell number. DNA microarray analysis showed that genes involved in the transport of cuticle precursors were downregulated in a defined region of the tetraploid hypocotyl when compared to the diploid hypocotyl. Cuticle permeability, as assessed by toluidine-blue staining, and cuticular structure, as visualized by electron microscopy, were altered in tetraploid plants. Taken together, these data indicate that promotion of cell elongation is responsible for ploidy-dependent size determination in the Arabidopsis hypocotyl, and that this process is directly or indirectly related to cuticular function.