Investigating the role of PURA in neurodevelopment using CRISPR/Cas9 saturation gene editing.

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Saturation genome editing, which uses CRISPR/Cas9 to systematically assay the effect of every possible amino acid substitution in a particular protein, has been successfully applied to investigate the function of important disease genes such as BRCA1. It is most successfully applied to the study of small proteins, where the total number of amino acid substitutions is small. PURA is one such small, single-exon gene which encodes an extremely well-conserved single stranded DNA and RNA binding protein with high affinity for purine-rich repeat, (GGN)n regions. PURA has multiple functions including in regulation of DNA replication, transcriptional activation, mRNA transport and translational repression. It is involved in neuronal proliferation, dendrite maturation and localised mRNA translation in neurons during neuronal development, cell growth and proliferation. De novo mutations in PURA cause a severe neurodevelopmental condition, first described by us, and PURA has been suggested to play a role in ALS pathology. However, how its function is affected in disease remains unclear. Saturation genome editing of PURA in cell lines, and further characterisation in frogs will provide insights into PURA dysfunction in disease, identifying therapeutic avenues for treatment of neurodevelopmental and neurodegenerative disease. This work will provide proof of principle that this approach can be used for understanding neurodevelopmental and neurodegenerative genes. The student will learn valuable cutting-edge molecular genetic techniques and join an experienced and supportive supervisory team with expertise in CRISPR/Cas gene editing, RNA biology and PURA-associated neurodevelopmental disorders.