CRISPR-Mediated Protein Tagging with Nanoluciferase to Investigate Native Chemokine Receptor Function and Conformational Changes

© 2020 The Authors G protein-coupled receptors are a major class of membrane receptors that mediate physiological and pathophysiological cellular signaling. Many aspects of receptor activation and signaling can be investigated using genetically encoded luminescent fusion proteins. However, the use of these biosensors in live cell systems requires the exogenous expression of the tagged protein of interest. To maintain the normal cellular context here we use CRISPR/Cas9-mediated homology-directed repair to insert luminescent tags into the endogenous genome. Using NanoLuc and bioluminescence resonance energy transfer we demonstrate fluorescent ligand binding at genome-edited chemokine receptors. We also demonstrate that split-NanoLuc complementation can be used to investigate conformational changes and internalization of CXCR4 and that recruitment of β-arrestin2 to CXCR4 can be monitored when both proteins are natively expressed. These results show that genetically encoded luminescent biosensors can be used to investigate numerous aspects of receptor function at native expression levels

A citizen science model for implementing statewide educational DNA barcoding.

Our aim was to develop a widely available educational program in which students conducted authentic research that met the expectations of both the scientific and educational communities. This paper describes the development and implementation of a citizen science project based on DNA barcoding of reptile specimens obtained from the Museums Victoria frozen tissue collection. The student program was run by the Gene Technology Access Centre (GTAC) and was delivered as a "one day plus one lesson" format incorporating a one-day wet laboratory workshop followed by a single lesson at school utilising online bioinformatics tools. The project leveraged the complementary resources and expertise of the research and educational partners to generate robust scientific data that could be analysed with confidence, meet the requirements of the Victorian state education curriculum, and provide participating students with an enhanced learning experience. During two 1-week stints in 2013 and 2014, 406 students mentored by 44 postgraduate university students participated in the project. Students worked mainly in pairs to process ~200 tissue samples cut from 53 curated reptile specimens representing 17 species. A total of 27 novel Cytochrome Oxidase subunit 1 (CO1) sequences were ultimately generated for 8 south-east Australian reptile species of the families Scincidae and Agamidae

Subtle changes in the levels of BCL-2 proteins cause severe craniofacial abnormalities

Apoptotic cell death removes unwanted cells and is regulated by interactions between pro-survival and pro-apoptotic members of the BCL-2 protein family. The regulation of apoptosis is thought to be crucial for normal embryonic development. Accordingly, complete loss of pro-survival MCL-1 or BCL-XL (BCL2L1) causes embryonic lethality. However, it is not known whether minor reductions in pro-survival proteins could cause developmental abnormalities. We explored the rate-limiting roles of MCL-1 and BCL-XL in development and show that combined loss of single alleles of Mcl-1 and Bcl-x causes neonatal lethality. Mcl-1(+/-); Bcl-x(+/-) mice display craniofacial anomalies, but additional loss of a single allele of pro-apoptotic Bim (Bcl2l11) restores normal development. These findings demonstrate that the control of cell survival during embryogenesis is finely balanced and suggest that some human craniofacial defects, for which causes are currently unknown, may be due to subtle imbalances between pro-survival and pro-apoptotic BCL-2 family members