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.
Efficient genetic engineering of lactic acid bacteria remains technically challenging due to their thick peptidoglycan cell wall, low transformation efficiency, strain-specific restriction–modification systems, and sensitivity to Cas9-induced double-strand breaks. In this study, we 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. The genetically modified strain exhibited upregulation of plnA, plnE, and plnF, accompanied by elevated antimicrobial activity. These findings underscore the feasibility of rationally reconfiguring a quorum-sensing-associated regulatory circuit and provide a practical strategy for successful genetic engineering in L. plantarum for elevated bacteriocin production.
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.
ABSTRACT Despite substantial advances in bacterial genome engineering, functional genetic analysis remains challenging in many non-model bacterial species, particularly among host-associated gram-positive bacteria. The fructophilic species Apilactobacillus kunkeei has been investigated for more than two decades and is a dominant member of the honeybee microbiome, where it contributes to pathogen resistance and colony fitness. Nevertheless, the mechanistic investigation of this ecologically important species has remained limited despite its growing probiotic relevance. To enable functional genomics in this organism, we developed an inducible genome-engineering platform that leverages its endogenous Type II-A CRISPR-Cas9 system. The system uses a sakacin-responsive dual-plasmid initiator–effector design in which phage-derived recombineering genes and a single-guide RNA are coordinately expressed, while DNA cleavage is mediated by natively expressed Cas9. Using this approach, we achieved scarless deletion of individual genes, including targets as large as ~25 kb, gene replacement with a fluorescent reporter, C-terminal epitope tagging, and precise nucleotide substitutions, with editing efficiencies approaching 100%. Both plasmids can be readily cured following modification, allowing recovery of clean mutant genotypes. We further demonstrate that endogenous Cas9 can be repurposed for CRISPR interference using a single, self-contained plasmid to enable targeted transcriptional repression. Together, this work establishes a robust strategy for genetic manipulation of A. kunkeei and expands the toolkit available for harnessing endogenous CRISPR-Cas systems in genetically recalcitrant, non-model gram-positive bacteria. IMPORTANCE Many ecologically and industrially important bacteria remain genetically recalcitrant, limiting functional genomic studies. As research increasingly extends beyond traditional model organisms, these limitations are especially apparent in non-model gram-positive bacteria from host-associated or environmental niches. Here, we establish an inducible genome-editing framework exploiting the endogenous Cas9 system of Apilactobacillus kunkeei, a key member of the honeybee microbiota. This approach enables reliable scarless gene deletions, precise nucleotide changes, large-scale genome modifications, and programmable transcriptional repression. By enabling genetic manipulation in A. kunkeei, this work facilitates experimental studies of its roles in honeybee health, microbial interactions, and host-associated adaptation, and highlights the potential of endogenous CRISPR-Cas systems for expanding genetic access in non-model bacteria. Many ecologically and industrially important bacteria remain genetically recalcitrant, limiting functional genomic studies. As research increasingly extends beyond traditional model organisms, these limitations are especially apparent in non-model gram-positive bacteria from host-associated or environmental niches. Here, we establish an inducible genome-editing framework exploiting the endogenous Cas9 system of Apilactobacillus kunkeei, a key member of the honeybee microbiota. This approach enables reliable scarless gene deletions, precise nucleotide changes, large-scale genome modifications, and programmable transcriptional repression. By enabling genetic manipulation in A. kunkeei, this work facilitates experimental studies of its roles in honeybee health, microbial interactions, and host-associated adaptation, and highlights the potential of endogenous CRISPR-Cas systems for expanding genetic access in non-model bacteria.
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.