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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. 
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while...
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Optimising Guide RNA Production for Multiplexed Cas9-Targeted Nanopore Sequencing to Detect Pathogens.

Gus R McFarlane1, Kim Whitaker2, Krista L Plett2

  • 1NSW Department of Primary Industries, Elizabeth Macarthur Agricultural Institute, Menangle, NSW, 2568, Australia. gus.mcfarlane@dpi.nsw.gov.au.

Molecular Biotechnology
|September 7, 2025
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Summary
This summary is machine-generated.

We evaluated six guide RNA (gRNA) production methods for Nanopore Cas9-targeted sequencing (nCATS) to detect plant pathogens. One method excelled, enabling accurate detection across tested ranges with high sequencing coverage.

Keywords:
CRISPR-Cas9Guide RNAMultiplexingNCATSNanoporePathogenPestTargeted

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Area of Science:

  • Molecular Biology
  • Genomics
  • Agricultural Science

Background:

  • Rapid and accurate detection of pathogens and pests is critical for agriculture, biosecurity, and human health.
  • Nanopore Cas9-targeted sequencing (nCATS) offers a promising approach for pathogen and pest identification.
  • Optimizing guide RNA (gRNA) production is essential for the efficiency and cost-effectiveness of nCATS.

Purpose of the Study:

  • To investigate and compare the sensitivity and practicality of six distinct gRNA production methods for nCATS.
  • To assess the impact of different gRNA production strategies on multiplexed detection of wheat fungal pathogens.
  • To identify the most effective and cost-efficient gRNA production method for nCATS applications.

Main Methods:

  • Generated libraries of eight guide RNAs (gRNAs) per method targeting conserved regions (5.8S rRNA-ITS2-28S rRNA) in five wheat fungal pathogens.
  • Evaluated commercially synthesized and in-house in vitro-transcribed gRNAs.
  • Assessed gRNA yield, integrity, performance in nCATS, and associated costs.

Main Results:

  • Significant differences were observed among the six gRNA production methods in terms of yield, integrity, performance, and cost.
  • The optimal gRNA production method successfully identified all target sequences across a range of 0.96 to 8.4 pg.
  • Achieved high sequencing coverage, ranging from 66X to 2037X, demonstrating robust detection capabilities.

Conclusions:

  • The choice of gRNA production method significantly impacts the success of nCATS for pathogen and pest detection.
  • The study identified a highly effective gRNA production strategy for nCATS, crucial for developing reliable diagnostic tools.
  • Findings provide valuable insights for optimizing cost-effective and sensitive gRNA production for widespread nCATS implementation in agriculture and biosecurity.