Methods for the Evasion of Restriction-Modification Systems During Genetic Engineering of Fusobacterium Species.

Enhanced Transformation Efficiency in Fusobacterium nucleatum by Bypassing Restriction-Modification Systems: A Methylation-Guided Protocol.

An optimized CRISPR/Cas9-based genome editing platform enhances bacteriocin production in Lactiplantibacillus plantarum

This study adapted an established CRISPR/Cas9 approach for the targeted disruption of plnD, a key negative regulatory gene within the plantaricin quorum-sensing network of Lactiplantibacillus plantarum 8P-A3 through extensive optimization of transformation and genome-editing conditions.

Advances in understanding of plasmid biology through the application of transposon insertion sequencing

Plasmids are extra-chromosomal DNA molecules capable of autonomous replication, stable inheritance in a bacterial population, and horizontal transfer to other bacteria. Plasmids can harbour auxiliary genetic material that contributes to host bacterial fitness, the most prominent example being antimicrobial resistance (AMR) determinants, which remain the greatest threat to modern medicine. Since their discovery in the early 1950s, plasmids have been extensively studied due to their diversity, their capacity to spread between bacterial hosts, and their ability to carry and disseminate multiple AMR genes simultaneously. Recent advances in sequencing technology have transformed plasmid research, with transposon–insertion sequencing (TIS) enabling simultaneous analysis of millions of mutants and providing unprecedented scale, speed and resolution for studying plasmid biology. Here, we briefly outline a recommended methodology for generating plasmid transposon mutant libraries, which can be combined with TIS to investigate plasmid replication, maintenance and conjugation. We further summarise data from nine comprehensive plasmid TIS studies to date on five distinct plasmids, discuss alternative uses for plasmid libraries, challenges and future perspectives.

CRISPR/Cas9-Mediated Site-Directed Mutagenesis of Genes in Klebsiella pneumoniae.

This protocol minimizes off-target editing, shortens experimental timelines, reduces screening workload, and provides a reliable platform for investigating resistance mechanisms, validating candidate genes, and supporting functional genomics studies in clinically relevant bacterial pathogens.

A Conditional Plasmid System for Markerless Gene Deletion in Genetically Recalcitrant Fusobacterium nucleatum subsp. animalis.

This chapter provides a detailed, step-by-step protocol for implementing a conditional plasmid system that enables efficient, markerless gene deletion in FNA strains and provides a powerful and adaptable tool for advancing genetic studies in this genetically recalcitrant subspecies.

Gene Silencing by CRISPR Interference in Fusobacterium nucleatum.

This chapter presents a step-by-step protocol for designing sgRNAs, constructing CRISPRi plasmids, transforming F. nucleatum ATCC 23726, and evaluating gene silencing phenotypes, using the nonessential gene ftsW, which encodes a protein required for peptidoglycan synthesis and cell division, as a model target.