Identifying essential long non-coding RNAs in cancer using CRISPRi-based dropout screens

Summary: Long non-coding RNAs (lncRNAs) are emerging as key regulators in the initiation, growth, and progression of cancer. High-throughput CRISPR-based techniques systematically assess the function of genes or regulatory elements present in the human genome. Here, we present a protocol for identifying essential lncRNAs in cancer using CRISPRi-based dropout screens. We describe steps to select target sites, design guide RNAs, and generate CRISPRi cell lines. We then detail the execution and analysis of CRISPRi-based dropout screens. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

An antisense RNA capable of modulating the expression of the tumor suppressor microRNA-34a

The microRNA-34a is a well-studied tumor suppressor microRNA (miRNA) and a direct downstream target of TP53 with roles in several pathways associated with oncogenesis, such as proliferation, cellular growth, and differentiation. Due to its broad tumor suppressive activity, it is not surprising that miR34a expression is altered in a wide variety of solid tumors and hematological malignancies. However, the mechanisms by which miR34a is regulated in these cancers is largely unknown. In this study, we find that a long noncoding RNA transcribed antisense to the miR34a host gene, is critical for miR34a expression and mediation of its cellular functions in multiple types of human cancer. We name this long noncoding RNA lncTAM34a, and characterize its ability to facilitate miR34a expression under different types of cellular stress in both TP53-deficient and wild-type settings

Area-wide management guidelines for Sterile Insect Technique developed through interdisciplinary modelling research

New technologies for insect pest management, such as Sterile Insect Technique (SIT), present an opportunity to reduce chemical use in agricultural landscapes. However, they require socio-economic and ecological evaluation to ensure they are effective. We present a decision-making framework for sterile Queensland fruit fly 'Qfly' (Bactrocera tryoni) release informed by both economic and biophysical models as part of an area-wide management (AWM) program with SIT. The conceptual framework presented here guided the development of economic and biophysical models to meet the objective of informing management strategies. Our economic modelling has provided an important assessment of the feasibility and opportunities for AWM and SIT at the scale of three case study regions in southern Australia (Sunraysia, Murray/Goulburn valley (MGV) and Riverland). We found that there are three potential economically-viable implementation strategies for AWM of Qfly including SIT: Outbreak eradication (and potentially maintenance of area freedom, should legislation allow); direct substitution for existing management techniques in spatially-isolated contexts (including in support of industry-specific market access); and more effective management in an urban context than existing ad hoc approaches. In terms of releases of sterile males, our biophysical research has generated valuable risk maps for three case study regions. An understanding of the spatial and temporal risk of Qfly occurrence for a given target region is essential to the development of a future tool or system for planning sterile fly releases, as well as for ongoing management and monitoring programs. As a minimum, knowledge of the land use, climatic suitability and host phenology in a region is required. Ideally, this should incorporate the construction of risk maps that can inform decision-making for releases. The spatial simulation modelling showed that complex landscapes, containing a higher diversity of fruiting hosts across different seasons, will pose greater challenges in achieving population suppression and adoption of AWM strategies in readiness for SIT. Urban areas pose a significant challenge, as these areas provide a reservoir of flies throughout the year with potential to move into nearby crops. Therefore, it is important that they are managed as part of an AWM approach (potentially employing SIT) in order to effectively suppress flies across a landscape. The model identifies potential hotspots and bottlenecks in space and time which can be used to develop more targeted and effective SIT release strategies. We show that an urban treatment is most effective when reducing populations in late winter/early spring before they can move into the agricultural area. Our work highlights the importance of a conceptual framework that provides a broad consideration of the economic, social, and biological feasibility of a SIT program prior to the release of sterile flies using modelling approaches, as well as an ongoing need to consider the socioeconomic and biophysical components of the system for sterile Qfly releases to be successful. This work led to the development of online guidelines for area-wide management (AWM) of Qfly incorporating Sterile Insect Technique (SIT) in Australia www.area-wide-management.com.au.</p