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

项目摘要 CRISPR-Cas系统通过采用向导RNA和免疫调节剂在细菌和古细菌中提供适应性免疫， 核酸内切酶效应物，特异性识别和切割侵入性核酸。特异性DNA靶向 已经采用并开发了CRISPR-Cas系统的切割活性用于基因组编辑， 各种其他应用，这是革命性的生物医学研究和超越。但是，安全 由于脱靶基因组编辑和这些系统对生物学的依赖， 内源性宿主DNA修复途径，阻碍临床应用。替代CRISPR-Cas的探索 自然界中的系统不仅提供了克服这些挑战的机会， 应用. CRISPR-Cas系统的结构和生物化学表征对于CRISPR-Cas系统的功能至关重要。 了解它们的机制并将其重新用于精确的基因组编辑。我们的长期目标是 阐明了V型介导的靶核酸识别和切割的潜在机制， 转座子相关的CRISPR-Cas系统，为更安全、更可靠的 用于治疗人类疾病。在本提案中，我们将研究四种 新发现的CRISRP-Cas系统，涵盖DNA靶向（Cas 12 i）、RNA靶向（Cas 12 g）和 CRISPR RNA引导的DNA转座（I-F型级联和Cas 12 k）。正如我们的初步数据显示， Cas 12 i比目前使用的Cas效应物容纳更长的crRNA-DNA异源双链体，因此可能是一种新的靶向分子。 提高基因组编辑的特异性。RNA引导的RNA酶Cas 12 g是紧凑的和热稳定的， 突出了其在RNA编辑和RNA靶向方面的潜力。此外，I-F型级联和Cas 12 k直接 RNA引导的DNA转座的转座机制，为基因组编辑开辟了新的范式 独立于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