Elucidating the Role of DNA Shape in CRISPR Target Discrimination

项目摘要 这一建议的核心是揭示DNA双链的内在物理性质(即DNA形状)和 阐明它们在CRSIPR Cas9和Cas12a靶标歧视中的作用。CRISPR(集群-定期- 间隔短回文重复)系统已被改编成通用和可编程的代理 以全基因组的方式操纵核酸靶标，引发了基因组编辑和 仍在快速推进的操纵行为。基于CRISPR的技术建立在对核的特定识别基础上 而阻碍其在治疗环境中应用的一个主要障碍是“靶外效应”，其中 CRISPR对异常基因靶点的不受控制和不受欢迎的行为会导致有害的后果。 需要显著提高CRISPR的专用性，这取决于进一步深入了解 CRISPR目标识别机制的研究。 拟议的工作重点是最广泛用于设计DNA基因组的Cas9和Cas12a。CAS9 和Cas12a都使用效应器蛋白质-RNA复合体来切割双链DNA，并选择他们的 基于：(I)RNA指南和DNA靶链片段之间的碱基配对的同源靶 指定为Protspacer和(Ii)靶DNA内的短Protspacer-相邻基序(PAM)。Ca9/Cas12a 目标识别依赖于固有的dna形状，而dna形状是由局部“核心”碱基对共同决定的(S) 以及许多其他因素，包括(远端)外围序列和拓扑约束(例如，超螺旋)。 然而，目前关于Cas9/Cas12a靶标选择的研究主要集中在跨越核心片段的DNA特征 PAM和ProtSpacer，还没有考虑到直接以外的外周序列的影响 RNA/DNA配对或DNA拓扑约束。 我们已经发展了独特的位置定向自旋标记方法来获得序列依赖的构象 (形状)自由双链以及由包括Cas9和Cas12a在内的蛋白质结合的DNA。我们最近的工作表明 没有参与RNA/DNA配对的DNA序列可以调节Cas9诱导的DNA解离和切割， 这些信息使具有短RNA导引的细胞中的基因编辑成为可能，众所周知，这种导引可以增强特异性。 在这些发现的基础上，我们将研究DNA外周序列和超螺旋是如何调制的 Cas9/Cas12a靶标识别以及影响游离DNA核心片段的形状。学到的信息 将被纳入到增强CRISPR目标特异性的算法中，以及用于预测三个 在全基因组环境中，从线性一维序列中获得的一维DNA形状。该项目将 增进对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队伍。

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.