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.

项目摘要/摘要： 拟议程序的长期目标是（i）在异国情调中发现V CRISPR/CAS系统 具有独特特征的微生物，（ii）阐明了对规则和机制的更深入的理解 CRISPR/CAS并将其应用于工程和改进其活动，（iii）将其应用于基因编辑和 各种疾病的诊断应用。虽然II型CRISPR/CAS9是研究最多的基因组 编辑工具，V型CRISPR/CAS12系统是最多样化的，具有各种功能不同的范围 单生cas12a-k核酸酶正在作为基因组编辑和 核酸检测。中心假设是（i）由于V型系统是最多样化的，相对相对 较新的，只有少数几个（<5％）的系统进行了适当的研究，而这些系统的绝大多数 经过研究且特征不佳，因此，对这些系统的系统研究将使新颖 基因组工程，染色质成像，基础编辑和诊断的工具。 （ii）对 工程crrna和CAS的序列结构活动关系将使能够发展 改进的基因组分析，组合酶学和基因组的多重策略的工具 编辑和诊断应用。虽然V CRISPR/CAS类型分享了交付差的挑战，但基因低 校正效率，以及与其他基于CRISPR的基因组编辑工具相关的高靶向裂解， 它们对诊断应用具有正交和重叠的挑战，包括a）低催化性 效率或敏感性不佳，b）不匹配或特异性较低的高耐受性，c）部署的稳定性不佳， d）缺乏控制，需要多重。在第一个程序中，V CRISPR/CAS类型的新型直系同源 系统将通过外来微生物的元基因组采矿来发现，这些微生物可以在极端繁衍生息 条件随后表达和纯化CAS酶和CRRNA，探针的鉴定 相邻的基序需求和以高通量方式测试酶活性。在第二个 程序，CRRNA和CAS蛋白将通过各种策略进行修改，以提高目标特异性和 活动。修改的CRRNA和CAS将允许阐明CRISPR/CAS系统的机制 进一步允许改进靶DNA或RNA的检测。最后，整合新颖和工程的CRISPR/CAS 使用模型系统将能够开发多路复用技术，这些技术将产生更广泛的影响 在检测和治疗多种疾病中。 Pi的实验室已经很重要 在所有三个提议的计划中的贡献，都有几个关键的合作和出版物，并得到了准备 开展成功的研究和培训计划。最大化支持的预期结果 早期研究人员的研究人员研究奖（MIRA）包括建立综合性 研究，理解和设计独特的CRISPR/CAS系统的研究计划以及专业的应对 他们的诊断和治疗传染病，癌症和遗传疾病的应用中的问题。