The accessibility to CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein) genetic tools has given rise to applications beyond site-directed genome editing for the detection of DNA and RNA. These tools include precise diagnostic detection of human disease pathogens, such as SARS-CoV-2 and Zika virus. Despite the technology being rapid and cost-effective, the use of CRISPR/Cas tools in the surveillance of the causative agents of wildlife diseases has not been prominent. This study presents the development of a minimally invasive, field-applicable and user-friendly CRISPR/Cas-based biosensor for the detection of Pseudogymnoascus destructans (Pd), the causative fungal agent of white-nose syndrome (WNS), an infectious disease that has killed more than five million bats in North America since its discovery in 2006. The biosensor assay combines a recombinase polymerase amplification (RPA) step followed by CRISPR/Cas12a nuclease cleavage to detect Pd DNA from bat dermal swab and guano samples. The biosensor had similar detection results when compared to quantitative PCR in distinguishing Pd-positive versus negative field samples. Although bat dermal swabs could be analysed with the biosensor without nucleic acid extraction, DNA extraction was needed when screening guano samples to overcome inhibitors. This assay can be applied to help with more rapid delineation of Pd-positive sites in the field to inform management decisions. With further optimization, this technology has broad translation potential to wildlife disease-associated pathogen detection and monitoring applications.

中文翻译：

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一种微创、现场适用的 CRISPR/Cas 生物传感器，有助于检测破坏性假裸囊菌（蝙蝠白鼻综合症的致病真菌）

CRISPR/Cas（成簇规则间隔短回文重复序列/CRISPR相关蛋白）遗传工具的可及性已经催生了除定点基因组编辑之外的用于检测DNA和RNA的应用。这些工具包括对人类疾病病原体（例如 SARS-CoV-2 和寨卡病毒）的精确诊断检测。尽管该技术快速且具有成本效益，但 CRISPR/Cas 工具在野生动物疾病病原体监测中的应用并不突出。这项研究展示了一种微创、现场适用且用户友好的基于 CRISPR/Cas 的生物传感器的开发，用于检测破坏性假裸囊菌( Pd )，白鼻综合征 (WNS) 的致病真菌，白鼻综合征 (WNS) 是一种传染病，自 2006 年发现以来，已在北美杀死了超过 500 万只蝙蝠。该生物传感器检测结合了重组酶聚合酶扩增 (RPA) 步骤和 CRISPR/Cas12a 核酸酶切割，可检测蝙蝠真皮拭子和鸟粪样本中的Pd DNA。与定量 PCR 相比，生物传感器在区分Pd阳性与阴性现场样品方面具有相似的检测结果。虽然蝙蝠真皮拭子可以在不提取核酸的情况下用生物传感器进行分析，但在筛选鸟粪样本以克服抑制剂时需要提取DNA。该测定可用于帮助在现场更快速地描绘Pd阳性位点，为管理决策提供信息。通过进一步优化，该技术在野生动物疾病相关病原体检测和监测应用中具有广泛的转化潜力。