Development and implementation of Crispr/Cas diagnostic methods for field detection of Citrus bark cracking viroid (CBCVd) in hops (Humulus lupulus L.)

Over the past 40 years, the world has been shaken by a number of epidemics caused by viruses such as HIV, SARS, H1N1, Ebola, Zika and most recently, SARS-CoV-2, known to all as the causative agent of COVID-19. In all cases, the public health response to the present viral threat was hampered by a lack of rapid, accessible, and reliable molecular diagnostic testing. Despite living in a highly developed society that has created many technological marvels, the rapid and accurate identification of pathogens still represents a critical point in both human and plant virology. The use of CRISPR system represents great potential in plant diagnostics, due to its sensitivity, specificity, speed, flexibility, robustness and ease of use. CRISPR in combination with RT-RPA and lateral flow assays (LFA) allows fast detection, which is also suitable for field use. The main goal of the project is to develop the CRISPR/Cas-RT-RPA method for the detection of CBCVd viroid, which will significantly shorten the analysis time and at the same time be sensitive, cost-effective and suitable for many applications, the most important of which will be field use. The CBCVd viroid causes an incurable severe hop stunt disease, which affected almost 500 ha (out of 1500 ha) of hop fields in Slovenia in the period 2007-2020. To control it, more than 330 ha have been grubbed-up, but it is still spreading to new hop fields. RT-PCR is currently in routine use, but does not allow detection of latent infections, so there is a need for a rapid and sensitive method. In previous research, we have developed sensitive molecular methods that allow multiplex detection and quantification, but are not flexible and fast. The development of new methods is a necessary step towards detection of new outbreaks and limitation of further spread that our expertise the most embrace. Within the project, we will develop three methods: RT-RPA (1), CRISPR/Cas (P2) and CRISPR/Cas-RT-RPA (P3), the use of which will be adjusted according to the needs of the analyses. We will first develop the RT-RPA method, which will be used to amplify the viroid, and at the same time we will optimize the detection, so we will be able to use it separately, as method with lower sensitivity (P1). In P1, we will design and test primers, compare different kits, and optimize the reaction. In the second part of the project (P2) we will develop a CRISPR/Cas system, design and test several crRNA molecules, ssDNA probes and compare Cas proteins. The method will be optimized without prior amplification, so that it can also be used as a separate system. The main part of the project will be the optimization of CRISPR/Cas-RT-RPA in which we will combine both developed methods into one (P3). An important goal of the project will be development of crude extract method (P4). By testing and optimizing various buffers and methods for homogenization of plant tissue, we will be able to determine CBCVd viroid by CRISPR/Cas-RT-RPA without RNA isolation. In P4, we will test LFAs, which will enable fast detection of CBCVd viroid with CRISPR/Cas-RT-RPA both in the laboratory and in the field. Using all the improvements, we expect to reduce the analysis time from 2 days, required for RT-PCR, to 1 hour. The developed method will therefore be introduced for field use, fast detection of outbreaks and testing of hop seedlings. In order to be able to use the CRISPR/Cas-RT-RPA method for routine testing, it will be validated accordingly (P5). The development of a method that will allow fast detection, without prior RNA isolation, while being sensitive and suitable for field use, will represent a major advance in hop research as well as plant diagnostics in general. The method will be used for fast detection of new outbreaks, testing of hop seedlings, evaluation of cultivar resistance and epidemiological studies. This will limit the further spread of the disease and mitigate the economic damage in hop growing.