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Construction of a Cosmid-Based Ultraefficient Genomic Library System for Filamentous Fungi of the Genus .
J Fungi (Basel)
February 2024
Department of Biotechnology and Life Sciences, Faculty of Biotechnology and Life Sciences, Sojo University, Ikeda 4-22-1, Nishi-ku, Kumamoto 860-0082, Japan.
Filamentous fungi of the genus include producers of industrially important organic acids, enzymes, and secondary metabolites, as well as pathogens of many plants and animals. Novel genes in the genome are potentially crucial for the fermentation and drug industries (e.g.
View Article and Find Full Text PDFReconstitution and Mutagenesis of Avian Infectious Laryngotracheitis Virus from Cosmid and Yeast Centromeric Plasmid Clones.
J Virol
April 2023
Institute of Molecular Virology and Cell Biology, Friedrich Loeffler-Institut, Federal Research Institute for Animal Health, Greifswald-Insel Riems, Germany.
The genomes of numerous herpesviruses have been cloned as infectious bacterial artificial chromosomes. However, attempts to clone the complete genome of infectious laryngotracheitis virus (ILTV), formally known as Gallid alphaherpesvirus-1, have been met with limited success. In this study, we report the development of a cosmid/yeast centromeric plasmid (YCp) genetic system to reconstitute ILTV.
View Article and Find Full Text PDFPreparation of Soil Metagenome Libraries and Screening for Gene-Specific Amplicons.
Methods Mol Biol
January 2022
School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand.
Cosmid libraries constructed from environmental metagenome samples are powerful tools for capturing the genomic diversity of complex microbial communities. The large insert size (∼35 kb) of such libraries means they are compatible with downstream expression of large biosynthetic gene clusters (BGCs). This allows the discovery of previously undescribed natural products that would be inaccessible using traditional culture-based discovery pipelines.
View Article and Find Full Text PDFConstruction of adenovirus vectors simultaneously expressing four multiplex, double-nicking guide RNAs of CRISPR/Cas9 and in vivo genome editing.
Sci Rep
February 2021
Laboratory of Virology, Institute of Microbial Chemistry (BIKAKEN), Microbial Chemistry Foundation, Shinagawa-ku, Tokyo, 141-0021, Japan.
Simultaneous expression of multiplex guide RNAs (gRNAs) is valuable for knockout of multiple genes and also for effective disruption of a gene by introducing multiple deletions. We developed a method of Tetraplex-guide Tandem for construction of cosmids containing four and eight multiplex gRNA-expressing units in one step utilizing lambda in vitro packaging. Using this method, we produced an adenovirus vector (AdV) containing four multiplex-gRNA units for two double-nicking sets.
View Article and Find Full Text PDFHighly multiplex guide RNA expression units of CRISPR/Cas9 were completely stable using cosmid amplification in a novel polygonal structure.
J Gene Med
November 2019
Laboratory of Virology, Institute of Microbial Chemistry (BIKAKEN), Microbial Chemistry Foundation, Tokyo, Japan.
Background: Genome editing using the CRISPR/Cas9 system is now well documented in basic studies and is expected to be applied to gene therapy. Simultaneous expression of multiplex guide RNA (gRNA) and Cas9/Cas9 derivative is attractive for the efficient knockout of genes and a safe double-nicking strategy. However, such use is limited because highly multiplex gRNA-expressing units are difficult to maintain stably in plasmids as a result of deletion via homologous recombination.
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