To Combine CRISPR/Cas9 Genome Editing, Nanotech and Chemical Genetics toward in vivo Protein Kinase Analysis

该项目正在开发一种活体“激酶分析”，它广泛适用于蛋白激酶。AKT1 Akt2是我们最初的型号。这两个蛋白有很高的序列同源性，但表现出功能性 发育、糖尿病、癌症等方面的差异。我们的动机是确定是否不同于 AKT1和Akt2底物的特异性和/或偏好是一种潜在的机制。该项目将培育 至少两名本科生和一名研究生进入生物医学下一阶段 职业生涯。 简而言之，我们正在结合碳纳米管传递ATP类似物，CRISPR/Cas9基因编辑和 化学遗传学，为了能够区分细胞的内源性磷酸化反应 从人类基因组中编码的那些&gt；500激酶中获得感兴趣的激酶--体内研究的先决条件 “激活法”。我们正在使用我们中的一位(蔡博士)开发的协议来使用碳纳米管来 ATP类似物A*TP-g-S的细胞内递送。AKT1和Akt2基因正在进行CRISPR/Cas9编辑，以 将看门人的蛋氨酸突变为甘氨酸，以扩大他们的ATP结合口袋。那是, 分别锚定Akt1M→G和Akt2M→G的两个HCT116细胞变体的创建。根据 由Kevin Shokat博士开发的成熟的化学遗传学方法，只有突变的激酶能够 适应体积较大的三磷酸腺苷类似物A*TP-g-S，这是由于其修改后的较大的腺嘌呤部分。类比 还具有被硫代磷酸取代的三磷酸腺苷g-磷酸。因此，纳米管提供了 类似物由突变的激酶专门用来标记其底物的硫代磷酸，这两种底物都是已知的 以及那些新的和以前没有被怀疑的。然后，可以使用该标记作为句柄来隔离 底物，然后可以用LC-MS/MS进行系统和无偏倚的鉴定和定量 举止。也就是说，底物的光谱--所有底物的特性和相对丰度--将是 分别为Akt1和Akt2识别。比较底物的Akt1和Akt2光谱 HCT116细胞将揭示不同底物的特异性和/或偏好是否是潜在的 它们的功能差异的机制。我们完成这些研究任务的目标是：1) CRISPR/Cas9编辑Akt1和Akt2以扩大其在HCT116人细胞中的ATP结合口袋，即， Akt1M→G细胞和Akt2M→G细胞的建立；2)鉴定和区分底物的光谱 这两种蛋白在HCT116细胞中均有表达。 意义：这一结果将为研究蛋白激酶建立一个新的、通用的范式。AKT1和 Akt2长期以来一直是药物和治疗开发的目标。识别他们各自的身份 底物光谱将有助于指导相关人类的进一步开发和改进工作 疾病。此外，该项目为学生提供了一个很好的跨学科平台来整合 课堂教育和研究活动。

This project is developing an in vivo “kinase assay”, which is widely applicable to protein kinases. Akt1 and Akt2 is our initial model. The two kinases share high sequence homology, but display functional difference in development, diabetics, cancers, and et al. Our motivation is to determine whether different Akt1 and Akt2 substrate specificity and/or preference are an underlying mechanism. The project will nurture the growth of at least two undergraduate and one graduate students to the next stage of their biomedical career. Briefly, we are combining carbon nanotube delivery of ATP analogues, CRISPR/Cas9 gene editing and chemical-genetics, in order to be able to distinguish the endogenous phosphorylation reactions of the kinase of interest from those of the > 500 kinases encoded in the human genome – a prerequisite for in vivo “kinase assay”. We are using a protocol developed by one of us (Dr. Cai) to use carbon nanotubes for intracellular delivery of the ATP analog A*TP-g-S. Akt1 and Akt2 genes are being CRISPR/Cas9 edited to mutate the gatekeeper methionine to a glycine, in order to enlarge their ATP binding pockets. That is, creation of two variants of HCT116 cells anchoring Akt1M→G and Akt2M→G, respectively. According to the well-established chemical-genetic method developed by Dr. Kevan Shokat, only the mutant kinase is able to accommodate the bulky ATP analogue A*TP-g-S due to its modified bulkier adenine moiety. The analogue also has the ATP g-phosphate group replaced by a thiophosphate group. Consequently, nanotube delivered analogue is used exclusively by the mutant kinase to thiophosphate-tag its substrates, both known ones and those that are new and previously unsuspected. The tag can then be used as a handle to isolate the substrates, which can then be identified and quantified with LC-MS/MS in a systematic and unbiased manner. That is, a spectrum of substrates – the identity and relative abundance of all substrates – will be identified respectively for Akt1 and Akt2. Comparing the Akt1 and the Akt2 spectrums of substrates in HCT116 cells will reveal whether differential substrate specificities and/or preferences are an underlying mechanism for their functional differences. Our aims to accomplish these research tasks are: 1) to CRISPR/Cas9 edit Akt1 and Akt2 to enlarge their ATP binding pockets in the HCT116 human cells, i.e., creation of Akt1M→G cells and Akt2M→G cells; and 2) to identify and differentiate the spectrums of substrates of the two kinases in HCT116 cells. Significance: The results will set a new and general paradigm for studying protein kinases. Akt1 and Akt2 have long been targeted for drug and therapeutic development. Identification of their respective spectrum of substrates will help guide further development and improvement efforts for relevant human diseases. Additionally, the project provides an excellent interdisciplinary platform for students to integrate classroom education and research activities.