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

饱和基因组编辑利用CRISPR/Cas9系统地分析特定蛋白质中每一种可能的氨基酸替换的影响，已成功地应用于研究BRCA1等重要疾病基因的功能。它最成功地应用于小蛋白质的研究，在这些蛋白质中，氨基酸替换的总数很少。PURA是这样一个小的、单外显子基因，它编码一个非常保守的单链DNA和RNA结合蛋白，与富嘌呤重复序列(GGN)n区域具有高亲和力。PURA具有多种功能，包括调节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、树突成熟和局部的mRNA翻译。PURA基因的从头突变导致严重的神经发育状况，这是我们首先描述的，PURA已被认为在ALS病理中发挥了作用。然而，它的功能在疾病中是如何受到影响的仍不清楚。Pura在细胞系中的饱和基因组编辑，以及在青蛙中的进一步特征将提供对疾病中Pura功能障碍的洞察，确定治疗神经发育和神经退行性疾病的途径。这项工作将提供原则性证据，证明这种方法可以用于理解神经发育和神经退化基因。学生将学习有价值的尖端分子遗传学技术，并加入一个经验丰富的支持性监督团队，该团队拥有CRISPR/Cas基因编辑、RNA生物学和Pura相关神经发育障碍方面的专业知识。

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.