CRISPR SHERLOCK system for CPLX1
Our goal was to create an indicative multi-component biosensor encompassing the CRISPR SHERLOCK system to detect an early stage Parkinson’s disease biomarker. Whilst the aggregation of the protein alpha-synuclein (SNCA) aids the characterisation of Parkinson’s Disease, this was not specific to Parkinson’s Disease and is associated with a group of neurodegenerative disorders called alpha - synucleinopathies . This led to our discovery of the mRNA CPLX1, which is suggested to specifically decrease in concentration during the development of Parkinson’s Disease. Through designing a CRISPR RNA that targets a constitutive exon of the CPLX1 mRNA, we are able to activate Cas13a in the presence of CPLX1 mRNA. Cas13a can be used for trans-cleavage of silenced fluorescent reporters, resulting in different degrees of fluorescence at different time points depending on the percentage activation state of Cas13a.
Results of the CRISPR SHERLOCK system for CPLX1 can be found here!
Glutathione is an antioxidant that protects cells by preventing cellular damage and disruption of metabolic pathways . It is strongly associated to Parkinson’s Disease; the lower the glutathione levels, the more severe the occurrence of Parkinson’s Disease. Glutathione is degraded into cysteine and glycine by the glutathione degradation pathway. Whilst glutathione cannot be detected by transcription factors, cysteine and glycine can be. We will develop a glycine biosensor by utilising the glycine degradation pathway native to E. coli. This will involve using transcription factor gcvA which becomes active when glycine levels elevate, which binds to the gcvBp promoter resulting in transcription. In a natural system, this would result in transcription of the small RNA molecule gcvB; however, we have swapped this gene for one that encodes a fluorescent reporter RFP.
Results of the glutathione biosensor can be found here!
Joy Milne was the basis of our inspiration for this project and her abilities led to the research at Manchester University. The research group involved in discovering the volatile biomarkers associated to what Milne could “smell” released a paper earlier in the year. Eicosane increased significantly in people living with PD which due to seborrhoeic dermatitis, a potential premotor feature associated with the increased risk of Parkinson’s Disease . We aimed to develop a biosensor to detect eicosane levels related to seborrhoeic dermatitis as a presymptom of Parkinson’s Disease. As eicosane was found on the surface of the skin, we aimed to convert this into 1-eicosanol. Utilising an alcohol dehydrogenase promoter for the expression of a fluorescence reporter RFP.
Results of the eicosane biosensor can be found here!
- Kim WS, Kågedal K and Halliday G. Alpha-synuclein biology in Lewy body diseases. Alzheimers Res Ther. 2014;6:73
- Forman HJ, Zhang H, Rinna A. Glutathione: an overview of it's protective roles, measurement, and biosynthesis. Mol Aspects Med. 2009;30:1-12
- Tanner C, Albers K, Goldman S, Fross R,Leimpeter A, Klingman J, Eeden S. Seborrheic dermatitis and risk of future Parkinson's Disease (PD). Neurology. 2012;78:S24.001