Department of Chemistry, University at Albany, State University of New York, 1400 Washington Avenue, Albany, New York 12222, United States.
Published: July 2021
AI Article Synopsis
Article Abstract
CRISPR-Cas12a is a powerful platform for DNA-based diagnostics. The detection scheme relies on unselective shredding of a fluorescent ssDNA reporter upon target DNA recognition. To extend the reporter library beyond ssDNAs, we discovered a fluorescent reporter type using a dsDNA template. In this design, the fluorescence of the dsDNA reporter is quenched contact-quenching mechanism. Upon detection, the quenched fluorescence recovers with the activation Cas12a complex. Here, we compared the probing performance of two dsDNA reporters with two ssDNA reporters. The rate of the Cas12a trans-cleavage reaction was studied using one of the dsDNA reporters under different settings. The detection of different sizes of dsDNA or ssDNA targets was studied systematically under three different temperatures. Lower thresholds for ssDNA and dsDNA target size were identified. The mismatch tolerance and target specificity were examined for both ssDNA and dsDNA targets, separately. The probing performance of the dsDNA reporter was evaluated in a random DNA pool with and without target strands. We report that dsDNA can serve as a tunable fluorescence reporter template expanding the toolbox for Cas12a-based diagnostics.
Extracellular vesicles (EVs) are cell-secreted lipid bilayer delimited particles that mediate cellular communication. These tiny sacs of cellular information play an important role in cell communication and alter the physiological process under both normal and pathological conditions. As such, tracking EVs can provide valuable information regarding the basic understanding of cell communication, the onset of early malignancy, and biomarker discovery.
Aim: This study explored the effect and mechanism of MAFF and HDAC6 on renal fibrosis and inflammation in lupus nephritis (LN).
Methods: IL-33 treated renal epithelial cells and MRL/lpr mice were respectively used for and experiments. The expressions of HDAC6, MAFF, and KLF5 were measured in cells and renal tissues.
School of Science and Engineering, Shenzhen Institute of Aggregate Science and Technology, The Chinese University of Hong Kong, Shenzhen, Guangdong, China.
CRISPR diagnostics face challenges like low signal efficiency and sensitivity due to their reliance on single-stranded nucleic acid fluorescent reporters.
The new CrisprAIE system combines CRISPR/Cas reactions with innovative aggregation-induced emission luminogens (AIEgens) for more effective fluorescence and improved clinical detection of norovirus and SARS-CoV-2.
CrisprAIE shows a substantial increase in sensitivity—up to 270 times better—compared to traditional CRISPR diagnostics and has potential for broader application in improving detection methods across various CRISPR-based techniques.
Cell-free transcription-translation (TXTL) systems expressing genes from linear dsDNA enable the rapid prototyping of genetic devices while avoiding cloning steps. However, repetitive inclusion of a reporter gene is an incompressible cost and sometimes accounts for most of the synthesized DNA length. Here we present reporter systems based on split-GFP systems that reassemble into functional fluorescent proteins and can be used to monitor gene expression in TXTL.
Scientists are trying to improve gene editing in cells by using new techniques that help fix DNA more effectively.
They developed smaller proteins (SlugCas9 and AsCas12a) that make it easier to use these techniques compared to bigger proteins that were hard to handle.
Tests showed that these new methods worked better and achieved higher gene editing efficiency, making it easier for researchers to use gene editing in their studies.
