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

项目总结 CRISPR-Cas系统通过利用引导RNA和 核酸内切酶效应器，用于特异性识别和切割侵袭性核酸。特异性DNA靶向 和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，正在给生物医学研究和其他领域带来革命性的变化。然而，安全 人们的担忧是因为非目标基因组编辑以及这些系统对 内源性宿主DNA修复途径，阻碍临床应用。CRISPR-CAS替代方案的探讨 自然界中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 申请。CRISPR-CAS系统的结构和生化特性对 了解它们的机制，并将它们重新用于精确的基因组编辑。我们的长期目标是 解开V型和TYPE介导的靶向核酸识别和切割的机制 转座子相关的CRISPR-CAS系统，为更安全和更可靠提供必要的知识 在治疗人类疾病方面的应用。在这项提案中，我们将研究四个分子机制 新发现的CRISRP-CAS系统，包括DNA靶向(Cas12i)、RNA靶向(Cas12g)和 CRISPR RNA引导的DNA转座(I-F型级联和Cas12k型)。根据我们的初步数据显示， 与目前使用的CAS效应器相比，Cas12i容纳了更长的crRNA-DNA异源双链，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，耐热性好， 突出了它在RNA编辑和RNA靶向方面的潜力。此外，I-F级联和Cas12k DIRECT RNA引导DNA转座的转座机制，开启了基因组编辑的新范式 不依赖于DNA修复途径。 1

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