Molecular Biology


    Protocols in Current Issue
    A Nucleocapsid-based Transcomplementation Cell Culture System of SARS-CoV-2 to Recapitulate the Complete Viral Life Cycle
    Authors:  Yanying Yu, Xiaohui Ju and Qiang Ding, date: 11/05/2021, view: 1438, Q&A: 0

    The ongoing COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). As this virus is classified as a biosafety level-3 (BSL-3) agent, the development of countermeasures and basic research methods is logistically difficult. Recently, using reverse genetics, we developed a BSL-2 cell culture system for production

    Dual sgRNA-based Targeted Deletion of Large Genomic Regions and Isolation of Heritable Cas9-free Mutants in Arabidopsis
    Authors:  Yu Jin and Sebastian Marquardt, date: 10/20/2020, view: 3973, Q&A: 0
    [Abstract] CRISPR/Cas9 system directed by a gene-specific single guide RNA (sgRNA) is an effective tool for genome editing such as deletions of few bases in coding genes. However, targeted deletion of larger regions generate loss-of-function alleles that offer a straightforward starting point for functional dissections of genomic loci. We present an ...
    Minigene Assay to Evaluate CRISPR/Cas9-based Excision of Intronic Mutations that Cause Aberrant Splicing in Human Cells
    Authors:  David J. Sanz and Patrick T. Harrison, date: 06/05/2019, view: 4683, Q&A: 0
    [Abstract] The construction of Hybrid minigenes provides a robust and simple strategy to study the effects of disease-causing mutations on mRNA splicing when biological material from patient cells is not available. Hybrid minigenes can be used as splicing reporter plasmids allow RNA expression and heterologous splicing reactions between synthetic splicing ...
    CRISPR/Cas9-mediated ssDNA Recombineering in Corynebacterium glutamicum
    Authors:  Jiao Liu, Yu Wang, Ping Zheng and Jibin Sun, date: 10/05/2018, view: 5916, Q&A: 0
    [Abstract] Corynebacterium glutamicum is a versatile workhorse for industrial bioproduction of many kinds of chemicals and fuels, notably amino acids. Development of advanced genetic engineering tools is urgently demanded for systems metabolic engineering of C. glutamicum. Recently unveiled clustered regularly interspaced short palindromic ...
    Method for CRISPR/Cas9 Mutagenesis in Candida albicans
    Authors:  Neta Dean and Henry Ng, date: 04/20/2018, view: 9978, 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 ...
    Single-step Precision Genome Editing in Yeast Using CRISPR-Cas9
    [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 ...
    Markerless Gene Editing in the Hyperthermophilic Archaeon Thermococcus kodakarensis
    Authors:  Alexandra M. Gehring, Travis J. Sanders and Thomas J. Santangelo, date: 11/20/2017, view: 6678, Q&A: 0
    [Abstract] The advent of single cell genomics and the continued use of metagenomic profiling in diverse environments has exponentially increased the known diversity of life. The recovered and assembled genomes predict physiology, consortium interactions and gene function, but experimental validation of metabolisms and molecular pathways requires more ...
    Method for Multiplexing CRISPR/Cas9 in Saccharomyces cerevisiae Using Artificial Target DNA Sequences
    Authors:  Rachael M. Giersch and Gregory C. Finnigan, date: 09/20/2017, view: 10297, Q&A: 0
    [Abstract] Genome manipulation has become more accessible given the advent of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) editing technology. The Cas9 endonuclease binds a single stranded (single guide) RNA (sgRNA) fragment that recruits the complex to a corresponding genomic target sequence where it induces a double stranded ...
    CRISPR-PCS Protocol for Chromosome Splitting and Splitting Event Detection in Saccharomyces cerevisiae
    Authors:  Yu Sasano and Satoshi Harashima, date: 05/20/2017, view: 9111, Q&A: 0
    [Abstract] Chromosome engineering is an important technology with applications in basic biology and biotechnology. Chromosome splitting technology called PCS (PCR-mediated Chromosome Splitting) has already been developed as a fundamental chromosome engineering technology in the budding yeast. However, the splitting efficiency of PCS technology is not high ...

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