Biology subjects

ZHENG, Y.

Publications and source records attributed to ZHENG, Y..

3 recordsLinked to original sources

bioRxiv2024

Discovery and Engineering of a New BvCas12a Nuclease for Mammalian Genome Editing and Nucleic Acid Detection

Cas12a is an RNA-guided endonuclease that has emerged as a powerful gene-editing tool. We have identified a novel Cas protein, BvCas12a, from Butyricimonas virosa with a 5-TYTN protospacer adjacent motif (PAM). BvCas12a exhibits double-stranded DNA cleavage activity in vitro and genome editing activity in eukaryotic cells. Though the editing efficiency of BvCas12a is marginally lower than that of AsCas12a, the editing specificity of BvCas12a in eukaryotic cells is comparable or superior to that of AsCas12a. Moreover, BvCas12a exhibits substantial collateral activity and can detect HPV DNA effectively and accurately in conjunction with isothermal amplification, highlighting its potential in nucleic acid diagnostics. Furthermore, we have engineered BvCas12a to create two variants, BvCas12a-R (N549R, T606P) and BvCas12a-RVR (N549R, K555V, C559R, T606P). These variants recognize expanded 5-YYN and 5-YN PAM in vitro, respectively. Additionally, they exhibit higher editing activity than wild-type BvCas12a and recognize 5-YYN PAM in vivo at all sites detected. In conclusion, we have identified a novel BvCas12a protein with high specificity and engineered two variants with broader PAM compatibility and improved genome editing efficiency. These findings offer a potent gene editing tool for application in scientific research, gene therapy, and nucleic acid diagnostics.

genomics↗

bioRxiv2024

Under pressure: altered endothelial flow response

Blood flow within the vasculature is a critical determinant of endothelial cell (EC) identity and functionality, yet the intricate interplay of various hemodynamic forces and their collective impact on endothelial and vascular responses are not fully understood. Specifically, the role of hydrostatic pressure in the EC flow response is understudied, despite its known significance in vascular development and disease. To address this gap, we developed in vitro models to investigate how pressure influences EC responses to flow. Our study demonstrates that elevated pressure conditions significantly modify shear-induced flow alignment and increase endothelial cell density. Bulk and single-cell RNA sequencing analyses revealed that, while shear stress remains the primary driver of flow-induced transcriptional changes, pressure modulates shear- induced signaling in a dose-dependent manner. These pressure-responsive transcriptional signatures identified in human ECs were conserved during the onset of circulation in early mouse embryonic vascular development, where pressure was notably associated with transcriptional programs essential to arterial and hemogenic EC fates. Our findings suggest that pressure plays a synergistic role with shear stress on ECs and emphasizes the need for an integrative approach to endothelial cell mechanotransduction, one that encompasses the effects induced by pressure alongside other hemodynamic forces.

bioengineering↗

bioRxiv2022

DNA packaging via hierarchical chromatin structures revealed by live-cell 3D imaging

Single-molecule localization microscopy is a powerful superresolution imaging technique to study biological questions by visualizing subcellular fine structures with nanometer-scale precision. However, its application in live-cell imaging studies has been impeded by the paucity of self-blinking organic fluorophores that enable high spatiotemporal resolution and labeling/localization density at a moderate laser intensity. Herein, we report a self-blinking Si-rhodamine dye 6-HESiR with a suitably increased "ON" fraction and a fluorogenic self-blinking dsDNA probe 6-HoeHESiR as a powerful tool for 3D superresolution imaging of native chromatin in eukaryotes without the use of photoswitching buffer and high laser intensity. With the probe 6-HoeHESiR, 3D superresolution imaging of in vitro reconstituted nucleosomal arrays and chromatin fibers yielded results consistent with EM analysis. Similar euchromatin and heterochromatin structures were visualized in fixed and live cells with high spatiotemporal resolution and labeling density, providing the first live-cell evidence for a hierarchical model of chromatin organization. 3D imaging results obtained in the presence of selective inhibitors of histone deacetylases also corroborate chromatin fiber decompaction upon hyperacetylation of histones.

cell biology↗