CRISPR/Cas12a-based detection is a novel approach for the efficient, sequence-specific identification of viruses. Here we adopt the use of CRISPR/Cas12a to identify the tomato brown rugose fruit virus (ToBRFV), a new and emerging tobamovirus which is causing substantial damage to the global tomato industry. Specific CRISPR RNAs (crRNAs) were designed to detect either ToBRFV or the closely related tomato mosaic virus (ToMV). This technology enabled the differential detection of ToBRFV and ToMV. Sensitivity assays revealed that viruses can be detected from 15–30 ng of RT-PCR product, and that specific detection could be achieved from a mix of ToMV and ToBRFV. In addition, we show that this method can enable the identification of ToBRFV in samples collected from commercial greenhouses. These results demonstrate a new method for species-specific detection of tobamoviruses. A future combination of this approach with isothermal amplification could provide a platform for efficient and user-friendly ways to distinguish between closely related strains and resistance-breaking pathogens.

We studied the ecology of the Lesser Kestrel Falco naumanni , a globally threatened species, to identify the factors causing its gradual decline in Israel, in order to stabilize and possibly restore its numbers. Lesser Kestrels in Israel breed in colonies, both adjacent to human settlements (rural and urban) and in the countryside, where they are found especially on cliffs. In this study, observations of Lesser Kestrels were carried out in three different breeding areas: (1) an urban colony in Jerusalem, (2) a rural colony in the Alona district and (3) a cliff in the Judean desert (open landscape colony) about 10 km east of Jerusalem. The number of fledglings per nest in Jerusalem (1.91) was lower than in Alona (2.44) and the cliff (3.16). As this lower productivity was associated neither with the clutch size, with hatching success, which were similar in all three regions (about four eggs per clutch, and 80% hatched, respectively), nor with egg fertility, it probably reflects factors operating during the nestling phase. We suggest that the two main factors limiting food availability and causing nestling deaths in Jerusalem are the relatively long flight distances between the breeding and hunting sites, and the use of pesticides in the city parks and lawns. Stochastic events superimposed on such factors, such as the drought of 1999, which markedly reduced productivity, may cause pronounced fluctuations eventually affecting long-term population persistence.

The ability to create nanoscaffolds within living cells using DNA has the potential to become a powerful tool in synthetic biology. However, to date, genetically encoded DNA nanostructures are limited to simple architecture due to the lack of genetic parts that can produce multiple ssDNAs in a single bacterium. Here, we develop a system that overcomes this challenge by using a single oligo gene mimicking operons. This was achieved by converting a noncoding RNA into a long ssDNA that self-cleaves into multiple ssDNAs using R3-DNAzymes (DNAzyme-based operon). We demonstrate the ability to apply the DNAzyme-based operon to produce a four-ssDNA crossover nanostructure (25 nm) that recruits split YFPs when properly assembled. This system enables the formation of more complex DNA nanostructures in vivo and thus paves the way to further integrate the field of DNA nanotechnology into living bacteria for basic biology, bioengineering, and medicine applications.

Bactrocera zonata, a major fruit pest species, is gradually spreading west from its native habitat in East Asia. In recent years it has become a major threat to the Mediterranean area, with the potential of invading Europe, the Americas, and Australia. To prevent its spreading, monitoring efforts in plantation sites and border controls are carried out. Despite these efforts, and due to morphological similarities between B. zonata and other pests in relevant developmental stages, the monitoring process is challenging, time-consuming, and requires external assistance from professional labs. CRISPR-Cas12a genetic diagnostics has been rapidly developing in recent years and provides an efficient tool for the genetic identification of pathogens, viruses, and other genetic targets. Here we design a CRISPR-Cas12a detection assay that differentially detects two major pest species, B. zonata and Ceratitis capitata. Our easy-to-use and affordable assay employs a simple DNA extraction technique together with isothermal amplification, and Cas12a-based detection. We demonstrate the specificity and high sensitivity of this method, and its relevance for on-site applications. This method is highly modular, and the presented target design method can be applied to a wide array of pests. Contribution: G.P conceived the project, D.M.A, G.P and D.B designed the experiments and wrote the manuscript, and D.M.A and T.P performed the experiments. Acknowledgments: We thank Dr. Liat Gidron, Dr. Yoav Gazit, and Dr. David Nestel for fly supplies and fruitful discussions. We thank Dr. Karin Mittelman for her insightful comments on the manuscript.