Project description:Our previous study shows that an anaerobic intestinal bacterium strain AJ110941P contributes to type 2 diabetes development in mice. Here we phylogenetically and physiologically characterized this unique mouse gut bacterium. The 16S rRNA gene analysis revealed that the strain belongs to the family Lachnospiraceae but shows low sequence similarities (?<?92.5%) to valid species, and rather formed a distinct cluster with uncultured mouse gut bacteria clones. In metagenomic database survey, the 16S sequence of AJ110941P also matched with mouse gut-derived datasets (56% of total datasets) with?>?99% similarity, suggesting that AJ110941P-related bacteria mainly reside in mouse digestive tracts. Strain AJ110941P shared common physiological traits (e.g., Gram-positive, anaerobic, mesophilic, and fermentative growth with carbohydrates) with relative species of the Lachnospiraceae. Notably, the biofilm-forming capacity was found in both AJ110941P and relative species. However, AJ110941P possessed far more strong ability to produce biofilm than relative species and formed unique structure of extracellular polymeric substances. Furthermore, AJ110941P cells are markedly long fusiform-shaped rods (9.0-62.5?µm) with multiple flagella that have never been observed in any other Lachnospiraceae members. Based on the phenotypic and phylogenetic features, we propose a new genus and species, Fusimonas intestini gen. nov., sp. nov. for strain AJ110941P (FERM BP-11443).
Project description:CRISPR effector proteins introduce double-stranded breaks into the mammalian genome, facilitating gene editing by non-homologous end-joining or homology-directed repair. Unlike the more commonly studied Cas9, the CRISPR effector protein Cas12a/Cpf1 recognizes a T-rich protospacer adjacent motif (PAM) and can process its own CRISPR RNA (crRNA) array, simplifying the use of multiple guide RNAs. We observed that the Cas12a ortholog of Lachnospiraceae bacterium MA2020 (Lb2Cas12a) edited mammalian genes with efficiencies comparable to those of AsCas12a and LbCas12a. Compared to these well-characterized Cas12a orthologs, Lb2Cas12a is smaller and recognizes a narrow set of PAM TTTV. We introduced two mutations into Lb2Cas12a, Q571K and C1003Y, that increased its cleavage efficiency for a range of target sequences beyond those of the commonly used Cas12a orthologs AsCas12a and LbCas12a. In addition to the canonical TTTV PAM, this variant, Lb2-KY, also efficiently cleaved target regions with CTTN PAMs. Finally, we demonstrated that Lb2-KY ribonucleoprotein (RNP) complexes edited two hemoglobin target regions useful for correcting common forms of sickle-cell anemia more efficiently than commercial AsCas12a RNP complexes. Thus, Lb2-KY has distinctive properties useful for modifying a range of clinically relevant targets in the human genome. Graphical Abstract CRISPR-Cas12a effector proteins edit mammalian genomes using a T-rich PAM with few off-targets. Tran et al. engineered a Cas12a nuclease from Lachnospiraceae bacterium MA2020. They demonstrated that this variant recognized a broadened PAM and edited mammalian genomes with higher efficiencies than commonly used orthologs, expanding Cas12a utility in genome engineering.
Project description:The aim of the present study was to identify bacteria that may contribute to the onset of metabolic dysfunctions. We isolated and identified a candidate bacterium belonging to Lachnospiraceae (strain AJ110941) in the feces of hyperglycemic obese mice. The colonization of germ-free ob/ob mice by AJ110941 induced significant increases in fasting blood glucose levels as well as liver and mesenteric adipose tissue weights, and decreases in plasma insulin levels and HOMA-? values. These results indicated that the specific gut commensal bacterium AJ110941 influenced the development of obesity and diabetes in ob/ob mice with genetic susceptibility for obesity.