Molecular basis for CRISPR RNA-guided nucleic acid cleavage and DNA transposition

CRISPR RNA引导的核酸切割和DNA转座的分子基础

• 批准号: 10389613
• 负责人: Leifu Chang
• 金额: $ 9.89万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10389613
• 关键词: Adopted; Archaea; Bacteria; Biochemical; Biomedical Research; CRISPR/Cas technology; Cleaved cell; Clustered Regularly Interspaced Short Palindromic Repeats; DNA; DNA Repair Pathway; Dependence; Endoribonucleases; Goals; Guide RNA; Heteroduplex DNA; Knowledge; Mediating; Molecular; Nature; Nucleic Acid Cleavage; Nucleic Acids; RNA; RNA Editing; Ribonucleases; Safety; Specificity; Structure; System; Technology; Work; adaptive immunity; clinical application; genome editing; human disease; improved; precise genome editing; thermostability; tool

PROJECT SUMMARY The CRISPR-Cas systems provide adaptive immunity in bacteria and archaea by employing guide RNAs and endonuclease effectors to specifically recognize and cleave invasive nucleic acids. The specific DNA targeting and cleavage activities of CRISPR-Cas systems have been adopted and developed for genome editing and various other applications, which are revolutionizing biomedical research and beyond. However, safety concerns are raised because of off-target genome editing and the dependence on endogenous DNA repair pathways, hindering clinical applications of CRISPR-Cas systems. Exploration of alternative CRISPR-Cas systems in nature not only offers an opportunity to overcome those challenges, but may also inspires new applications. Structural and biochemical characterizations of CRISPR-Cas systems are critical for understanding of their mechanisms and repurposing them for precise genome editing. Our long-term goals are to unravel the mechanisms underlying target nucleic acid recognition and cleavage mediated by diverse CRISPR-Cas systems, which provide essential knowledge for safe and reliable use of this technology in treating human diseases. In this proposal, we will work on the molecular mechanisms for four newly discovered CRISRP-Cas systems, covering DNA targeting (Cas12i), RNA targeting (Cas12g) and CRISPR RNA guided DNA transposition (VcCascade and Cas12k). As revealed in our structure, Cas12i accommodates longer crRNA-DNA heteroduplex than currently used Cas effectors, thus potentially improve the specificity for genome editing. The RNA-guided RNase Cas12g is compact and thermostable, and thus potentially expand the toolkits for RNA editing and RNA-targeting applications. VcCascade and Cas12k direct transposition machinery for RNA-guided DNA transposition, opening a new paradigm for genome editing independent of DNA repair pathways. 1

项目概要 CRISPR-Cas系统通过使用引导RNA和 核酸内切酶效应子特异性识别和切割侵入性核酸。特异性 DNA 靶向 CRISPR-Cas系统的切割活性已被采用和开发用于基因组编辑和 各种其他应用正在彻底改变生物医学研究及其他领域。不过，安全 由于脱靶基因组编辑和对内源 DNA 修复的依赖而引起担忧 途径，阻碍了 CRISPR-Cas 系统的临床应用。替代 CRISPR-Cas 的探索 自然界的系统不仅提供了克服这些挑战的机会，而且还可能激发新的 应用程序。 CRISPR-Cas系统的结构和生化特征对于 了解它们的机制并重新利用它们进行精确的基因组编辑。我们的长期目标是 揭示不同介导的靶核酸识别和切割的潜在机制 CRISPR-Cas 系统，为安全可靠地使用该技术提供了必要的知识 治疗人类疾病。在本提案中，我们将研究四种新发现的分子机制 CRISRP-Cas系统，涵盖DNA靶向（Cas12i）、RNA靶向（Cas12g）和CRISPR RNA引导 DNA 转座（VcCascade 和 Cas12k）。正如我们的结构所示​​，Cas12i 可以容纳更长的时间 crRNA-DNA 异源双链体比目前使用的 Cas 效应器更复杂，因此有可能提高基因组的特异性 编辑。 RNA 引导的 RNase Cas12g 结构紧凑且热稳定，因此有可能扩展工具包 用于 RNA 编辑和 RNA 靶向应用。 VcCascade 和 Cas12k 直接换位机械 RNA引导的DNA转座，开启了独立于DNA修复的基因组编辑的新范式 途径。 1