Optimising Guide RNA Production for Multiplexed Cas9-Targeted Nanopore Sequencing to Detect Pathogens

In agriculture, biosecurity, and human health, the rapid and accurate detection of pathogens and pests is crucial. Our study investigates the sensitivity and practicality of six guide RNA (gRNA) production methods for use in Nanopore Cas9-targeted sequencing (nCATS), focusing on their implications for multiplexed pathogen and pest detection. Each production method generated a library of eight gRNAs capable of excising ~ 1.6 kb fragments of the 5.8S_rRNA-ITS2-28S_rRNA regions of five economically significant wheat fungal pathogens. Through comparative analysis, we evaluated the efficacy of commercially synthesised and in-house in vitro-transcribed gRNAs, assessing their impact on sequencing enrichment outcomes. Our findings highlight differences amongst the methods in terms of gRNA yield, integrity, performance, and costs. Our best performing gRNA production method was able to successfully identify all target sequences across the 0.96 to 8.4 pg ranges we tested with coverage ranging from 66 to 2037X. This study highlights the challenges and opportunities in implementing nCATS for multiplexed pathogen and pest detection, offering insights into the development of cost-effective and reliable gRNA production strategies for nCATS.