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

将 CRISPR/Cas9 基因组编辑、纳米技术和化学遗传学结合起来进行体内蛋白激酶分析

基本信息

批准号：
9813823
负责人：
Degeng Wang
金额：
$ 8.41万
依托单位：
TEXAS TECH UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-07-01 至 2022-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9813823
关键词：
AKT1 gene AKT2 gene Adenine Apoptosis Binding Biological Assay CRISPR/Cas technology Carbon Nanotubes Cells Development Drug Targeting Education Environment Gatekeeping Generations Genes Genetic Genomics Glycine Goals Growth HCT116 Cells Human Human Cell Line Human Genome In Vitro Knock-in Knockout Mice Malignant Neoplasms Metabolism Methionine Methods Mitotic Cell Cycle Modeling Motivation Mutate Mutation Nanotechnology Nanotubes Neoplasm Metastasis PTEN gene Phosphorylation Phosphotransferases Process Protein Kinase Protocols documentation Reaction Research Research Activity Research Personnel Sequence Homology Signal Transduction Students Substrate Specificity Testing Variant Western Blotting analog career chemical genetics diabetic drug development education research genome editing graduate student hands on research homologous recombination human disease improved in vivo inhibitor/antagonist innovation inorganic phosphate interest mutant next generation power analysis preference promoter screening therapeutic development thiophosphate undergraduate student

项目摘要

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.

本项目正在开发一种广泛适用于蛋白激酶的体内“激酶测定法”。AKT1 Akt 2是我们的初始模型。这两种激酶具有高度的序列同源性，但显示出功能性。在发展，糖尿病，癌症等方面的差异。我们的动机是确定是否不同 Akt 1和Akt 2底物特异性和/或偏好性是潜在的机制。该项目将培养至少两名本科生和一名研究生的成长到他们的生物医学的下一阶段事业简而言之，我们正在将ATP类似物的碳纳米管递送，CRISPR/Cas9基因编辑和化学遗传学，为了能够区分内源性磷酸化反应的从人类基因组中编码的> 500种激酶中筛选出感兴趣的激酶-这是体内研究的先决条件。 “激酶测定”。我们正在使用我们中的一位（蔡博士）开发的协议，将碳纳米管用于 ATP类似物A*TP-g-S的细胞内递送。Akt 1和Akt 2基因正在被CRISPR/Cas9编辑，将门控甲硫氨酸突变为甘氨酸，以扩大它们的ATP结合口袋。也就是说，创建分别锚定Akt 1 M →G和Akt 2 M →G的HCT 116细胞的两种变体。根据由Kevan Shokat博士开发的成熟的化学遗传方法，只有突变激酶能够由于其经修饰的较大的腺嘌呤部分，其容纳较大的ATP类似物A*TP-g-S。模拟也具有被硫代磷酸基团取代的ATP g-磷酸基团。因此，类似物仅由突变激酶用于硫代磷酸标记其底物，这两种底物都是已知的以及那些新的和以前未被怀疑的。然后，标记可以用作句柄，底物，然后可以用LC-MS/MS以系统和无偏的方式进行鉴定和定量。方式也就是说，底物的光谱-所有底物的身份和相对丰度-将是分别鉴定了Akt 1和Akt 2。比较Akt 1和Akt 2底物的光谱， HCT 116细胞将揭示差异底物特异性和/或偏好是否是潜在的功能差异的机制。我们完成这些研究任务的目标是：1） CRISPR/Cas9编辑Akt 1和Akt 2以扩大它们在HCT 116人类细胞中的ATP结合口袋，即， Akt 1 M →G细胞和Akt 2 M →G细胞的创建;和2）鉴定和区分底物的光谱在HCT 116细胞中的两种激酶。意义：本研究结果将为蛋白激酶的研究提供一个新的、通用的范式。akt 1和 Akt 2长期以来一直是药物和治疗开发的目标。识别各自的底物谱将有助于指导相关人类疾病此外，该项目为学生提供了一个优秀的跨学科平台来整合课堂教学和研究活动。