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

基本信息

批准号：
10222734
负责人：
Leifu Chang
金额：
$ 33.17万
依托单位：
PURDUE UNIVERSITY
依托单位国家：
美国
项目类别：
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/10222734
关键词：
Adopted Archaea Bacteria Base Pairing Biochemical Biological Assay Biomedical Research CRISPR/Cas technology Cleaved cell Clustered Regularly Interspaced Short Palindromic Repeats Complex Coupled Cryoelectron Microscopy DNA DNA Integration DNA Repair DNA Repair Pathway Data Deoxyribonucleases Dependence Development Endoribonucleases Family Genetic Genome Goals Guide RNA Heteroduplex DNA In Vitro Knowledge Life Mediating Mobile Genetic Elements Molecular Mutation Nature Nucleic Acid Cleavage Nucleic Acids Plasmids Process Proteins RNA RNA Editing RNA Processing RNA Sequences Resolution Ribonucleases Safety Site-Directed Mutagenesis Specificity Structural Models Structure System Technology Testing Transposase Ursidae Family Virus Work adaptive immunity base clinical application complex data ds-DNA endonuclease genome editing human disease improved insight novel particle precise genome editing recruit targeted nucleases thermostability tool virtual

项目摘要

PROJECT SUMMARY 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 of these systems on endogenous host DNA repair pathways, hindering clinical application. Exploration of alternative CRISPR-Cas systems in nature not only offers an opportunity to overcome those challenges but may also inspire new applications. Structural and biochemical characterizations of CRISPR-Cas systems are critical for understanding 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 type V and transposon-associated CRISPR-Cas systems, which provide essential knowledge for safer and more reliable application in treating human disease. 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 (type I-F Cascade and Cas12k). As revealed in our preliminary data, Cas12i accommodates a longer crRNA-DNA heteroduplex than currently used Cas effectors, thus potentially improving specificity for genome editing. The RNA-guided RNase Cas12g is compact and thermostable, highlighting its potential for RNA editing and RNA targeting. Furthermore, type I-F Cascade 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以及这些系统对内源性宿主DNA修复途径，阻碍了临床应用。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V型和介导的靶核酸识别和切割的潜在机制转座子相关 CRISPR-Cas 系统，为更安全、更可靠提供必要的知识在治疗人类疾病中的应用。在本提案中，我们将研究四个方面的分子机制新发现的CRISRP-Cas系统，涵盖DNA靶向（Cas12i）、RNA靶向（Cas12g）和 CRISPR RNA 引导的 DNA 转座（I-F 级联和 Cas12k 型）。正如我们的初步数据所示， Cas12i 可容纳比目前使用的 Cas 效应器更长的 crRNA-DNA 异源双链体，因此有可能提高基因组编辑的特异性。 RNA 引导的 RNase Cas12g 结构紧凑且耐热，强调了其在 RNA 编辑和 RNA 靶向方面的潜力。此外，I-F Cascade 型和 Cas12k direct 用于RNA引导DNA转座的转座机器，开启基因组编辑的新范式独立于 DNA 修复途径。 1