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

项目总结 CRISPR-Cas系统通过使用GUIDE RNAs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 由于非靶标基因组编辑和对内源性DNA修复的依赖而引起的担忧 途径，阻碍CRISPR-CAS系统的临床应用。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，并将其重新用于精确的基因组编辑。我们的长期目标是 解开多种基因介导的靶核酸识别和切割的机制 CRISPR-CAS系统，为安全可靠地使用这项技术在 治疗人类疾病。在这个提案中，我们将研究四个新发现的分子机制 CRISRP-CAS系统，包括DNA靶向(Cas12i)、RNA靶向(Cas12g)和CRISPR RNA引导 DNA转座(VcCascade和Cas12k)。如我们的结构所示，Cas12i可以容纳更长的时间 与目前使用的Cas效应器相比，crRNA-DNA异源双链可能提高了基因组的特异性 正在编辑。RNA引导的RNaseCas12g结构紧凑，耐热性好，因此有可能扩展工具箱 用于RNA编辑和RNA靶向应用。VcCascade和Cas12k直接移位机 RNA引导的DNA转座，开启了一种独立于DNA修复的基因组编辑新范式 小路。 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