Discovery and engineering of CRISPR/Cas systems

项目摘要/摘要： 拟议计划的长期目标是：(I)发现外来环境中的V型CRISPR/CAS系统 具有独特特征的微生物，(Ii)阐明了对生物多样性的规律和机制的更深层次的理解 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。虽然II型CRISPR/Cas9是研究最多的基因组 编辑工具，类型V CRISPR/CAS12系统是最多样化的，具有广泛的不同功能 单效应子Cas12a-k核酸酶作为下一代基因组编辑和 核酸检测。中心假设是：(一)由于V型系统是最多样化和相对 较新的是，只有少数(5%)这些系统得到了适当的研究，而这些系统中的绝大多数 研究不足，特征不佳，因此，对这些系统的系统研究将使 用于基因组工程、染色质成像、碱基编辑和诊断的工具。(二)深入认识 工程crRNA和Cas的序列-结构-活性关系将使开发 改进的元基因组分析工具、组合酶学和基因组多重策略 编辑和诊断应用程序。而V型CRISPR/Cas则面临着传递差、基因低的挑战 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， 它们在诊断应用方面既具有正交性又具有重叠性，包括：)低催化 效率或敏感性差，b)对错配的容忍度高或特异性低，c)部署的稳定性差， 以及d)缺乏控制，这对于多路传输来说是可取的。在第一个程序中，V型CRISPR/CAS的新同源基因 系统将通过对外来微生物的元基因组挖掘而被发现，这些微生物可以在极端情况下茁壮成长 Cas酶和crRNAs的表达、纯化条件及原核表达产物的鉴定 相邻基序要求，以及高通量方式的酶活性测试。在第二个 计划中，crRNAs和Cas蛋白将采用各种策略进行修饰，以提高靶标特异性和 活动。经过修饰的crRNAs和Cas将有助于阐明CRISPR/Cas系统的机制，从而 进一步允许改进对目标DNA或RNA的检测。最后，集成了新型和工程化的CRISPR/CAS 使用模型系统将使多路技术的发展能够产生更广泛的影响 在检测和治疗各种疾病方面发挥了重要作用。私家侦探的实验室已经取得了重大进展 在所有三个拟议的计划中做出贡献，并与几个关键的合作伙伴和出版物合作，并准备 进行成功的研究和培训计划。最大限度支持的预期结果 早期调查人员研究奖(MIRA)包括建立一个综合性的 研究计划，以发现、了解和设计独特的CRISPR/CAS系统并解决主要问题 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。

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.