Elucidating the Role of DNA Shape in CRISPR Target Discrimination

阐明 DNA 形状在 CRISPR 靶标识别中的作用

• 批准号: 10597689
• 负责人: Peter Z Qin
• 金额: $ 41.25万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-01 至 2027-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10597689
• 关键词: 3-Dimensional; Affect; Algorithms; Base Pairing; Basic Science; Biology; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; DNA; DNA Sequence; DNA-Binding Proteins; Development; Discrimination; Distal; Engineering; Gene Targeting; Genes; Genome; Guide RNA; Learning; Methods; Molecular Conformation; Nucleic Acids; Peripheral; Proteins; RNA; Role; Shapes; Site; Specificity; Spin Labels; System; Technology; Therapeutic; Work; ds-DNA; genome editing; genome-wide; improved; next generation; physical property; research and development; therapeutic development

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.

项目概要 该提案的重点是揭示 DNA 双链体的内在物理特性（即 DNA 形状）和 阐明它们在 CRSIPR Cas9 和 Cas12a 靶点识别中的作用。 间隔短回文重复）系统已适应多功能和可编程代理 以全基因组方式操纵核酸靶标，引发基因组编辑和 仍在快速发展的基于 CRISPR 的技术是建立在核酸的特异性识别基础上的。 酸，阻碍其在治疗环境中应用的一个主要障碍是“脱靶效应”，其中 CRISPR 对异常基因靶标的不受控制和不良作用会导致有害后果。 CRISPR特异性需要显着提高，这有赖于进一步深入的了解 CRISPR 靶点识别机制。 拟议的工作重点是最广泛用于 DNA 基因组工程的 Cas9 和 Cas12a。 和Cas12a都使用效应蛋白-RNA复合物来切割双链DNA，并选择它们的 同源靶标基于：(i) RNA 向导和 DNA 靶标链片段之间的碱基配对 指定为原型间隔子和 (ii) 靶 DNA 内的短原型间隔子相邻基序 (PAM)。 目标辨别依赖于内在的 DNA 形状，这是由局部“核心”碱基对共同决定的 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，目前对 Cas9/Cas12a 靶点选择的研究主要集中在跨核心片段的 DNA 特征上。 PAM 和 protospacer，尚未考虑到直接以外的外围序列的影响 RNA/DNA 配对或 DNA 拓扑限制。 我们开发了独特的定点自旋标记方法来获得序列依赖性构象 我们最近的工作表明，游离双链体以及与 Cas9 和 Cas12a 等蛋白质结合的 DNA 的形状。 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， 这些信息使得能够利用已知能够增强特异性的短RNA向导在细胞中进行基因编辑。 基于这些发现，我们将研究 DNA 外围序列和超螺旋如何调节 Cas9/Cas12a 目标歧视以及影响游离 DNA 核心片段的形状 学到的信息。 将被纳入算法中，以增强 CRISPR 靶向特异性，以及预测三 该项目将在全基因组范围内从线性一维序列中提取三维 DNA 形状。 对 DNA 特异性识别的理解是生物学的基础，并将有助于 培养下一代科学劳动力。