Digital CRISPR/Cas12a-based platform for precise quantification of telomerase activity

Luo, Xinyi; Wang, Ke; Wei, Qidong; Yu, Zhaoning; Chen, Lei; Zhou, Jianhua; Wang, Jiasi

doi:10.1016/j.snb.2023.134374

Cited by 3 publications

(2 citation statements)

References 22 publications

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“…An advantage will be the possibility to determine with extreme resolution the length of telomeres of individual specific chromosomes using fluorescence spectroscopy (as few as 100 bp [81]), single-molecule real-time (SMRT) sequencing-based methods (at nucleotide resolution; [82]), or nanopore sequencing (as few as 75 bp at specific ends of chromosomes; [83]). Moreover, efforts are underway to quantitatively determine the TL and telomerase activity by harnessing the CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated proteins) system [84]. Regarding telomere dynamics in livestock, the goals and experimental design of the project should be the overriding factors in deciding which of the current and emergent methods to apply.…”

Section: Methods To Determine Tlmentioning

confidence: 99%

Telomere Dynamics in Livestock

Zhang,

Baker,

Welsh

et al. 2023

Biology

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Telomeres are repeated sequences of nucleotides at the end of chromosomes. They deteriorate across mitotic divisions of a cell. In Homo sapiens this process of lifetime reduction has been shown to correspond with aspects of organismal aging and exposure to stress or other insults. The early impetus to characterize telomere dynamics in livestock related to the concern that aged donor DNA would result in earlier cell senescence and overall aging in cloned animals. Telomere length investigations in dairy cows included breed effects, estimates of additive genetic control (heritability 0.12 to 0.46), and effects of external stressors on telomere degradation across animal life. Evaluation of telomeres with respect to aging has also been conducted in pigs and horses, and there are fewer reports of telomere biology in beef cattle, sheep, and goats. There were minimal associations of telomere length with animal productivity measures. Most, but not all, work in livestock has documented an inverse relationship between peripheral blood cell telomere length and age; that is, a longer telomere length was associated with younger age. Because livestock longevity affects productivity and profitability, the role of tissue-specific telomere attrition in aging may present alternative improvement strategies for genetic improvement while also providing translational biomedical knowledge.

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Section: Methods To Determine Tlmentioning

confidence: 99%

Telomere Dynamics in Livestock

Zhang,

Baker,

Welsh

et al. 2023

Biology

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“…Clustered regularly interspaced short palindromic repeats (CRISPR)-based detection is a novel and promising approach for nucleic acid diagnostics. − The CRISPR-associated (Cas) enzyme relies on CRISPR RNA (crRNA) that directs sequence-specific recognition of target nucleic acids to form a ribonucleoprotein (RNP) complex and then amplifies signal via the trans-cleavage of fluorogenic reporters. − The test result can be read by measuring the released reporter by using a fluorescence detector. Though CRISPR-based systems have been repurposed for a variety of applications, strategies that only rely on the trans-cleavage activity suffer from limited sensitivity in clinical application.…”

mentioning

confidence: 99%

DECODE: Contamination-Free Digital CRISPR Platform for Point-of-Care Detection of Viral DNA/RNA

Li,

Yin,

Zheng

et al. 2024

ACS Sens.

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Rapid and precise nucleic acid testing at the pointof-care (POC) is essential for effective screening and management of infectious diseases. Current polymerase-based molecular diagnostics often suffer from potential cross-contamination issues, particularly in POC settings. Here, we introduce DECODE, a contamination-free nucleic acid detection platform integrating digital microfluidics (DMF) for nucleic acid extraction and a digital CRISPR amplification-free assay for pathogen detection. The digital CRISPR assay demonstrates sensitivity, detecting target DNA and RNA in the reaction mixture at concentrations of 10 and 5 copies/μL, respectively. Leveraging DMF-extracted samples enhances the performance of the digital CRISPR amplification-free assay. DECODE offers a sample-to-result workflow of 75 min using compact devices. Validation studies using clinical samples confirm DECODE's robust performance, achieving 100% sensitivity and specificity in detecting HPV18 from cervical epithelial cells and influenza A from nasal swabs. DECODE represents a versatile, contamination-free detection platform poised to enhance integrated public health surveillance efforts.

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Terminal Chemical Modifications of crRNAs Enable Improvement in the Performance of CRISPR-Cas for Point-of-Care Nucleic Acid Detection

Wan,

Li,

et al. 2024

Anal. Chem.

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CRISPR-Cas systems, harnessing their precise nucleic acid recognition via CRISPR RNA (crRNA), offer promise for the accurate testing of nucleic acids in the field. However, the inherent susceptibility of crRNA to degradation poses challenges for accurate detection in lowresource settings. Here, we utilized the chemically modified crRNA for the CRISPR-Cas-based assay (CM-CRISPR). We found that the extension and chemical modification to crRNA significantly enhanced the trans-cleavage activity of LbCas12a. The chemically modified crRNA was resistant to degradation, and CM-CRISPR showed superior detection capability in complex environments. CM-CRISPR could be combined with recombinase polymerase amplification (RPA) and applied in a droplet digital platform, enabling attomolar-level sensitivity. We also developed a portable and automated device for a digital CRISPR assay, which is amenable to point-ofcare testing (POCT). The extraction-free procedure was integrated with this assay to streamline the workflow, and clinical samples were successfully detected. This work finds a simple and efficient way to improve the performance of CRISPR-Cas and develops a portable platform for POCT, representing a significant advance toward practical applications of CRISPR-based diagnostics.

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Digital CRISPR/Cas12a-based platform for precise quantification of telomerase activity

Cited by 3 publications

References 22 publications

Telomere Dynamics in Livestock

Telomere Dynamics in Livestock

DECODE: Contamination-Free Digital CRISPR Platform for Point-of-Care Detection of Viral DNA/RNA

Terminal Chemical Modifications of crRNAs Enable Improvement in the Performance of CRISPR-Cas for Point-of-Care Nucleic Acid Detection

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