Discovery and engineering of CRISPR/Cas systems

CRISPR/Cas系统的发现和工程

• 批准号: 10664042
• 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/10664042
• 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; Malignant Neoplasms; Metagenomics; Mining; Orthologous Gene; Outcome; Publications; RNA; RNA Editing; Research; Research Personnel; Running; Specificity; Structure-Activity Relationship; System; Technology; Testing; Training Programs; United States National Institutes of Health; base editing; 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.

项目总结/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。 具有独特功能的微生物，（ii）阐明了更深层次的理解的规则和机制， CRISPR/Cas并将其应用于工程化和改善其活性，以及（iii）将它们应用于基因编辑和 用于诊断一系列疾病。尽管II型CRISPR/Cas9是研究最多的基因组， 作为一种编辑工具，V型CRISPR/Cas 12系统是最多样化的，具有广泛的功能上的不同， 单效应子Cas 12 a-k核酸酶正在成为用于基因组编辑和 核酸检测核心假设是：（i）由于V型系统是最多样化和相对 较新的，只有少数（<5%）的这些系统已得到适当的研究，而绝大多数这些系统， 是研究不足和缺乏特点，因此，这些系统的系统研究将使新的 基因组工程、染色质成像、碱基编辑和诊断工具。(ii)更深入地了解 通过工程化crRNA和Cas的序列-结构-活性关系将使得能够开发 用于宏基因组分析、组合酶学和基因组多重策略的改进工具 编辑和诊断应用程序。虽然V型CRISPR/Cas面临着递送不良、基因表达水平低和基因表达水平低的挑战， 校正效率，以及与其他基于CRISPR的基因组编辑工具相关的高脱靶切割， 它们在诊断应用中具有正交和重叠的挑战，包括a）低催化活性， 效率或灵敏度差，B）错配的高耐受性或低特异性，c）部署的稳定性差， 以及d）缺乏控制，这是多路复用所希望的。在第一个项目中，V型CRISPR/Cas的新型直系同源物 通过对可以在极端条件下繁衍生息的外来微生物进行宏基因组挖掘，将发现系统 条件，然后表达和纯化Cas酶和crRNA，鉴定前间区序列 相邻基序要求，以及以高通量方式测试酶活性。在第二 根据该计划，crRNA和Cas蛋白将用各种策略进行修饰，以提高靶特异性， 活动修饰的crRNA和Cas将允许阐明CRISPR/Cas系统的机制， 进一步允许改进的靶DNA或RNA的检测。最后，将新型和工程化的CRISPR/Cas 与模型系统相结合，将能够开发具有更广泛影响的多路复用技术 检测和治疗多种疾病。私家侦探的实验室已经在 在所有三个拟议的计划与几个关键的合作和出版物的贡献，并准备 成功开展研究和培训项目最大限度地提高妇女地位委员会支助的预期成果 早期研究者研究奖（MIRA）包括建立一个综合的 研究计划，以发现，理解和工程师独特的CRISPR/Cas系统和解决主要的 它们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。