: Bacteroides, as one of the most abundant and diverse genera in the human gut, is regarded as a window into the study of gut microbiota-host interactions. Currently, CRISPR/Cas-based gene editing systems targeting Bacteroides have been widely applied, while the large size of Cas nucleases limits their potential application scenarios (such as in situ gut Bacteroides editing based on phage delivery). Therefore, this study aims to develop a compact and highly efficient genetic editing tool in Bacteroides., We developed a miniaturized CRISPR/Cas gene editing system for human gut Bacteroides. First, the editing capabilities of different miniaturized CRISPR/Cas systems, including AsCas12f, CasΦ2, and ISDge10, were evaluated in Bacteroides fragilis. Subsequently, the editing capability of AsCas12f was assessed across various Bacteroides species, and the size of this system was further optimized. The results demonstrated that the CRISPR/AsCas12f genome editing system exhibited the highest editing efficiency in B. fragilis. The CRISPR/AsCas12f system achieved efficient genome editing in B. fragilis, Bacteroides thetaiotaomicron, and Phocaeicola vulgatus. Furthermore, with a repair template of 500 bp homologous arms, the editing efficiency remained as high as 94.7%. In conclusion, CRISPR/AsCas12f can serve as a chassis tool enzyme for the development of Bacteroides-based miniature gene editors and derivative technologies, laying a foundation for the further development of gene editing technology for Bacteroides.
CRISPR-Cas Systems/genetics* ; Gene Editing/methods* ; Bacteroides/genetics* ; Humans ; Gastrointestinal Microbiome/genetics* ; Bacteroides fragilis/genetics*

OBJECTIVE: To explore the clinical features and genetic basis of a patient with glycogen storage disease type VI (GSD-VI). METHODS: Clinical data of the patient was collected. Genomic DNA was extracted from peripheral blood samples of the proband and his parents. Genetic variants were detected by using whole exome sequencing. Candidate variants were verified by Sanger sequencing followed by bioinformatics analysis. RESULTS: The proband presented fasting hypoglycemia, hepatomegaly, growth retardation, transaminitis, metabolic acidosis and hyperlactatemia. Liver biopsy indicated GSD. Novel compound heterozygous PYGL gene variants (c.2089A>G/c.158_160delACT) were detected in the proband. Compound heterozygosity was confirmed by Sanger sequencing of the patient's genomic DNA. Provean and MutationTaster predicted the two variants as deleterious and the variant sites are highly conserved. CONCLUSION The compound heterozygous variants (c.2089A>G/c.158_160delACT) of PYGL gene probably underlay the GSD in the patient. The two novel variants have expanded the spectrum of PYGL gene variants and provided the basis for genetic counseling of the family.
Child ; Family ; Genetic Testing ; Glycogen Storage Disease Type VI/genetics* ; Humans ; Mutation ; Whole Exome Sequencing

Objective:To explore the effects of serum triglyceride (STAG) level within 48 hours after hospitalization on the complications of acute pancreatitis (AP).Methods:From January 2012 to June 2016, 1 006 hospitalized patients diagnosed with AP at the Third People′s Hospital of Chengdu were collected. According to the STAG level within 48 hours after hospitalization, AP patients were divided into normal to mild hypertriglyceridemia (HTG) group(STAG <2.3 mmol/L, 877 cases), moderate HTG group(STAG: 2.3 to <8.5 mmol/L, 82 cases) and severe HTG group (≥8.5 mmol/L, 47 cases). The general clinical data and the incidence of local complications of AP including acute necrotizing pancreatitis, pancreatic necrosis, acute peripancreatic fluid collection (APFC) and acute necrotic collection (ANC) and AP-associated gastrointestinal abnormal changes were compared in the three groups. The severity of the complications of AP was scored by modified-magnetic resonance severity index (M-MRSI). Wilcoxon rank sum test and chi-square test were used for statistical analysis, and multivariate logistic regression analysis was used to analyze the correlation between STAG level and persistent organ failure (POF).Results:Compared with that of the normal to mild HTG group and moderate HTG group, the age of the patients of the severe HTG group was the youngest (52 years old, 19 to 82 years old and 47 years old, 21 to 62 years old vs. 35 years old, 18 to 43 years old), the proportion of male was the highest (46.3%, 406/877 and 64.6%, 53/82 vs. 85.1%, 40/47), and the differences were statistically significant( Z=3.943, 2.841, χ2=26.912, 6.224, all P<0.017). The proportion of body mass index (BMI)≥30 kg/m 2 in severe HTG group was higher than that in normal to mild HTG group (38.3%, 18/47 vs. 20.2%, 177/877), and the difference was statistically significant ( χ2=8.792, P=0.003). The proportions of patients with history of diabetes and severe alcohol intake of moderate HTG group and severe HTG group were all higher than those of normal to mild HTG group (31.7%, 26/82 and 29.8%, 14/47 vs. 15.4%, 135/877; 37.8%, 31/82 and 46.8%, 22/47 vs. 9.6%, 84/877), and the differences were statistically significant ( χ2=14.286, 6.833, 56.613 and 60.844, all P<0.017). Compared with those of the normal to mild HTG group and moderate HTG group, the incidences of pancreatic necrosis, APFC, and the M-MRSI score of the severe HTG group were all the highest (8.2%, 72/877 and 15.9%, 13/82 vs. 38.3%, 18/47; 17.8%, 156/877 and 36.6%, 30/82 vs. 59.6%, 28/47; 2, 0 to 10 and 3, 0 to 10 vs. 5, 0 to 10), and the differences were statistically significant( χ2=45.936, 8.244, 48.842 and 6.381, Z=2.711 and 3.049, all P<0.017). The incidence rates of acute necrotizing pancreatitis and ANC of moderate HTG group and severe HTG group were all higher than those of normal to mild HTG group(28.0%, 23/82 and 48.9%, 23/47 vs. 13.3%, 117/877; 26.8%, 22/82 and 42.6%, 20/47 vs. 13.3%, 117/877), and the differences were statistically significant ( χ2=13.011, 43.965, 11.008 and 30.144, all P<0.017). The incidence rate of POF of severe HTG group was higher than those of normal to mild HTG group and moderate HTG group (46.8%, 22/47 vs.14.8%, 130/877 and 24.4%, 20/82), and the differences were statistically significant ( χ2=33.205 and 6.838, both P<0.017). The results of multivariate logistic regression analysis showed that age ≥ 60 years old (odds ratio ( OR)=1.84, 95% confidence interval ( CI) 1.26 to 3.03), BMI≥30 kg/m 2 ( OR=2.41, 95% CI 1.61 to 3.77), alcohol intake ( OR=3.81, 95% CI 2.09 to 5.47), moderate HTG( OR=1.89, 95% CI 1.78 to 5.23) and severe HTG ( OR=3.65, 95% CI 1.98 to 6.49) were independent risk factors of POF(all P<0.05). Conclusion:The STAG level is related to the complications of AP, and moderate HTG and severe HTG(STAG ≥2.3 mmol/L) are independently associated with the risk of POF.