Precise editing of pathogenic nucleotide repeat expansions in iPSCs using paired prime editor.

Nucleotide repeat expansion disorders, a group of genetic diseases characterized by the expansion of specific DNA sequences, pose significant challenges to treatment and therapy development. Here, we present a precise and programmable method called prime editor-mediated correction of nucleotide repeat expansion (PE-CORE) for correcting pathogenic nucleotide repeat expansion. PE-CORE leverages a prime editor and paired pegRNAs to achieve targeted correction of repeat sequences. We demonstrate the effectiveness of PE-CORE in HEK293T cells and patient-derived induced pluripotent stem cells (iPSCs). Specifically, we focus on spinal and bulbar muscular atrophy and spinocerebellar ataxia type, two diseases associated with nucleotide repeat expansion. Our results demonstrate the successful correction of pathogenic expansions in iPSCs and subsequent differentiation into motor neurons. Specifically, we detect distinct downshifts in the size of both the mRNA and protein, confirming the functional correction of the iPSC-derived motor neurons. These findings highlight PE-CORE as a precision tool for addressing the intricate challenges of nucleotide repeat expansion disorders, paving the way for targeted therapies and potential clinical applications.

Gαs slow conformational transition upon GTP binding and a novel Gαs regulator.

G proteins are major signaling partners for G protein-coupled receptors (GPCRs). Although stepwise structural changes during GPCR-G protein complex formation and guanosine diphosphate (GDP) release have been reported, no information is available with regard to guanosine triphosphate (GTP) binding. Here, we used a novel Bayesian integrative modeling framework that combines data from hydrogen-deuterium exchange mass spectrometry, tryptophan-induced fluorescence quenching, and metadynamics simulations to derive a kinetic model and atomic-level characterization of stepwise conformational changes incurred by the ß2-adrenergic receptor (ß2AR)-Gs complex after GDP release and GTP binding. Our data suggest rapid GTP binding and GTP-induced dissociation of Gαs from ß2AR and Gßγ, as opposed to a slow closing of the Gαs α-helical domain (AHD). Yeast-two-hybrid screening using Gαs AHD as bait identified melanoma-associated antigen D2 (MAGE D2) as a novel AHD-binding protein, which was also shown to accelerate the GTP-induced closing of the Gαs AHD.

Infection of Brain Organoids and 2D Cortical Neurons with SARS-CoV-2 Pseudovirus.

Since the global outbreak of SARS-CoV-2 (COVID-19), infections of diverse human organs along with multiple symptoms continue to be reported. However, the susceptibility of the brain to SARS-CoV-2, and the mechanisms underlying neurological infection are still elusive. Here, we utilized human embryonic stem cell-derived brain organoids and monolayer cortical neurons to investigate infection of brain with pseudotyped SARS-CoV-2 viral particles. Spike-containing SARS-CoV-2 pseudovirus infected neural layers within brain organoids. The expression of ACE2, a host cell receptor for SARS-CoV-2, was sustained during the development of brain organoids, especially in the somas of mature neurons, while remaining rare in neural stem cells. However, pseudotyped SARS-CoV-2 was observed in the axon of neurons, which lack ACE2. Neural infectivity of SARS-CoV-2 pseudovirus did not increase in proportion to viral load, but only 10% of neurons were infected. Our findings demonstrate that brain organoids provide a useful model for investigating SARS-CoV-2 entry into the human brain and elucidating the susceptibility of the brain to SARS-CoV-2.