Targeting Promoter G-quadruplexes with CRISPR-dCas9 for Transcription Regulation

使用 CRISPR-dCas9 靶向启动子 G 四链体进行转录调控

• 批准号: 10438973
• 负责人: Hamza Balci
• 金额: $ 45.43万
• 依托单位: KENT STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10438973
• 关键词: Anabolism; Binding; Biological Assay; Cell Line; Cell Proliferation; Circular Dichroism; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Complex; DNA; DNA Sequence; DNA-Directed RNA Polymerase; Data; Dopamine; Electrophoretic Mobility Shift Assay; Engineering; Enzymes; Fluorescence; Fluorescence Resonance Energy Transfer; G-Quartets; GTP-Binding Protein alpha Subunits, Gs; Gene Expression; Genes; Genetic Transcription; Genome; Guidelines; Heterogeneity; Human Genome; In Vitro; KRAS2 gene; Kinetics; Knowledge; Luciferases; Malignant Neoplasms; Measurement; Mediating; Medical; Methods; Modeling; Molecular Conformation; Mutation; Nucleotides; Oncogenes; Organism; Physiological; Polymerase; Polymerase Chain Reaction; Proteins; Proto-Oncogenes; Publications; RNA; Reverse Transcription; Signal Transduction; Site; Site-Directed Mutagenesis; Specificity; Structure; Technology; Time; Transcription Initiation Site; Transcriptional Regulation; Tyrosine 3-Monooxygenase; Work; biophysical analysis; c-myc Genes; cancer therapy; ds-DNA; endonuclease; genome editing; mRNA Expression; mutant; novel; programs; promoter; protein expression; single molecule; small molecule; tool; transcription factor

Project Summary: Clustered Regularly Interspaced Palindromic Repeats (CRISPR) and CRISPR- associated (Cas) proteins, particularly Cas9, have provided unprecedented programmable control on targeting specific sequences. Cas9 and its engineered mutant endonuclease-dead Cas9 (dCas9), use CRISPR-RNA (crRNA) as a guide to target a 20-nucleotide long DNA sequence. An active CRISPR complex requires near-perfect complementarity and R-loop formation between crRNA and the target DNA. Despite its frequent use in different applications and extensive knowledge about it, the capabilities and limitations of Cas9 for targeting sequences that are prone to folding into secondary structures are not known. Secondary structures, such as G-quadruplexes (GQs), are abundant in the human genome and are physiologically and medically significant. In two recent publications, we demonstrated examples of how GQs could inhibit target recognition and R-loop formation, distort the structure, and inhibit Cas9-mediated DNA cleavage. We propose to perform systematic single molecule and bulk biophysical studies to determine how GQs located in the vicinity of Cas9 target site impact critical aspects of its function. Targeting potentially GQ forming sequences (PQS) also provides new application venues for CRISPR and could solve a long-standing problem in achieving transient and sequence-specific transcription regulation through PQS. PQS have been identified in promoters of prominent oncogenes and transcription factors that are upregulated in many cancers. Stabilizing GQs in promoters of these oncogenes with small molecules has resulted in suppression of their transcription, which made this approach a potential anti- cancer therapy. However, despite their structural specificity, these small molecules are not sequence specific. Site-directed mutagenesis has also been used to modify PQS to control gene expression; however, such changes are permanent. Targeting PQS with sequence-specificity and modulating gene expression temporarily have not been possible with alternative approaches, which we propose can be achieved with dCas9. We hypothesize that gene expression can be up or down regulated by targeting PQS or their vicinity with dCas9. We present data on tyrosine hydroxylase (TH) transcription which supports this hypothesis. The proposal has two specific aims: (1) To determine how GQs in target strand or non-target strand of DNA influence target recognition, binding, conformational states, kinetics, and cleavage activities of Cas9 (and dCas9) using in vitro single molecule fluorescence, particularly FRET, and ensemble methods. (2) To target PQS in promoters of c-MYC, KRAS, and TH genes by dCas9 to determine whether their mRNA and protein expressions can be modulated in relevant cell lines. While c-MYC codes for a transcription factor that is up-regulated in many cancers, KRAS is an oncogene that is significant in signaling and cell proliferation. TH is the rate limiting enzyme in dopamine biosynthesis.

项目摘要：成簇规则间隔回文重复序列（CRISPR）和CRISPR- 相关的（Cas）蛋白，特别是Cas9，已经提供了前所未有的可编程控制， 针对特定序列。Cas9及其工程化突变体核酸内切酶-死Cas9（dCas 9），用途 CRISPR-RNA（crRNA）作为靶向20个核苷酸长的DNA序列的向导。一个活跃的CRISPR 复合物需要crRNA和靶DNA之间近乎完美的互补性和R环形成。 尽管它在不同的应用中频繁使用，并且对它有广泛的了解，但它的功能和 Cas9用于靶向易于折叠成二级结构的序列的限制是未知的。 二级结构，如G-四链体（GQ），在人类基因组中是丰富的， 具有生理和医学意义。在最近的两份出版物中，我们展示了如何 GQs可以抑制靶点识别和R环形成，扭曲结构，并抑制Cas9介导的细胞凋亡。 DNA裂解。我们建议进行系统的单分子和批量生物物理研究， 确定位于Cas9靶位点附近的GQ如何影响其功能的关键方面。 靶向潜在的GQ形成序列（PQS）也为CRISPR提供了新的应用场所 并且可以解决实现瞬时和序列特异性转录的长期问题 通过PQS监管。PQS已被确定为主要癌基因和转录的启动子 在许多癌症中上调的因子。稳定这些癌基因启动子中的GQ， 分子导致了它们的转录抑制，这使得这种方法成为一种潜在的抗- 癌症治疗。然而，尽管它们的结构特异性，这些小分子不是序列 特定.定点诱变也已用于修饰PQS以控制基因表达;然而， 这种变化是永久性的。以序列特异性靶向PQS并调节基因表达 暂时还不可能用替代方法，我们建议可以用 dCas9。我们推测，基因表达可以通过靶向PQS或其附近来上调或下调 dCas9我们目前的数据酪氨酸羟化酶（TH）转录支持这一假设。 该建议有两个具体目标：（1）确定GQ在靶链或非靶链中的作用， DNA影响Cas9的靶标识别、结合、构象状态、动力学和切割活性 (and dCas 9），使用体外单分子荧光，特别是FRET和系综方法。(2)到 通过dCas 9靶向c-MYC、KRAS和TH基因启动子中的PQS，以确定它们的mRNA和 可以在相关细胞系中调节蛋白质表达。而c-MYC编码转录因子 在许多癌症中上调，KRAS是一种在信号传导和细胞凋亡中重要的癌基因。 增殖TH是多巴胺生物合成的限速酶。