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

饱和基因组编辑使用CRISPR/Cas9系统地分析特定蛋白质中每种可能的氨基酸替代的影响，已成功应用于研究重要疾病基因（如BRCA1）的功能。它最成功地应用于小蛋白质的研究，其中氨基酸取代的总数很少。PURA就是这样一个小的单外显子基因，它编码一种非常保守的单链DNA和RNA结合蛋白，对富含嘌呤的重复（GGN）n区域具有高亲和力。PURA具有多种功能，包括调控DNA复制、转录激活、mRNA转运和翻译抑制。在神经元发育、细胞生长和增殖过程中，它参与神经元的增殖、树突成熟和神经元的局部mRNA翻译。PURA的新生突变引起严重的神经发育疾病，这是我们首次描述的，PURA被认为在ALS病理中起作用。然而，其功能在疾病中如何受到影响仍不清楚。细胞系中PURA的饱和基因组编辑，以及青蛙中PURA的进一步表征，将为疾病中的PURA功能障碍提供见解，为治疗神经发育和神经退行性疾病确定治疗途径。这项工作将为这种方法可以用于理解神经发育和神经退行性基因提供原理证明。学生将学习有价值的尖端分子遗传技术，并加入一个经验丰富、支持的监督团队，他们在CRISPR/Cas基因编辑、RNA生物学和pura相关的神经发育障碍方面具有专业知识。

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.