Discovery and engineering of CRISPR/Cas systems

CRISPR/Cas 系统的发现和工程

• 批准号: 10511620
• 负责人: Piyush K Jain
• 金额: $ 36.72万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10511620
• 关键词: Address; Award; BCAR1 gene; Biological Models; CRISPR/Cas technology; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Communicable Diseases; Communities; DNA; Detection; Development; Diagnosis; Diagnostic; Discipline; Disease; Engineering; Enzymatic Biochemistry; Enzymes; Genes; Genetic Diseases; Genome engineering; Image; Institutes; Metagenomics; Mining; Orthologous Gene; Outcome; Publications; RNA; RNA Editing; Research; Research Personnel; Research Training; Running; Specificity; Structure-Activity Relationship; System; Technology; Testing; Training Programs; United States National Institutes of Health; base; base editing; cancer genetics; combinatorial; gene correction; genome editing; improved; microorganism; next generation; novel; nuclease; nucleic acid detection; programs; tool

PROJECT SUMMARY/ABSTRACT: The long-term objectives of the proposed program are to (i) discover type V CRISPR/Cas systems in exotic microorganisms with unique features, (ii) elucidate a deeper understanding of the rules and mechanisms of CRISPR/Cas and apply it for engineering and improving its activity, and (iii) apply them for gene editing and diagnostic applications for a range of diseases. Although the type II CRISPR/Cas9 is the most studied genome editing tool, the type V CRISPR/Cas12 systems are the most diverse with a wide range of functionally distinct single-effector Cas12a-k nucleases that are emerging as next-generation tools for both genome editing and nucleic acid detection. The central hypothesis is that (i) since the type V systems are most diverse and relatively newer, only a handful (<5%) of these systems have been properly studied, while a vast majority of these systems are understudied and poorly characterized and therefore, a systematic study of these systems will enable novel tools for genome engineering, chromatin imaging, base editing, and diagnostics. (ii) A deeper understanding of the sequence-structure-activity relationship by engineering crRNA and Cas will enable the development of improved tools for metagenomic analysis, combinatorial enzymology, and multiplexing strategies for genome editing and diagnostic applications. While the type V CRISPR/Cas share challenges of poor delivery, low gene correction efficiency, and high off-target cleavage associated with other CRISPR-based genome editing tools, they possess both orthogonal and overlapping challenges for diagnostic applications, including a) low catalytic efficiency or poor sensitivity, b) high tolerance of mismatches or low specificity, c) poor stability for deployment, and d) lack of control, desirable for multiplexing. In the first program, novel orthologs of type V CRISPR/Cas systems will be discovered by metagenomic mining of exotic microorganisms that can thrive at extreme conditions followed by expression and purification of Cas enzymes and crRNAs, identification of protospacer adjacent motif requirement, and testing of enzymatic activity in a high-throughput fashion. In the second program, crRNAs and Cas proteins will be modified with various strategies to improve target specificity and activity. Modified crRNAs and Cas would allow elucidation of mechanisms of CRISPR/Cas systems that could further allow improved detection of target DNA or RNA. Finally, integrating novel and engineered CRISPR/Cas with model systems would enable the development of multiplexed technologies that will have broader impacts in the detection and treatment of a wide range of diseases. The PI's lab has already made significant contributions in all three proposed programs with several key collaborations and publications and is poised to run a successful research and training program. The expected outcomes of the support from the Maximizing Investigators' Research Award (MIRA) for Early Stage Investigators include the establishment of an integrative research program to discover, understand, and engineer unique CRISPR/Cas systems and addressing of major problems in their applications for diagnosing and treating infectious diseases, cancers, and genetic disorders.

项目概要/摘要： 该计划的长期目标是 (i) 在外来物种中发现 V 型 CRISPR/Cas 系统 具有独特特征的微生物，（ii）阐明对微生物的规则和机制有更深入的了解 CRISPR/Cas 并将其应用于工程和提高其活性，以及​​ (iii) 将其应用于基因编辑和 一系列疾病的诊断应用。尽管 II 型 CRISPR/Cas9 是研究最多的基因组 编辑工具中，V 型 CRISPR/Cas12 系统是最多样化的，具有广泛的功能不同 单效应 Cas12a-k 核酸酶正在成为基因组编辑和基因组编辑的下一代工具 核酸检测。中心假设是 (i) 由于 V 型系统最为多样化且相对 较新的，只有少数（<5%）这些系统得到了适当的研究，而绝大多数这些系统 对这些系统的研究不足且特征较差，因此，对这些系统的系统研究将能够实现新颖的 用于基因组工程、染色质成像、碱基编辑和诊断的工具。 （二）更深入的了解 通过改造 crRNA 和 Cas 来确定序列-结构-活性关系将有助于开发 改进的宏基因组分析、组合酶学和基因组多重策略工具 编辑和诊断应用程序。而 V 型 CRISPR/Cas 则面临递送不良、基因含量低等挑战。 与其他基于 CRISPR 的基因组编辑工具相关的校正效率和高脱靶切割， 它们在诊断应用方面面临着正交和重叠的挑战，包括a）低催化 效率或灵敏度差，b) 错配容忍度高或特异性低，c) 部署稳定性差， d) 缺乏控制，对于多路复用来说是理想的。在第一个程序中，V 型 CRISPR/Cas 的新型直向同源物 通过对能够在极端条件下繁衍生息的外来微生物进行宏基因组挖掘，可以发现系统 Cas 酶和 crRNA 的表达和纯化、原型间隔子的鉴定 相邻主题的要求，以及以高通量方式测试酶活性。在第二个 计划中，crRNA 和 Cas 蛋白将通过各种策略进行修饰，以提高目标特异性和 活动。修饰的 crRNA 和 Cas 将有助于阐明 CRISPR/Cas 系统的机制 进一步改进了目标 DNA 或 RNA 的检测。最后，整合新颖且工程化的 CRISPR/Cas 模型系统将有助于开发具有更广泛影响的多重技术 用于检测和治疗多种疾病。 PI 实验室已经取得了重大成果 对所有三个拟议计划的贡献以及多项关键合作和出版物的贡献，并已做好准备 开展成功的研究和培训计划。最大化支持的预期成果 为早期研究人员提供的研究人员研究奖（MIRA）包括建立一个综合性研究中心 研究计划旨在发现、理解和设计独特的 CRISPR/Cas 系统并解决主要问题 其在诊断和治疗传染病、癌症和遗传性疾病的应用中存在的问题。