CRISPR Screen Multiplexing for Uncharacterised Region Function (SMURF)

药物开发过程的初步阶段需要依赖于潜在的细胞药物靶标，如蛋白质，以在特定疾病环境中进行验证。这一发展阶段被称为“靶标验证”，它为特定靶标的调节可能给患者带来治疗益处提供了证据。然而，目前的靶标验证方法经常识别假阳性靶标，然后进一步进行药物开发。因此，开发的候选药物化合物可能很少或根本没有治疗效果。这导致开发过程的大量成本和时间延迟，并使患者没有适当的治疗选择。减少有缺陷的目标假设数量的一种方法是在验证过程中改进目标的结构和功能数据。CRISPR-Cas基因编辑系统来源于细菌和古细菌，其天然功能是作为对病毒感染的免疫防御。现在，这种方法已被用于编辑哺乳动物细胞的基因组，并已被证明在研究和治疗环境中都是无价的。最近发表的基于crispr的筛选数据表明，基因序列的某些部分对蛋白质功能并不重要，因此不太可能作为特定的药物靶点赋予任何治疗益处。该项目的重点是确定CRISPR系统是否适用于高通量筛选格式，并能够产生与目标结构和功能相关的可靠数据。这将允许CRISPR-Cas在药物发现中更大规模地用于验证具有临床潜力的新型未表征药物靶点。这个项目有两个不同的目的。首先，将在靶蛋白内进行精确的DNA编辑，以诱导对特定抑制剂化合物的差异敏感性。其次，将进行更大规模的随机基因敲除筛选，以确定对目标蛋白功能至关重要的核苷酸序列。随着表达必需的CRISPR-Cas组件的稳定细胞系的设计和开发，将使用基于细胞的检测来验证协议，以识别受干扰的蛋白质功能。还需要对目标进行基因测序，以说明已经引入的确切DNA变化。最初，这些研究将在一个简单的细胞系系统中进行，然后再发展到更复杂的人类癌细胞系。周期蛋白依赖性激酶2最初将被用作模型靶点，因为它的结构和功能已经在文献中得到了很好的表征。备选模型目标也将在项目过程中进行分析。如果成功，该研究可以通过帮助鉴定蛋白质上相关药物靶标位点，为制药行业提供改进的靶标验证方法。最终，这种方法可能有助于识别对危及生命的疾病有效的药物。

A preliminary stage of the drug development process requires a dependency on a potential cellular drug target, such as a protein, to be validated in a specific disease setting. This development stage is termed 'target validation' and it provides evidence that modulation of a specific target may confer therapeutic benefit to patients. However current methods in target validation frequently identify false positive targets which are then pursued further for drug development. As a result, candidate drug compounds are developed that may confer little or no therapeutic benefit. This incurs substantial cost and time delays to the development process and leaves patients without appropriate treatment options. One approach to reducing the number of flawed target hypotheses is to improve the structural and functional data on targets during their validation. The CRISPR-Cas gene editing system has been derived from bacteria and archaea where its native function is as an immune defence against viral infection. This has now been adapted to edit the genome of mammalian cells and has proven invaluable for use within both research and therapeutic settings. Recently published CRISPR-based screening data has demonstrated that certain sections of a gene sequence are not critical to protein function and are therefore unlikely to confer any therapeutic benefit as specific drug target sites. The focus of this project is to determine whether the CRISPR system is amenable to high-throughput screening formats and capable of producing reliable data relating to a target's structure and function. This would allow CRISPR-Cas to be utilised on a larger scale within drug discovery for the validation of novel uncharacterised drug targets that have clinical potential.This project has two distinct aims. Firstly, precise DNA editing will be undertaken within a target protein to induce differential sensitivity to specific inhibitor compounds. Secondly, larger randomised gene knockout screens will be conducted to identify those nucleotide sequences that are critical to the function of a target protein. Following the design and development of stable cell lines which express the requisite CRISPR-Cas components, protocols will be validated using cell-based assays to identify perturbed protein function. Genetic sequencing of the target will also be required to illustrate the exact DNA changes that have been introduced. Initially these studies will be conducted in a simplified cell line system before progressing to more complex human cancer cell lines. Cyclin-dependent kinase 2 will initially be used as a model target since its structure and function have been well characterised in the literature. Alternative model targets will also undergo analysis during the course of the project. If successful, this research may provide the pharmaceutical industry with improved methods for target validation by aiding the identification of relevant drug target sites on proteins. Ultimately, this approach may facilitate the identification of effective drugs for life-threatening diseases.