CRISPR/Pepper‐tDeg: A Live Imaging System Enables Non‐Repetitive Genomic Locus Analysis with One Single‐Guide RNA

Advanced Science, Journal Year: 2024, Volume and Issue: 11(32)

Published: June 26, 2024

Abstract CRISPR‐based genomic‐imaging systems have been utilized for spatiotemporal imaging of the repetitive genomic loci in living cells, but they are still challenged by limited signal‐to‐noise ratio (SNR) at a non‐repetitive locus. Here, an efficient system is proposed, termed CRISPR/Pepper‐tDeg, engineering CRISPR sgRNA scaffolds with degron‐binding Pepper aptamers binding fluorogenic proteins fused Tat peptide derived degron domain (tDeg). The target‐dependent stability switches both and protein allow this to image telomeres sensitively 5‐fold higher SNR than conventional CRISPR/MS2‐MCP using “always‐on” fluorescent tag. Subsequently, CRISPR/Pepper‐tDeg applied simultaneously label track two different loci, centromeres, cells combining systems. Given further improved split design, extended sequence only one aptamer insertions. Neither complex design nor difficult plasmid construction required, greatly reducing technical barriers define organization dynamics thus demonstrating large application potential biological research, clinical diagnosis therapy.

Language: Английский

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Engineered CRISPR/Cas Ribonucleoproteins for Enhanced Biosensing and Bioimaging

Analytical Chemistry, Journal Year: 2025, Volume and Issue: unknown

Published: March 11, 2025

CRISPR-Cas systems represent a highly programmable and precise nucleic acid-targeting platform, which has been strategically engineered as versatile toolkit for biosensing bioimaging applications. Nevertheless, their analytical performance is constrained by inherent functional activity limitations of natural CRISPR/Cas systems, underscoring the critical role molecular engineering in enhancing capabilities. This review comprehensively examines recent advancements ribonucleoproteins (RNPs) to enhance capabilities advanced detection cellular imaging. We explore innovative strategies developing enhanced RNPs, including Cas protein through mutagenesis fusion techniques, guide RNA via chemical structural modifications. Furthermore, we evaluate these RNPs' applications sensitive biomarker live-cell genomic DNA monitoring, while analyzing current challenges prospective developments RNP bioimaging.

Language: Английский

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Establishment of CRISPR‐STAR System to Realise Simultaneous Transcriptional Activation and Repression in Yarrowia lipolytica

Microbial Biotechnology, Journal Year: 2025, Volume and Issue: 18(4)

Published: April 1, 2025

ABSTRACT The ability to regulate gene expression in multiple directions is crucial maximise the production of microbial cell factories. However, lack a regulatory tool that can simultaneously activate and repress genes restricts manipulation diversity Yarrowia lipolytica , which an industrial workhorse for bioproduction. To address this issue, we developed CRISPR s caffold RNAs (scRNAs)‐mediated t ranscriptional ctivation r epression (CRISPR‐STAR) platform. Firstly, evaluated different methods bidirectional regulation using on both endogenous synthetic promoters Y. chose utilisation orthogonal scRNAs recruit activation inhibition domains. Secondly, CRISPR‐STAR was optimised by introduction alternative dCas proteins, scRNA structures activators. 2.6‐fold 54.9‐fold were achieved promoters, respectively, when VPR transcriptional activator recruited via MS2 hairpin. repression several successfully achieved, with levels ranging from 3% 32%, MXI1 repressor PP7 Finally, applied enhance fatty alcohol activating FAR (encodes acyl‐CoA reductase) PEX10 integral membrane protein required peroxisome biogenesis matrix import). Compared non‐targeting control, bidirectionally regulated strain showed 55.7% increase yield 778.8 mg/L. Our findings demonstrate platform enables multi‐mode genes, offering engineering opportunities improve productive performance Y .

Language: Английский

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Aptamer–ODN Chimeras: Enabling Cell-Specific ODN Targeting Therapy

Cells, Journal Year: 2025, Volume and Issue: 14(10), P. 697 - 697

Published: May 12, 2025

Oligonucleotides (ODNs) such as siRNA, saRNA, and miRNA regulate gene expression through a variety of molecular mechanisms show unique potential in the treatment genetic diseases rare diseases, but their clinical application is still limited by efficiency delivery system, especially problem insufficient targeting extrahepatic tissues. As homologous nucleic acid molecules, aptamers have become key tool to improve targeted ODNs. Aptamer-ODN chimeras can not only bind multiple proteins on cell surface with high specificity selectivity, they also internalize into cells. Furthermore, outperform traditional systems terms cost-effectiveness chemical modification flexibility. This review systematically summarizes origin progress aptamer-ODN chimera therapy, discusses some innovative design strategies, proposes views future direction chimeras.

Language: Английский

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