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http://dx.doi.org/10.3724/abbs.2025050 | DOI Listing |
J Vis Exp
June 2025
Clinical Research Institute, The Affiliated Nanhua Hospital, Hengyang Medical School, University of South China;
The development of genome-wide plasmid libraries using existing genomic repositories serves as a pivotal prerequisite for systematic functional characterization of genes across diverse biological processes. Current high-throughput methodologies for inter-vector DNA fragment transfer, however, necessitate PCR amplification of target sequences prior to cloning, rendering the generation of genome-scale plasmid collections technically demanding and time-intensive. By leveraging a CRISPRshuttle cassette, we developed a new high-throughput cloning method, CRISPR-based shuttle cloning (CRISPRshuttle cloning), which facilitates the transfer of many DNA fragments from donor plasmids sharing identical backbone sequences to a CRISPRshuttle-compatible vector without PCR amplification of the DNA fragments.
View Article and Find Full Text PDFHum Gene Ther
August 2025
Department of Chronic Diseases and Metabolism, Laboratory of Respiratory Diseases and Thoracic Surgery (BREATHE), KU Leuven, Leuven, Belgium.
The advent of genome editing has kindled the hope to cure previously uncurable, life-threatening genetic diseases. However, whether this promise can be ultimately fulfilled depends on how efficiently gene editing agents can be delivered to therapeutically relevant cells. Over time, viruses have evolved into sophisticated, versatile, and biocompatible nanomachines that can be engineered to shuttle payloads to specific cell types.
View Article and Find Full Text PDFActa Biochim Biophys Sin (Shanghai)
April 2025
Clinical Research Institute, the Affiliated Nanhua Hospital, Hengyang Medical School, University of South China, Hengyang 421002, China.
Appl Environ Microbiol
July 2024
Department of Civil, Construction and Environmental Engineering, San Diego State University, San Diego, California, USA.
Methanogenic archaea, which are integral to global carbon and nitrogen cycling, currently face challenges in genetic manipulation due to unique physiology and limited genetic tools. This review provides a survey of current and past developments in the genetic engineering of methanogens, including selection and counterselection markers, reporter systems, shuttle vectors, mutagenesis methods, markerless genetic exchange, and gene expression control. This review discusses genetic tools and emphasizes challenges tied to tool scarcity for specific methanogenic species.
View Article and Find Full Text PDFLancet Microbe
January 2024
Fujian Provincial Key Laboratory of Soil Environmental Health and Regulation, College of Resources and Environment, Fujian Agriculture and Forestry University, Fuzhou, China; Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, China. Electronic address:
Background: The global spread of plasmid-borne carbapenem resistance is an ongoing public health challenge; however, the nature of such horizontal gene transfer events among complex bacterial communities remains poorly understood. We examined the in-situ transfer of the globally dominant New Delhi metallo-β-lactamase (NDM)-5-positive IncX3 plasmid (denoted pX3_NDM-5) in hospital wastewater to simulate a real-world, One Health antimicrobial resistance context.
Methods: For this transmission study, we tagged pX3_NDM-5 with the green fluorescent protein gene, gfp, using a CRISPR-based method and transferred the plasmid to a donor Escherichia coli strain.