Elucidating the Role of DNA Shape in CRISPR Target Discrimination

项目摘要 该提议集中于揭示DNA双链体的内在物理性质（即，DNA形状）和 阐明了它们在CRSIPR Cas9和Cas 12 a靶标辨别中的作用。CRISPR（定期检测） 间隔短回文重复）系统已被改编成多功能和可编程的代理 以全基因组方式操纵核酸靶点，引发基因组编辑的革命， 操纵仍在迅速推进。基于CRISPR的技术建立在对核酸的特异性识别上。 酸，并且阻碍其在治疗环境中应用的主要障碍是“脱靶效应”，其中 CRISPR对异常基因靶标的不受控制和不期望的作用导致有害后果。 需要显著提高CRISPR特异性，而这取决于进一步的深入了解 CRISPR靶点区分机制的研究。 拟议的工作重点是Cas9和Cas 12 a，它们最广泛地用于DNA基因组工程。Cas9 和Cas 12 a都使用效应蛋白-RNA复合物来切割双链DNA，并选择它们的 （i）RNA引导物与DNA靶链的区段之间的碱基配对 命名为原型间隔区，和（ii）靶DNA内的短原型间隔区邻近基序（PAM）。Ca9/Cas12a 目标辨别依赖于内在的DNA形状，其由局部“核心”碱基对共同决定 以及包括（远端）外围序列和拓扑约束的许多其它因素（例如，supercoiling）。 然而，目前关于Cas9/Cas 12 a靶选择的研究集中在核心片段的DNA特征， PAM和原型间隔区，并且还没有解释除了直接序列之外的外周序列的影响。 RNA/DNA配对或DNA拓扑约束。 我们发展了独特的定点自旋标记方法，获得序列依赖构象 图1示出了游离双链体以及与包括Cas9和Cas 12 a的蛋白质结合的DNA的形状。我们最近的研究表明 不参与RNA/DNA配对的DNA序列可以调节Cas9诱导的DNA解旋和切割， 这些信息使细胞中的基因编辑能够使用已知可以增强特异性的短RNA指导。 基于这些发现，我们将研究DNA外周序列和超螺旋如何调节 Cas9/Cas 12 a靶向区分以及影响游离DNA核心片段的形状。学习的信息 将被纳入增强CRISPR靶向特异性的算法中，以及预测三个- 在全基因组范围内，线性一维序列的三维DNA形状。该项目将 推进对DNA特异性识别的理解，这是生物学的基础，并将有助于 培养下一代科学人才。

Project Summary This proposal centers on uncovering intrinsic physical properties of DNA duplex (i.e., DNA shape) and elucidating their roles in CRSIPR Cas9 and Cas12a target discrimination. CRISPR (Clustered-Regularly- Interspaced-Short-Palindromic-Repeats) systems have been adapted into versatile and programmable agents for manipulating nucleic acid targets in a genome-wide fashion, unleashing a revolution in genome editing and manipulation that is still rapidly advancing. CRISPR-based technology is built upon specific recognition of nucleic acids, and a major obstacle hampering its applications in therapeutic settings is the “off-target effect”, in which uncontrolled and undesired actions of CRISPR on aberrant gene targets result in deleterious consequences. Significant improvement of CRISPR specificity is required, and this depends on further in-depth understanding of mechanism of CRISPR target discrimination. Proposed work focuses on Cas9 and Cas12a that are most widely used for engineering DNA genomes. Cas9 and Cas12a both use an effector protein-RNA complex to cleave double-stranded DNAs, and select their cognate targets based on: (i) base-pairing between the RNA guide and a segment of the DNA target-strand designated as protospacer and (ii) a short protospacer-adjacent-motif (PAM) within the target DNA. Ca9/Cas12a target discrimination rely on intrinsic DNA shape, which is determined collectively by the local “core” base-pair(s) and many other factors including (distal) peripheral sequences and topological constraints (e.g., supercoiling). However, current studies on Cas9/Cas12a target selection focus on DNA features at the core segment spanning the PAM and protospacer, and have not yet accounted for impacts of peripheral sequences beyond direct RNA/DNA pairing or DNA topological constraints. We have developed unique site-directed spin labeling methods to obtain sequence-dependent conformation (shape) of free duplexes as well as DNAs bound by proteins including Cas9 and Cas12a. Our recent work shows that DNA sequences not involved in RNA/DNA pairing can modulate Cas9-induced DNA unwinding and cleavage, and the information enables gene editing in cells with short RNA guides that are known to enhance specificity. Building on these findings, we will investigate how DNA peripheral sequences and supercoiling modulate Cas9/Cas12a target discrimination as well as affect the shape of free DNA core segments. Information learned will be incorporated into algorithms for enhancing CRISPR targeting specificity, as well as for predicting three- dimensional DNA shape from the linear one-dimensional sequence in a genome-wide setting. The project will advance understanding on DNA specific recognition that is fundamental to biology, and will contribute to development of the next generation of scientific workforce.