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对内源性DNA修复的依赖， 这阻碍了CRISPR-Cas系统的临床应用。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，而且还可能激发新的 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，并将它们重新用于精确的基因组编辑。我们的长期目标是 为了阐明靶核酸识别和切割的机制， CRISPR-Cas系统为安全可靠地使用该技术提供了必要的知识， 治疗人类疾病在这个建议中，我们将研究四个新发现的分子机制， CRISRP-Cas系统，涵盖DNA靶向（Cas 12 i）、RNA靶向（Cas 12 g）和CRISPR RNA引导 DNA转座（VcCascade和Cas 12 k）。正如我们的结构所揭示的那样，Cas 12 i容纳更长的时间， 与目前使用的Cas效应物相比，crRNA-DNA异源双链体，因此潜在地提高了对基因组的特异性 编辑. RNA引导的RNase Cas 12 g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Cas 12 k直接换位机， RNA引导的DNA转座，为独立于DNA修复的基因组编辑开辟了新的范式 途径。 1

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