CRISPR (clustered regularly interspaced short palindromic repeats), an ancient defense mechanism used by prokaryotes to cleave nucleic acids from invading viruses and plasmids, is currently being harnessed by researchers worldwide to develop new point-of-need diagnostics. In CRISPR diagnostics, a CRISPR RNA (crRNA) containing a "spacer" sequence that specifically complements with the target nucleic acid sequence guides the activation of a CRISPR effector protein (Cas13a, Cas12a or Cas12b), leading to collateral cleavage of RNA or DNA reporters and enormous signal amplification. CRISPR function can be disrupted by some types of sequence mismatches between the spacer and target, according to previous studies. This poses a potential challenge in the detection of variable targets such as RNA viruses with a high degree of sequence diversity, since mismatches can result from target variations. To cover viral diversity, we propose in this study that during crRNA synthesis mixed nucleotide types (degenerate sequences) can be introduced into the spacer sequence positions corresponding to viral sequence variations. We test this crRNA design strategy in the context of the Cas13a-based SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) technology for detection of Crimean-Congo hemorrhagic fever virus (CCHFV), a biosafety level 4 pathogen with wide geographic distribution and broad sequence variability. The degenerate-sequence CRISPR diagnostic proves functional, sensitive, specific and rapid. It detects within 30-40 minutes 1 copy/μl of viral RNA from CCHFV strains representing all clades, and from more recently identified strains with new mutations in the CRISPR target region. Also importantly, it shows no cross-reactivity with a variety of CCHFV-related viruses. This proof-of-concept study demonstrates that the degenerate sequence-based CRISPR diagnostic is a promising tool of choice for effective detection of highly variable viral pathogens.
Download full-text PDF |
Source |
|---|---|
| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8939784 | PMC |
| http://dx.doi.org/10.1371/journal.pntd.0010285 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
A genome-wide atlas of human cell morphology.
Nat Methods
January 2025
Broad Institute of MIT and Harvard, Cambridge, MA, USA.
A key challenge of the modern genomics era is developing empirical data-driven representations of gene function. Here we present the first unbiased morphology-based genome-wide perturbation atlas in human cells, containing three genome-wide genotype-phenotype maps comprising CRISPR-Cas9-based knockouts of >20,000 genes in >30 million cells. Our optical pooled cell profiling platform (PERISCOPE) combines a destainable high-dimensional phenotyping panel (based on Cell Painting) with optical sequencing of molecular barcodes and a scalable open-source analysis pipeline to facilitate massively parallel screening of pooled perturbation libraries.
View Article and Find Full Text PDFProximity-activated guide RNA of CRISPR-Cas12a for programmable diagnostic detection and gene regulation.
Nucleic Acids Res
January 2025
Beijing Key Laboratory for Bioengineering and Sensing Technology, University of Science and Technology Beijing, Beijing 100083, China.
The flexibility and programmability of CRISPR-Cas technology have made it one of the most popular tools for biomarker diagnostics and gene regulation. Especially, the CRISPR-Cas12 system has shown exceptional clinical diagnosis and gene editing capabilities. Here, we discovered that although the top loop of the 5' handle of guide RNA can undergo central splitting, deactivating CRISPR-Cas12a, the segments can dramatically restore CRISPR function through nucleic acid self-assembly or interactions with small molecules and aptamers.
View Article and Find Full Text PDFA Pair of Fluorescent Probes Enabling Precise Diagnosis of Liver Cancer by Complementary Imaging.
ACS Cent Sci
January 2025
Department of Chemistry, Pohang University of Science and Technology (POSTECH), Pohang 37673, Republic of Korea.
Hepatocellular carcinoma (HCC) is by far the predominant malignant liver cancer, with both high morbidity and mortality. Early diagnosis and surgical resections are imperative for improving the survival of HCC patients. However, limited by clinical diagnosis methods, it is difficult to accurately distinguish tumor tissue and its boundaries in the early stages of cancer.
View Article and Find Full Text PDFSelective In Situ Analysis of Hepatogenic Exosomal microRNAs via Virus-Mimicking Multifunctional Magnetic Vesicles.
Adv Healthc Mater
January 2025
The Institute of Chinese Medicine of Nanjing University, Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing University Drum Tower Hospital Clinical College, Nanjing University of Chinese Medicine, Nanjing, 210008, China.
Drug-induced liver injury (DILI) is a common clinical problem with urgent respect to demanding early diagnosis. Exosomal miRNAs are reliable and noninvasive biomarkers for the early diagnosis of DILI. However, accurate and feasible detection of exosomal miRNAs is often hampered by the low abundance of miRNAs, inefficient exosome separation techniques, and the requirement for RNA extraction from large sample volumes.
View Article and Find Full Text PDFTopologically constrained DNA-mediated one-pot CRISPR assay for rapid detection of viral RNA with single nucleotide resolution.
EBioMedicine
January 2025
Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Tsinghua University, New Cornerstone Science Foundation, Beijing, 100084, China. Electronic address:
Background: The widespread and evolution of RNA viruses, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), highlights the importance of fast identification of virus subtypes, particularly in non-laboratory settings. Rapid and inexpensive at-home testing of viral nucleic acids with single-base resolution remains a challenge.
Methods: Topologically constrained DNA ring is engineered as substrates for the trans-cleavage of Cas13a to yield an accelerated post isothermal amplification.
Want AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!