A HIT-trapping strategy for rapid generation of reversible and conditional alleles using a universal donor.

Targeted mutagenesis in model organisms is key for gene functional annotation and biomedical research. Despite technological advances in gene editing by the CRISPR-Cas9 systems, rapid and efficient introduction of site-directed mutations remains a challenge in large animal models. Here, we developed a robust and flexible insertional mutagenesis strategy, homology-independent targeted trapping (HIT-trapping), which is generic and can efficiently target-trap an endogenous gene of interest independent of homology arm and embryonic stem cells. Further optimization and equipping the HIT-trap donor with a site-specific DNA inversion mechanism enabled one-step generation of reversible and conditional alleles in a single experiment. As a proof of concept, we successfully created mutant alleles for 21 disease-related genes in primary porcine fibroblasts with an average knock-in frequency of 53.2%, a great improvement over previous approaches. The versatile HIT-trapping strategy presented here is expected to simplify the targeted generation of mutant alleles and facilitate large-scale mutagenesis in large mammals such as pigs.

ASGR1 is a promising target for lipid reduction in pigs with PON2 as its inhibitor.

Although the role of asialoglycoprotein receptor 1 (ASGR1) in lowering lipid levels is well established, recent studies indicate that ASGR1 inhibition can cause unexpected liver damage in pigs, raising a serious issue about whether ASGR1 can be a good target for treating ASCVD. Here, we utilized the CRISPR-Cas9 system to regenerate ASGR1-knockout pigs, who displayed decreased lipid profiles without observable liver damage. This was confirmed by the lower levels of serum ALT and AST, reduced expression of inflammation markers, and normal histological morphology. Also, we implemented immunoprecipitation combined with mass spectrometry (IP-MS) and discovered that paraoxonase-2 (PON2) can interact with and significantly degrade ASGR1 in a dose-dependent manner. This degradation reduced lipid levels in mice, accompanied by little inflammation. Our study highlights the effectiveness and safety of degrading ASGR1 to reduce lipid levels in pigs and provides a potential inhibitor of ASGR1.

A Genome-Wide CRISPR Screen Identifies Factors Regulating Pluripotency Exit in Mouse Embryonic Stem Cells.

Pluripotency maintenance and exit in embryonic stem cells is a focal topic in stem cell biology. However, the effects of screening under very stringent culture conditions (e.g., differentiation medium, no leukemia inhibitory factor, no chemical inhibitors such as PD0325901 and CHIR99021, and no feeder cells) and of prolonging culture for key factors that regulate pluripotency exit, have not yet been reported. Here, we used a genome-wide CRISPR library to perform such a screen in mouse embryonic stem cells. Naïve NANOG-GFP mESCs were first transfected with a mouse genome-wide CRISPR knockout library to obtain a mutant mESCs library, followed by screening for two months in a strict N2B27 differentiation medium. The clones that survived our stringent screening were analyzed to identify the inserted sgRNAs. In addition to identifying the enriched genes that were reported in previous studies (Socs3, Tsc1, Trp53, Nf2, Tcf7l1, Csnk1a1, and Dhx30), we found 17 unreported genes, among which Zfp771 and Olfr769 appeared to be involved in pluripotency exit. Furthermore, Zfp771 knockout ESCs showed a differentiation delay in embryonic chimera experiments, indicating Zfp771 played an important role in pluripotency exit. Our results show that stringent screening with the CRISPR library can reveal key regulators of pluripotency exit.

Overcoming the instability of aerobic granular sludge under nitrogen deficiency through shortening settling time.

This study investigated the short settling time strategy to overcome the instability of aerobic granular sludge (AGS) under nitrogen deficiency. AGS variations in its physical and chemical characteristics and microbial community were investigated. Results showed that nitrogen deficiency led to the instability of AGS, while short settling time strategy could overcome the instability of AGS under nitrogen deficiency. Extracellular polymeric substances (EPS), especially, the increased secretion of polysaccharide and proteins with amide III groups at the short settling time enhanced the stability of the granules under nitrogen deficiency. Unclassified_f_Microbacteriaceae shifted to be the major bacteria group at short settling time, along with the decrease of Meganema and Rhodobacter and the increase of Lysobacter, which may play an important role in enhancing AGS stability. Therefore, shortening settling time supports an effective strategy for applications of AGS under nitrogen deficiency.

Effect of nitrogen deficiency on the stability of aerobic granular sludge.

To assess the stability of aerobic granular sludge (AGS) under nitrogen deficiency conditions, three sequence batch reactors were operated with chemical oxygen demand (COD) to nitrogen (N) ratios of 100/5, 100/2.5, and 100/2, while COD concentration was kept consistent. AGS variations in its physicochemical characteristics, microbial community, and treatment performance were investigated. The results indicated that good treatment performance and stable AGS were achieved under nitrogen deficiency. Extracellular polymeric substances (EPS) regulating mechanism preserved AGS stability under nitrogen deficiency, especially through increased secretion of polysaccharide. In addition, members of the Anaerolineaceae were the major filamentous bacteria, which are strictly anaerobic organism, providing a possible explanation to the lack of filamentous bacteria outgrowth under N deficient condition. Insights from this study could help lower chemical costs in AGS applications for specific industrial wastewater treatments.

Simultaneous nitrification/denitrification and stable sludge/water separation achieved in a conventional activated sludge process with severe filamentous bulking.

This study investigated the long-term treatment performance of a conventional activated sludge (AS) process operating at a microaerobic DO level (0.5-1.0mg·L-1) in the aeration tank and a long settling time of >10h in the clarification tank for sewage treatment. The microaerobic DO conditions led to severe sludge bulking. However, good sludge/water separation and excellent pollutant removal performance (COD, 95±2%; NH4+-N, 99±1%; and TN, 69±6%) were stably achieved in the microaerobic AS system during its 150days of continuous operation. This is the first report to demonstrate that a long settling time effectively overcame the effect of severe filamentous bulking in conventional AS process, and that microaerobic DO conditions achieved excellent simultaneous nitrification and denitrification reactions in the aeration tank. The process characteristics of the microaerobic AS system differed substantially from those existing biological denitrification processes, including A/O, CANON, and OLAND processes.

Effect of long-term organic removal on ion exchange properties and performance during sewage tertiary treatment by conventional anion exchange resins.

This study evaluated the long-term dissolved organic matter (DOM), phosphorus and nitrogen removal performance of a commercially available conventional anion exchange resin (AER) from actual secondary effluent (SE) in a sewage treatment plant based on a pilot-scale operation (2.2 m(3) d(-1), 185 cycles, 37,000 bed volume, 1.5 years). Particular emphasis was given to the potential effect of DOM fouling on the ion exchange properties and performance during the long-term operation. Despite the large range of COD (15.6-33.5 mg L(-1)), BOD5 (3.0-5.6 mg L(-1)), DOC (6.5-24.2 mg L(-1)), and UV254 (UV absorption at 254 nm) (0.108-0.229 cm(-1)) levels in the SE, the removal efficiencies of the AER for the aforementioned parameters were 43±12%, 46±15%, 45±9%, and 72±4%, respectively. Based on three-dimensional fluorescence excitation-emission matrix data, i.e., the fluorescence intensities of four regions (peaks A-D), all organic components of the SE were effectively removed (peak A 74%, peak B 48%, peak C 55%, and peak D 45%) following the adsorption. The AER effluent still has considerable polycyclic aromatic hydrocarbons' ecological hazard on freshwater fishes when they were significantly removed from SE. The obvious DOM fouling on the AER, identified by color change, had no significant influence on the long-term removal of the representative inorganic anions (averaging 95±4% phosphate, 100±0% SO4(2-), and 62±17% NO3(-)) and AER properties (including total exchange capacity, moisture content, and true density). The conventional AER can produce high quality reclaimed water from SE at a low operational cost.