Protocol for efficient CRISPR/Cas9/AAV-mediated homologous recombination in mouse hematopoietic stem and progenitor cells

Mutations that accumulate in self-renewing hematopoietic stem and progenitor cells (HSPCs) can cause severe blood disorders. To model such disorders in mice, we developed a CRISPR/Cas9/adeno-associated virus (AAV)-based system to knock in and repair genes by homologous recombination in mouse HSPCs. Here, we provide a step-by-step protocol to achieve high efficiency of gene knockin in mouse HSPCs, while maintaining engraftment capacity. This approach enables the functional study of hematopoietic disease mutations in vivo, without requiring germline mutagenesis

Potential reuse of coal mine wastewater: a case study in Quang Ninh, Vietnam

In Vietnam, the local regulation and environmental impact are driving coal mining industry to reuse the large volume of wastewater it produces. The co-research project between National University of Civil Engineering (NUCE) and Mitsubishi Rayon Corporation (MRC) has started early 2013 to evaluate if the MRC membranes could be a great tool for treatment of coal mine wastewater for reuse. The experiment were conducted at one of coal mine plants in Quang Ninh province, Vietnam. It was found that pre-treatment of coal mine wastewater was an important part in the treatment process. The MRC membrane was a significant barrier to maintain stable and high quality effluent to meet the requirement of Vietnam national technical standard for domestic use

Hematological parameters of red tilapia (Oreochromis sp.) fed lemongrass essential oil (Cymbopogon citratus) after challenge with Streptococcus agalactiae

The study involved feeding lemongrass essential oil (LEO) supplements to red tilapia (Oreochromis sp.) at concentrations including Control - 0 mg, T1 – 200 mg, T2 – 300 mg, and T3 – 400 mg per kg of feed. The research investigated changes in hematological (HCT, Hb, RBC, WBC & thrombocytes) and erythrocyte’s morphological (major/minor axis; perimeter, and area of erythrocyte) parameters before infection, 5- and 10-days post-infection (DPI). According to analytical findings, a diet containing LEO enhanced the synthesis of both erythrocytes and leukocytes in the peripheral blood of red tilapia after 20 days of being used. Therefore, the indicators of this group of fish showed better performance than those that did not use LEO supplement five days after bacterial infection. Fish fed 200 mg/kg of LEO after being challenged with S. agalactiae for ten days showed an improved effect on red blood cell production. White blood cells decreased at all concentrations because of citral’s immunomodulatory properties

Factors influencing farmers' forestland-use changes over 15 years (2005–2020) in Thua Thien Hue province, Vietnam

Over the last decades, Vietnam has seen substantial shifts in forest landscape uses and associated livelihoods. We document the livelihood transformations in Nam Dong, a mountainous district of Central Vietnam, where land uses have changed from the utilisation of products from natural forests and shifting cultivation (swidden agriculture) to acacia tree-dominated plantation forestry. Forestry policies (forestland allocation, plantation development agendas), the increase in the economic value of acacia, and household livelihood assets are the primary factors driving these changes. We also found that there are differences in the access to and ownership of forestland with regard to households of different communities and between poor vs wealthy households. Therefore, careful attention needs to be paid to guide future land use policies in the area to foster social and ecological sustainability. HIGHLIGHTS • Major livelihood and forestland-use changes have taken place in central Vietnam over the last two decades. • There has been widespread conversion of forestland (degraded natural forests, swidden land) and cropland to acacia plantations. • Household-scale forestland use changes were primarily driven by forestry policies, the market for woodchips, and land resource access. • There is inequality in access to and ownership of forestland between poor and wealthier households in the mountain district of Vietnam. • Cases of illegal forestland conversions pose challenges to ensuring sustainable forest landscapes

Shape dependent protein‐induced stabilization of gold nanoparticles: From a protein corona perspective

Gold nanoparticles (AuNPs) are promising materials for many bioapplications. However, upon contacting with biological media, AuNPs undergo changes. The interaction with proteins results in the so-called protein corona (PC) around AuNPs, leading to the new bioidentity and optical properties. Understanding the mechanisms of PC formation and its functions can help us to utilise its benefits and avoid its drawbacks. To date, most of the previous works aimed to understand the mechanisms governing PC formation and focused on the spherical nanoparticles, although non-spherical nanoparticles are designed for a wide range of applications in biosensing. In this work, we investigated the differences in PC formation on spherical and anisotropic AuNPs (nanostars in particular) from the joint experimental (extinction spectroscopy, zeta potential and surface-enhanced Raman scattering [SERS]) and computational methods (the finite element method and molecular dynamics [MD] simulations). We discovered that protein does not fully cover the surface of anisotropic nanoparticles, leaving SERS hot-spots at the tips and high curvature edges ‘available’ for analyte binding (no SERS signal after pre-incubation with protein) while providing protein-induced stabilization (indicated by extinction spectroscopy) of the AuNPs by providing a protein layer around the particle's core. The findings are confirmed from our MD simulations, the adsorption energy significantly decreases with the increased radius of curvature, so that tips (adsorption energy: 2762.334 kJ/mol) would be the least preferential binding site compared to core (adsorption energy: 11819.263 kJ/mol). These observations will help the development of new nanostructures with improved sensing and targeting ability

Efficient CRISPR/Cas9-mediated gene knockin in mouse hematopoietic stem and progenitor cells

Mutations accumulating in hematopoietic stem and progenitor cells (HSPCs) during development can cause severe hematological disorders. Modeling these mutations in mice is essential for understanding their functional consequences. Here, we describe an efficient CRISPR/Cas9-based system to knock in and repair genes in mouse HSPCs. CRISPR/Cas9 ribonucleoproteins, in combination with recombinant adeno-associated virus (rAAV)-DJ donor templates, led to gene knockin efficiencies of up to 30% in the Lmnb1 and Actb loci of mouse HSPCs in vitro. The targeted HSPCs engraft and reconstitute all immune cell lineages in the recipient mice. Using this approach, we corrected a neomycin-disrupted Rag2 gene. The Rag2-corrected HSPCs restore B and T cell development in vivo, confirming the functionality of the approach. Our method provides an efficient strategy to study gene function in the hematopoietic system and model hematological disorders in vivo, without the need for germline mutagenesis

Efficient and precise CRISPR/Cas9-mediated MECP2 modifications in human-induced pluripotent stem cells

Patients with Rett syndrome (RTT) have severe mental and physical disabilities. The majority of RTT patients carry a heterozygous mutation in methyl-CpG binding protein 2 (MECP2), an X-linked gene encoding an epigenetic factor crucial for normal nerve cell function. No curative therapy for RTT syndrome exists, and cellular mechanisms are incompletely understood. Here, we developed a CRISPR/Cas9-mediated system that targets and corrects the disease relevant regions of the MECP2 exon 4 coding sequence. We achieved homologous recombination (HR) efficiencies of 20% to 30% in human cell lines and iPSCs. Furthermore, we successfully introduced a MECP2(R270X) mutation into the MECP2 gene in human induced pluripotent stem cells (iPSCs). Consequently, using CRISPR/Cas9, we were able to repair such mutations with high efficiency in human mutant iPSCs. In summary, we provide a new strategy for MECP2 gene targeting that can be potentially translated into gene therapy or for iPSCs-based disease modeling of RTT syndrome

Application of a spacer-nick gene-targeting approach to repair disease-causing mutations with increased safety

The CRISPR/Cas9 system is a powerful tool for gene repair that holds great potential for gene therapy to cure monogenic diseases. Despite intensive improvement, the safety of this system remains a major clinical concern. In contrast to Cas9 nuclease, Cas9 nickases with a pair of short-distance (38-68 bp) PAM-out single-guide RNAs (sgRNAs) preserve gene repair efficiency while strongly reducing off-target effects. However, this approach still leads to efficient unwanted on-target mutations that may cause tumorigenesis or abnormal hematopoiesis. We establish a precise and safe spacer-nick gene repair approach that combines Cas9(D10A) nickase with a pair of PAM-out sgRNAs at a distance of 200-350 bp. In combination with adeno-associated virus (AAV) serotype 6 donor templates, this approach leads to efficient gene repair with minimal unintended on- and off-target mutations in human hematopoietic stem and progenitor cells (HSPCs). Here, we provide detailed protocols to use the spacer-nick approach for gene repair and to assess the safety of this system in human HSPCs. The spacer-nick approach enables efficient gene correction for repair of disease-causing mutations with increased safety and suitability for gene therapy

Tuberculosis Acquired Outside of Households, Rural Vietnam

Using population-based data from rural Vietnam, we assessed tuberculosis (TB) transmission within and outside of households. Eighty-three percent of persons with recent household TB were infected by different strains of Mycobacterium tuberculosis than were their household members. This result argues against the effectiveness of active TB case finding among household members

Efficient CRISPR-mediated mutagenesis in primary immune cells using CrispRGold and a C57BL/6 Cas9 transgenic mouse line

Applying clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9)-mediated mutagenesis to primary mouse immune cells, we used high-fidelity single guide RNAs (sgRNAs) designed with an sgRNA design tool (CrispRGold) to target genes in primary B cells, T cells, and macrophages isolated from a Cas9 transgenic mouse line. Using this system, we achieved an average knockout efficiency of 80% in B cells. On this basis, we established a robust small-scale CRISPR-mediated screen in these cells and identified genes essential for B-cell activation and plasma cell differentiation. This screening system does not require deep sequencing and may serve as a precedent for the application of CRISPR/Cas9 to primary mouse cells