Jun 2026· Cell Systems· Vol 17, pp.
101650
· 0 citations· 86 references
Medicine
TL;DR
STIB (ShCAST-based transient insertion system for Bacteroides), an efficient genome-editing tool derived from CRISPR-associated transposases that enables rapid and site-specific insertions independent of homologous recombination is developed.
Abstract
Gut Bacteroides are abundant and critical to human health, yet most are genetically cumbersome, non-model microbes. A widely applicable editing tool for Bacteroides is essential for gut microbiome manipulation. Here, we develop STIB (ShCAST-based transient insertion system for Bacteroides), an efficient genome-editing tool derived from CRISPR-associated transposases that enables rapid and site-specific insertions independent of homologous recombination. By fusing a nicking homing endonuclease to the transposase and an ATPase to Cas12k, we systematically optimize STIB to minimize plasmid cointegration and achieve >97% on-target insertion. STIB exhibits broad applicability across different genomic loci in diverse Bacteroides species, including non-model species. Finally, we apply STIB to achieve species- and site-specific editing of distinct Bacteroides species within a complex synthetic gut microbiota. Overall, STIB expands the toolbox for the functional investigation and engineering of the human microbiome. A record of this paper's transparent peer review process is included in the supplemental information.
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.
Mahesh S Iyer, Erik Hagström, Kristina Näslund et al.· Applied and Environmental Mi...· 0 citations
This article synthesizes contemporary advancements in CRISPR-mediated mammalian genome modification, detailing core mechanisms – such as guide RNA and the Cas9 endonuclease – alongside next-generation modalities, including base and prime editing.
Olga Aldoshina, Dmitriy Lazarev, E. Smirnova· Veterinariya, Zootekhniya i...· 0 citations
Five previously uncharacterized MG102-like Cas9d orthologs are identified that share the hallmark genomic, sequence, and structural features of type II-D Cas9 and establish compact MG102-like Cas9d orthologs as robust and specific genome editors and provide promising, single-AAV– compatible scaffolds for in vivo therapeutic genome editing.
Qiaochu Wang, Ahmed Saleh, G. S. Rao et al.· bioRxiv· 0 citations
The results identify Pgm3 as the most promising candidate for further development as a Ciona-based model of human disease and demonstrate the utility of tissue-specific CRISPR screening for prioritizing candidate disease gene orthologs identified through comparative genomics platforms like Zoogle.
Sabrina A. Hernandez, Christopher J. Johnson, Alberto Stolfi· bioRxiv· 0 citations
A consolidated guide for selecting suitable CRISPR-Cas technologies and underscoring important considerations for their continued development in leishmaniasis research is offered, highlighting the transition of CRISPR-Cas systems from proof-of-concept tools to versatile platforms for functional genomics, target validation and translational research in Leishmania.
A. Ata, Derya Topuz Ata· Molecular Biology Reports· 0 citations
Genome-wide viability catalogs produced by transposon sequencing (Tn-seq) and CRISPR interference (CRISPRi) have successfully mapped the essential genome of Streptococcus mutans. However, particularly for genes annotated as “hypothetical” or uncharacterized, translating these findings into mechanistic biological functions remains a significant bottleneck. In this study, we developed an integrated functional genomics pipeline combining predictive bioinformatics, tunable CRISPRi transcriptional silencing, transmission electron microscopy, transcriptomics, and genetic suppressor screens to characterize nine legacy hypothetical essential genes in S. mutans. Comparative transcriptomics and proteomics revealed a conserved baseline stress signature across diverse essential pathways, marked by the coordinated downregulation of the citZ-citB-idh metabolic locus and insoluble matrix synthesis enzymes (gtfBC), paired with the robust activation of the integrative and conjugative element TnSmu1. Against this backdrop of systemic stress, we successfully resolved the function of SMU_393, defining it as a functional equivalent of the pneumococcal regulator of chromosome segregation, RocS. Depletion of SMU_393 resulted in abnormal cell widening, hypersensitivity to DNA damage, and a significant subpopulation of anucleate cells. Remarkably, these phenotypes were bypassed by a spontaneous surface-exposed missense mutation (dnaAQ197E) within the AAA+ ATPase domain of the replication initiator. Together, this work uncovers an important cell cycle regulator and provides a framework for exploring uncharacterized essential genes of the oral microbiome. Importance Although genome sequencing has identified thousands of genes required for bacterial survival, the precise biological roles for many of them remain completely unknown. This study implements an integrated functional genomics pipeline to resolve the molecular functions of legacy uncharacterized essential genes in the oral pathogen Streptococcus mutans. We discovered a critical molecular checkpoint that acts as a physical anchor, linking the bacterial chromosome to the cell envelope to ensure that chromosome replication is synchronized with cell division. Remarkably, a single mutation in the replication machinery can fully bypass the loss of this anchor, maintaining proper genetic inheritance even during severe cellular stress. Ultimately, this study provides a pipeline for uncovering highly specific physiological vulnerabilities that can be exploited for targeted therapeutics against oral pathogens.
Courtney Dover, Kipa Tamrakar, Bikash Dwivedi et al.· bioRxiv· 0 citations