Engineered CRISPR-OsCas12f1 and RhCas12f1 with robust activities and expanded target range for genome editing.

The type V-F CRISPR-Cas12f system is a strong candidate for therapeutic applications due to the compact size of the Cas12f proteins. In this work, we identify six uncharacterized Cas12f1 proteins with nuclease activity in mammalian cells from assembled bacterial genomes. Among them, OsCas12f1 (433 aa) from Oscillibacter sp. and RhCas12f1 (415 aa) from Ruminiclostridium herbifermentans, which respectively target 5' T-rich Protospacer Adjacent Motifs (PAMs) and 5' C-rich PAMs, show the highest editing activity. Through protein and sgRNA engineering, we generate enhanced OsCas12f1 (enOsCas12f1) and enRhCas12f1 variants, with 5'-TTN and 5'-CCD (D = not C) PAMs respectively, exhibiting much higher editing efficiency and broader PAMs, compared with the engineered variant Un1Cas12f1 (Un1Cas12f1_ge4.1). Furthermore, by fusing the destabilized domain with enOsCas12f1, we generate inducible-enOsCas12f1 and demonstate its activity in vivo by single adeno-associated virus delivery. Finally, dead enOsCas12f1-based epigenetic editing and gene activation can also be achieved in mammalian cells. This study thus provides compact gene editing tools for basic research with remarkable promise for therapeutic applications.

The association between intracompartmental pressure and skeletal muscle recovery after tibial diaphyseal fractures: an ambispective cohort study.

BACKGROUND: Due to the special anatomy of the lower leg, tibial diaphyseal fracture causes increased intracompartmental pressure (ICP). Not only is this increased ICP the manifestation of skeletal muscle injury, but it induces further deterioration of the injury. The aim of this study was to assess the association between short-term ICP elevation and long-term skeletal muscle recovery after severe limb trauma. METHODS: In this single-center ambispective cohort study, we retrospectively screened and recruited a cohort of tibial diaphyseal fracture patients with integrated ICP data during the early post-traumatic period, and performed a prospective observational study to evaluate their skeletal muscle recovery through long-term follow-up and MR imaging after the removal of the implants. We analyzed the association between ICP elevation and skeletal muscle recovery using statistical methods. RESULTS: A total of 46 patients with healed fractures underwent intramedullary nail removal and MR imaging. The absolute values of the Pearson product-moment correlation coefficients between various ICP parameters and the cross-sectional area ratio (CSAR) ranged from 0.588 to 0.793, and the correlation coefficients between the ICP parameters and the average T2-weighted signal intensity ratio (T2SIR) varied from 0.566 to 0.775. Statistically significant associations were observed between the ICP parameters and the MR imaging parameters when simple linear regression analysis was performed. Among the ICP parameters, the accumulated ΔP (ΔP = diastolic blood pressure minus ICP) had the highest determination coefficient and explained 62.1% and 59.1% of the variance in CSAR and T2SIR, respectively. CONCLUSIONS: Short-term ICP elevation was associated with long-term skeletal muscle recovery following tibial diaphyseal fracture, especially for ICP data that integrated time factors. LEVEL OF EVIDENCE: Level 3.

[Effects of sawdust dosage on the diversity and community structure of bacteria associated with the hyphae of Pleurotus eryngii]. / æœ¨å±‘æ·»åŠ å¯¹åˆºèŠ¹ä¾§è€³èŒä¸å ±ç”Ÿç»†èŒå¤šæ ·æ€§å’Œç¾¤è½ç»“æž„çš„å½±å“.

Some fungi engage symbiosis with bacteria, which can effectively promote the metabolism and growth of fungi. The diversity and community structure of bacteria can reflect the growth and substrate utilization of fungi. In this study, we analyzed the effects of different sawdust dosages on the community structure and diversity of bacteria associated with the hyphae of Pleurotus eryngii using high-throughput sequencing technology (HST) based on PCR-amplified 16S rRNA V3-V4 fragments. The results showed that the high-quality sequences from five groups of mycelia samples were clustered into 25 phyla, 52 classes, 114 orders, 199 families, and 406 genera. Proteobacteria (35.0%-85.9%) and Firmicutes (6.5%-38.4%) were the most abundant bacterial phyla, while Acinetobacter (14.8%-71.6%) and Pseudomonas (1.7%-22.3%) were the dominant symbiotic genera. Compared with the mycelia grown on the complete culture medium, sawdust addition could increase the diversity of bacteria coexisting with P. eryngii mycelia, and change the community structure of 10 dominant phyla and 9 dominant genera. P. eryngii cultivated in substrate containing 5 g sawdust had the fastest mycelium growth rate, thick mycelia, and neatly edges. Furthermore, judging from the abundance and diversity, Pseudomonas and Lactobacillus became the dominant genera, which were positively correlated with the mycelia growth vigour. Sawdust, as an important carbon source, could affect the growth and development of P. eryngii and the community structure and diversity of bacteria coexisting with mycelia. This study would lay a theoretical foundation for exploring the molecular mechanism of sawdust and mycelium symbiosis affecting the growth and development of P. eryngii.

Study on the community structure and function of symbiotic bacteria from different growth and developmental stages of Hypsizygus marmoreus.

BACKGROUND: The symbiotic bacteria associated with edible fungi are valuable microbial resources worthy of in-depth exploration. It is important to analyze the community structure and succession of symbiotic bacteria in mushrooms. This can assist in the isolation of growth-promoting strains that have an essential relationship with the cultivation cycle as well as the agronomic traits and yields of fruiting bodies. RESULTS: In all of the samples from cultivation bags of Hypsizygus marmoreus, 34 bacterial phyla were detected. Firmicutes was the most abundant bacterial phylum (78.85%). The genus Serratia showed an exponential increase in abundance in samples collected from the cultivation bags in the mature period, reaching a peak abundance of 55.74% and the dominant symbiotic flora. The most predominant strain was Serratia odorifera HZSO-1, and its abundance increased with the amount of hyphae of H. marmoreus. Serratia odorifera HZSO-1 could reside in the hyphae of H. marmoreus, promote growth and development, shorten the fruiting cycle by 3-4 days, and further increase the fruiting body yield by 12%. CONCLUSIONS: This study is a pioneering demonstration of the community structure of the symbiotic microbiota and bacteria-mushroom interaction in the growth and development of edible fungi. This work lays a theoretical foundation to improve the industrial production of mushrooms with symbiotic bacteria as assisting agents.