TL;DR
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.
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.
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.
Compared with conventional homologous recombination, the CRISPR-Cas9 system substantially improved gene disruption efficiency, thereby overcoming a major limitation in the genetic manipulation of lichen-forming fungi.
An inducible genome-editing framework exploiting the endogenous Cas9 system of Apilactobacillus kunkeei, a key member of the honeybee microbiota, is established and the toolkit available for harnessing endogenous CRISPR-Cas systems in genetically recalcitrant, non-model gram-positive bacteria is expanded.
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.
This chapter describes strategies to evade RM defenses and improve transformation efficiencies across diverse Fusobacterium lineages, employing a sequence-based "RM-silencing" method that has enabled successful delivery of replicative plasmids, linear recombination templates, and transposon cassettes into previously intractable Fusobacterium clinical isolates.
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