TL;DR
Improved the previously developed tool by designing a single-plasmid variant instead of the two-plasmid one and by adapting the tetracycline resistance gene as an additional selective marker in L. lactis, significantly simplifies and accelerates the delivery of the required genetic material into the chromosome.
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.
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 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.
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 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.
A novel marker recycling cassette is reported for Aspergillus species that enables highly efficient gene disruption and marker recycling by combining a doxycycline-inducible Cre/ loxP system with a uracil biosynthesis pathway and counterselection using uracil analogs.
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