Cell Biology

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    Protocols in Current Issue
    In vitro Generation of CRISPR-Cas9 Complexes with Covalently Bound Repair Templates for Genome Editing in Mammalian Cells
    [Abstract] The CRISPR-Cas9 system is a powerful genome-editing tool that promises application for gene editing therapies. The Cas9 nuclease is directed to the DNA by a programmable single guide (sg)RNA, and introduces a site-specific double-stranded break (DSB). In mammalian cells, DSBs are either repaired by non-homologous end joining (NHEJ), generating ...
    Method for CRISPR/Cas9 Mutagenesis in Candida albicans
    Authors:  Neta Dean and Henry Ng, date: 04/20/2018, view: 3708, Q&A: 0
    [Abstract] Candida albicans is the most prevalent and important human fungal pathogen. The advent of CRISPR as a means of gene editing has greatly facilitated genetic analysis in C. albicans. Here, we describe a detailed step-by-step procedure to construct and analyze C. albicans deletion mutants. This protocol uses plasmids that ...
    Generating Loss-of-function iPSC Lines with Combined CRISPR Indel Formation and Reprogramming from Human Fibroblasts
    Authors:  Andrew M. Tidball, Preethi Swaminathan, Louis T. Dang and Jack M. Parent, date: 04/05/2018, view: 3930, Q&A: 1
    [Abstract] For both disease and basic science research, loss-of-function (LOF) mutations are vitally important. Herein, we provide a simple stream-lined protocol for generating LOF iPSC lines that circumvents the technical challenges of traditional gene-editing and cloning of established iPSC lines by combining the introduction of the CRISPR vector ...
    Cytosolic and Nuclear Delivery of CRISPR/Cas9-ribonucleoprotein for Gene Editing Using Arginine Functionalized Gold Nanoparticles
    Authors:  Rubul Mout and Vincent M. Rotello, date: 10/20/2017, view: 5396, Q&A: 1
    [Abstract] In this protocol, engineered Cas9-ribonucleoprotein (Cas9 protein and sgRNA, together called Cas9-RNP) and gold nanoparticles are used to make nanoassemblies that are employed to deliver Cas9-RNP into cell cytoplasm and nucleus. Cas9 protein is engineered with an N-terminus glutamic acid tag (E-tag or En, where n = the number of glutamic acid in ...
    Using CRISPR/Cas9 for Large Fragment Deletions in Saccharomyces cerevisiae
    Authors:  Huanhuan Hao, Jing Huang, Tongtong Liu, Hui Tang and Liping Zhang, date: 07/20/2017, view: 5500, Q&A: 0
    [Abstract] CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9) systems have emerged as a powerful tool for genome editing in many organisms. The wide use of CRISPR/Cas9 systems may be due to the fact that these systems contain a simple guide RNA (sgRNA) that is relatively easy to design and they are very ...
    Multiplex Gene Editing via CRISPR/Cas9 System in Sheep
    Authors:  Yiyuan Niu, Yi Ding, Xiaolong Wang and Yulin Chen, date: 07/05/2017, view: 4829, Q&A: 0
    [Abstract] Sheep is a major large animal model for studying development and disease in biomedical research. We utilized CRISPR/Cas9 system successfully to modify multiple genes in sheep. Here we provide a detailed protocol for one-cell-stage embryo manipulation by co-injecting Cas9 mRNA and RNA guides targeting three genes (MSTN, ASIP, and ...
    Targeted Nucleotide Substitution in Mammalian Cell by Target-AID
    Authors:  Takayuki Arazoe, Keiji Nishida and Akihiko Kondo, date: 06/05/2017, view: 5342, Q&A: 0
    [Abstract] Programmable RNA-guided nucleases based on CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated protein) systems have been applied to various type of cells as powerful genome editing tools. By using activation-induced cytidine deaminase (AID) in place of the nuclease activity of the CRISPR/Cas9 system, we have ...
    DNA-free Genome Editing of Chlamydomonas reinhardtii Using CRISPR and Subsequent Mutant Analysis
    Authors:  Jihyeon Yu, Kwangryul Baek, EonSeon Jin and Sangsu Bae, date: 06/05/2017, view: 6332, Q&A: 0
    [Abstract] We successfully introduced targeted knock-out of gene of interest in Chlamydomonas reinhardtii by using DNA-free CRISPR. In this protocol, the detailed procedures of an entire workflow cover from the initial target selection of CRISPR to the mutant analysis using next generation sequencing (NGS) technology. Furthermore, we introduce a ...
    A Protocol for Production of Mutant Mice Using Chemically Synthesized crRNA/tracrRNA with Cas9 Nickase and FokI-dCas9
    Authors:  Satoshi Hara, Miho Terao and Shuji Takada, date: 06/05/2017, view: 4908, Q&A: 0
    [Abstract] The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is the most widely used genome editing tool. A common CRISPR/Cas9 system consists of two components: a single-guide RNA (sgRNA) and Cas9. Both components are required for the introduction of a double-strand break (DSB) at a specific ...
    Generation of Mutant Pigs by Direct Pronuclear Microinjection of CRISPR/Cas9 Plasmid Vectors
    Authors:  Chin-kai Chuang, Ching-Fu Tu and Chien-Hong Chen, date: 06/05/2017, view: 5008, Q&A: 0
    [Abstract] A set of Cas9 and single guide CRISPR RNA expression vectors was constructed. Only a very simple procedure was needed to prepare specific single-guide RNA expression vectors with high target accuracy. Since the de novo zygotic transcription had been detected in mouse embryo at the 1-cell stage, the plasmid DNA vectors encoding Cas9 and ...



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