Standard approaches for dCas9-based modification of gene expression are limited in the ability to multiplex targets, establish streamlined cassettes, and utilize commonly studied Pol II promoters. In this work, we repurpose the dCas9-VPR activator to act as a dual-mode activator/repressor that can be programmed solely on the basis of target position at gene loci. Furthermore, we implement this approach using a streamlined Pol II-ribozyme system that allows expression of many sgRNAs from a single transcript. By "stepping" dCas9-VPR within the promoter region and ORF we create graded activation and repression (respectively) of target genes, allowing precise control over multiplexed gene modulation. Expression from the Pol II system increased the net amount of sgRNA production in cells by 3.88-fold relative to the Pol III SNR52 promoter, leading to a significant improvement in dCas9-VPR repression strength. Finally, we utilize our Pol II system to create galactose-inducible switching of gene expression states and multiplex constructs capable of modulating up to 4 native genes from a single vector. Our approach represents a significant step toward minimizing DNA required to assemble CRISPR systems in eukaryotes while enhancing the efficacy (greater repression strength), scale (more sgRNAs), and scope (inducibility) of dCas9-mediated gene regulation.
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http://dx.doi.org/10.1021/acssynbio.7b00163 | DOI Listing |
Endocr Relat Cancer
January 2025
X Zheng, Thyroid and Neck Tumor, Tianjin Medical University Cancer Institute and Hospital, Tianjin, China.
Anaplastic Thyroid Cancer (ATC) is an aggressive form of cancer with poor prognosis, heavily influenced by its tumor immune microenvironment (TIME). Understanding the cellular and gene expression dynamics within the TIME is crucial for developing targeted therapies. This study analyzes the immune microenvironment of ATC and Papillary Thyroid Cancer (PTC) using single-cell RNA sequencing (scRNA-seq).
View Article and Find Full Text PDFJ Agric Food Chem
January 2025
School of Food Science and Technology, International Joint Laboratory on Food Safety, Synergetic Innovation Center of Food Safety and Quality Control, Jiangnan University, Wuxi, Jiangsu 214122, P.R. China.
Aflatoxins (AFs), notorious mycotoxins that pose significant risks to human and animal health, make biodegradation extremely crucial as they offer a promising approach to managing and reducing their harmful impacts. In this study, we identified a manganese peroxidase from (Mnp) through protein similarity analysis, which has the capability to degrade four AFs (AFB, AFB, AFG, and AFG) simultaneously. The gene encoding this enzyme was subject to codon optimization, followed by cold shock induction expression using the pColdII vector, leading to the soluble expression of manganese peroxidase (Mnp) in .
View Article and Find Full Text PDFTransl Stroke Res
January 2025
Department of Neurology, McGovern Medical School, The University of Texas Health Science Center at Houston, 6431 Fannin Street, Houston, TX, 77030, USA.
The role of chromatin biology and epigenetics in disease progression is gaining increasing recognition. Genes that escape X chromosome inactivation (XCI) can impact neuroinflammation through epigenetic mechanisms. Our previous study has suggested that the X escapee genes Kdm6a and Kdm5c are involved in microglial activation after stroke in aged mice.
View Article and Find Full Text PDFHum Cell
January 2025
Infectious Disease Laboratory, Chengdu Public Health Clinical Center, Chengdu, 610061, People's Republic of China.
Hepatocellular carcinoma (HCC) is a primary malignant neoplasm exhibiting a high mortality rate. Taxifolin is a naturally occurring flavonoid compound that exhibits a range of pharmacological properties. The effects of taxifolin on HCC remain largely unexplored.
View Article and Find Full Text PDFMar Biotechnol (NY)
January 2025
Liaoning Key Laboratory of Marine Animal Immunology, Dalian Ocean University, Dalian, 116023, China.
The Pacific oyster Crassostrea gigas is rich in taurine, a conditionally essential amino acid functioning in anti-oxidation, anti-inflammation, anti-aging, osmoregulation, and neuromodulation. Breeding oyster varieties with enhanced taurine content is significant to meet people's demand for high-quality oysters. In the present study, polymorphisms in the oyster cysteamine dioxygenase (CgADO) gene that encodes the central enzyme of the cysteamine pathway for taurine synthesis were investigated, and their association with taurine content was assessed in the Changhai (CH) and Qinhuangdao (QHD) populations.
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