Efficient photoactivatable Dre recombinase for cell type-specific spatiotemporal control of genome engineering in the mouse.

Precise genetic engineering in specific cell types within an intact organism is intriguing yet challenging, especially in a spatiotemporal manner without the interference caused by chemical inducers. Here we engineered a photoactivatable Dre recombinase based on the identification of an optimal split site and demonstrated that it efficiently regulated transgene expression in mouse tissues spatiotemporally upon blue light illumination. Moreover, through a double-floxed inverted open reading frame strategy, we developed a Cre-activated light-inducible Dre (CALID) system. Taking advantage of well-defined cell-type-specific promoters or a well-established Cre transgenic mouse strain, we demonstrated that the CALID system was able to activate endogenous reporter expression for either bulk or sparse labeling of CaMKIIα-positive excitatory neurons and parvalbumin interneurons in the brain. This flexible and tunable system could be a powerful tool for the dissection and modulation of developmental and genetic complexity in a wide range of biological systems.

Amelioration of an Inherited Metabolic Liver Disease through Creation of a De Novo Start Codon by Cytidine Base Editing.

Base editing technology efficiently generates nucleotide conversions without inducing excessive double-strand breaks (DSBs), which makes it a promising approach for genetic disease therapy. In this study, we generated a novel hereditary tyrosinemia type 1 (HT1) mouse model, which contains a start codon mutation in the fumarylacetoacetate hydrolase (Fah) gene by using an adenine base editor (ABE7.10). To investigate the feasibility of base editing for recombinant adeno-associated virus (rAAV)-mediated gene therapy, an intein-split cytosine base editor (BE4max) was developed. BE4max efficiently induced C-to-T conversion and restored the start codon to ameliorate HT1 in mice, but an undesired bystander mutation abolished the effect of on-target editing. To solve this problem, an upstream sequence was targeted to generate a de novo in-frame start codon to initiate the translation of FAH. After treatment, almost all C-to-T conversions created a start codon and restored Fah expression, which efficiently ameliorated the disease without inducing off-target mutations. Our study demonstrated that base editing-mediated creation of de novo functional elements would be an applicable new strategy for genetic disease therapy.

Development of a novel isolation unit for entomophthoralean fungi.

Entomophthoralean fungi are important natural enemies of pests and highly co-evolved with their hosts. However, successful isolation of entomophthoralean fungi can be difficult due to their fastidious culture requirements; this is an important obstacle to research on Entomophthorales. In this study, we designed an isolation unit and evaluated it against the conventional 'descending conidia' isolation method. There was no difference in contamination rate between the methods (78% and 76% clean isolations) despite the isolation unit not requiring laminar-flow facilities. Furthermore, more conidia were collected in the new isolation unit than using a standard method. The isolation unit is efficient, convenient and is operational in the field.

[Effects of Oxygen Carriers on Submerged Fermentation of Poria cocos].

OBJECTIVE: To explore the regulation effects of oxygen carriers on Poria cocos submerged fermentation system which usually can be seriously inhibited by dissolved oxygen limitation. METHODS: One-factor-at-a-time design was employed to determine the oxygen carrier addition strategy through analyzing the effects of different oxygen carries, concentration and adding time of oxygen carrier on Poria cocos submerged fermentation. Then the oxygen carrier addition strategy was established and the metabolic processes of Poria cocos submerged fermentation were investigated comprehensively. RESULTS: The optimal oxygen carrier addition strategy was adding 1% (V/V) Tween-80 at 48 h after inoculation. Under this optimized condition, dry cell weight of Poria cocos reached 13.43 g/L in a 10 L bioreactor, while yields of exopolysaccharides and pachymic acid were 8.58 g/L and 989.52 µg/L, respectively, which exhibited obvious promoting effects compared with no addition oxygen carrier fermentation process. CONCLUSION: Tween-80 can remarkably increase the levels of cell growth, exopolysaccharides biosynthesis and pachymic acid in Poria cocos submerged fermentation system, which may provide new reference for further exploring dissolved oxygen limitation in high density fermentation of medical fungi efficiently.

[Biosorption of heavy metals in fluoritum decoction by fungal mycelium].

OBJECTIVE: To explore the biosorption technology of heavy metals in Fluoritum decoction by fungal mycelium. METHODS: Four factors including fungal mycelium amount, adsorption time, pH value and temperature were employed to estimate the fungal biomass adsorption conditions for removing the heavy metals in Fluoritum decoction. Then an orthogonal experimental design was taken to optimize the biosorption process, and the removal efficiency was also evaluated. RESULTS: Under the optimized conditions of 1.0 g/50 mL Fluoritum decoction, 3 hours adsorption time, pH 5.0 and 40 degrees C, a result of 70.12% heavy metals removal rate was accomplished with 35.99% calcium ion loss. CONCLUSION: The study indicates that removing of heavy metals in Fluoritum decoction through fungal mycelium is feasible, and the experiment results can also provide a basis for further research on biosorption of heavy metals in traditional Chinese medicine

Enhanced genome editing to ameliorate a genetic metabolic liver disease through co-delivery of adeno-associated virus receptor.

Genome editing through adeno-associated viral (AAV) vectors is a promising gene therapy strategy for various diseases, especially genetic disorders. However, homologous recombination (HR) efficiency is extremely low in adult animal models. We assumed that increasing AAV transduction efficiency could increase genome editing activity, especially HR efficiency, for in vivo gene therapy. Firstly, a mouse phenylketonuria (PKU) model carrying a pathogenic R408W mutation in phenylalanine hydroxylase (Pah) was generated. Through co-delivery of the general AAV receptor (AAVR), we found that AAVR could dramatically increase AAV transduction efficiency in vitro and in vivo. Furthermore, co-delivery of SaCas9/sgRNA/donor templates with AAVR via AAV8 vectors increased indel rate over 2-fold and HR rate over 15-fold for the correction of the single mutation in PahR408W mice. Moreover, AAVR co-injection successfully increased the site-specific insertion rate of a 1.4 kb Pah cDNA by 11-fold, bringing the HR rate up to 7.3% without detectable global off-target effects. Insertion of Pah cDNA significantly decreased the Phe level and ameliorated PKU symptoms. This study demonstrates a novel strategy to dramatically increase AAV transduction which substantially enhanced in vivo genome editing efficiency in adult animal models, showing clinical potential for both conventional and genome editing-based gene therapy.