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Related Concept Videos

RNA-seq03:21

RNA-seq

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RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases. 
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Rapid Amplification of cDNA Ends, or RACE, is one of the most effective methods to obtain a full-length cDNA from an mRNA sequence between a known internal region to the unknown sequence at the 5’ or 3’ end. The unknown region is cloned in the cDNA by a gene-specific primer that binds the known end, and a hybrid primer that attaches a predefined anchor sequence to the unknown end of the cDNA. The sequence in between is amplified by PCR with an anchor primer and a gene-specific...
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Updated: May 5, 2026

Field-Deployable Candidatus Liberibacter asiaticus Detection Using Recombinase Polymerase Amplification Combined with CRISPR-Cas12a
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A Modular and Programmable Cas13d Platform for RNA Single Nucleotide Variant Detection.

Zeyu Wang1, Jiahao Li1, Zhuying Yue1,2

  • 1State Key Laboratory of Systems Medicine For Cancer, Shanghai Cancer Institute, Renji Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China.

Advanced Science (Weinheim, Baden-Wurttemberg, Germany)
|May 4, 2026
PubMed
Summary
This summary is machine-generated.

This study introduces a configurable CRISPR-Cas13d diagnostic tool for precise single-nucleotide variant detection in RNA. The framework enables ultra-sensitive, amplification-free detection down to 0.6% variant allele fraction, improving cancer diagnostics.

Keywords:
CRISPR platformCas13d RNA diagnosticsprecision oncologyprogrammablesingle nucleotide variant detection

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • CRISPR-based diagnostics show promise but struggle with single-nucleotide variant (SNV) discrimination due to targetability constraints and specificity-sensitivity trade-offs.
  • Existing methods often require flanking sequences and face challenges in distinguishing between wild-type and mutant alleles without compromising assay performance.

Purpose of the Study:

  • To develop a flexible CRISPR-Cas13d framework for enhanced SNV detection in RNA.
  • To engineer a Cas13d system with improved allelic discrimination and sensitivity for diverse diagnostic applications.

Main Methods:

  • Developed a scenario-guided Cas13d framework with pre-defined operating modes.
  • Mapped mismatch-sensitive windows for rule-based crRNA design to enhance allelic discrimination.
  • Engineered a miniaturized Cas13d scaffold by inserting RNA binding domains (RBDs) to restore assay performance.

Main Results:

  • Achieved ultra-sensitive, amplification-free detection of ~0.6% variant allele fraction (VAF) using a dual-RBD variant and optimized crRNAs.
  • Established a robust amplified mode with optional loop-mediated isothermal amplification for broader low-VAF detection.
  • Demonstrated high concordance in classifying mutation status (KRAS G12D, IDH1 R132C, BRAF V600E) in 45 clinical tumor RNA specimens.

Conclusions:

  • The configurable Cas13d toolkit and rule-guided strategy enable CRISPR-based RNA SNV diagnostics tailored to specific performance objectives.
  • The system offers significant potential for sensitive and accurate detection of clinically relevant mutations in oncology.
  • Validated sub-1% detection capabilities in a clinical-matrix spike-in, highlighting its utility without pre-amplification.