Targeted disruption of the BCR-ABL fusion gene by Cas9/dual-sgRNA inhibits proliferation and induces apoptosis in chronic myeloid leukemia cells.

The BCR-ABL fusion gene, formed by the fusion of the breakpoint cluster region protein ( BCR) and the Abl Oncogene 1, Receptor Tyrosine Kinase ( ABL) genes, encodes the BCR-ABL oncoprotein, which plays a crucial role in leukemogenesis. Current therapies have limited efficacy in patients with chronic myeloid leukemia (CML) because of drug resistance or disease relapse. Identification of novel strategies to treat CML is essential. This study aims to explore the efficiency of novel CRISPR-associated protein 9 (Cas9)/dual-single guide RNA (sgRNA)-mediated disruption of the BCR-ABL fusion gene by targeting BCR and cABL introns. A co-expression vector for Cas9 green fluorescent protein (GFP)/dual-BA-sgRNA targeting BCR and cABL introns is constructed to produce lentivirus to affect BCR-ABL expression in CML cells. The effects of dual-sgRNA virus-mediated disruption of BCR-ABL are analyzed via the use of a genomic sequence and at the protein expression level. Cell proliferation, cell clonogenic ability, and cell apoptosis are assessed after dual sgRNA virus infection, and phosphorylated BCR-ABL and its downstream signaling molecules are detected. These effects are further confirmed in a CML mouse model via tail vein injection of Cas9-GFP/dual-BA-sgRNA virus-infected cells and in primary cells isolated from patients with CML. Cas9-GFP/dual-BA-sgRNA efficiently disrupts BCR-ABL at the genomic sequence and gene expression levels in leukemia cells, leading to blockade of the BCR-ABL tyrosine kinase signaling pathway and disruption of its downstream molecules, followed by cell proliferation inhibition and cell apoptosis induction. This method prolongs the lifespan of CML model mice. Furthermore, the effect is confirmed in primary cells derived from patients with CML.

SARS-CoV-2 and Its Omicron Variants Detection with RT-RPA -CRISPR/Cas13a-Based Method at Room Temperature.

Background: The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered a global health crisis, with genetic mutations and evolution further creating uncertainty about epidemic risk. It is imperative to rapidly determine the nucleic acid sequence of SARS-CoV-2 and its variants to combat the coronavirus pandemic. Our goal was to develop a rapid, room-temperature, point-of-care (POC) detection system to determine the nucleic acid sequences of SARS-CoV-2 isolates, especially omicron variants. Methods: Based on the conserved nucleotide sequence of SARS-CoV-2, bioinformatics software was used to analyze, design, and screen optimal enzymatic isothermal amplification primers and efficient CRISPR RNAs (crRNAs) of CRISPR/Cas13a to the target sequences. Reverse transcription-recombinase polymerase amplification (RT-RPA) was used to amplify the virus, and CRISPR/Cas13a-crRNA was used to cleave the SARS-CoV-2 target sequence. The sensitivity of nucleic acid detection was assessed by serial dilution of plasmid templates. All reactions were performed at room temperature. Results: RT-RPA, combined with CRISPR/Cas13a, can detect the SARS-CoV-2 with a minimum content of 102 copies/µL, and can effectively distinguish between the original strain and the Omicron variant with a minimum limit of detection (LOD) of 103 copies/µL. Conclusions: The method developed in this study has potential application in clinical detection of SARS-CoV-2 and its omicron variants.

The Biomechanical Mechanism of Upper Airway Collapse in OSAHS Patients Using Clinical Monitoring Data during Natural Sleep.

Obstructive sleep apnea hypopnea syndrome (OSAHS) is a common sleep disorder characterized by repeated pharyngeal collapse with partial or complete obstruction of the upper airway. This study investigates the biomechanics of upper airway collapse of OSASH patients during natural sleep. Computerized tomography (CT) scans and data obtained from a device installed on OSASH patients, which is comprised of micro pressure sensors and temperature sensors, are used to develop a pseudo three-dimensional (3D) finite element (FE) model of the upper airway. With consideration of the gravity effect on the soft palate while patients are in a supine position, a fluid-solid coupling analysis is performed using the FE model for the two respiratory modes, eupnea and apnea. The results of this study show that the FE simulations can provide a satisfactory representation of a patient's actual respiratory physiological processes during natural sleep. The one-way valve effect of the soft palate is one of the important mechanical factors causing upper airway collapse. The monitoring data and FE simulation results obtained in this study provide a comprehensive understanding of the occurrence of OSAHS and a theoretical basis for the individualized treatment of patients. The study demonstrates that biomechanical simulation is a powerful supplementation to clinical monitoring and evaluation.