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 (Cas) 蛋白，特别是 Cas9，提供了前所未有的可编程控制 靶向特定序列。 Cas9 及其工程突变体核酸内切酶死亡 Cas9 (dCas9)，使用 CRISPR-RNA (crRNA) 作为靶向 20 核苷酸长 DNA 序列的指导。活跃的 CRISPR 复合物需要 crRNA 和目标 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程序中经常使用并且对其有广泛的了解，但它的功能和 Cas9 对于易于折叠成二级结构的靶向序列的局限性尚不清楚。 二级结构，例如 G-四链体 (GQ)，在人类基因组中含量丰富，并且 具有生理和医学意义。在最近的两篇出版物中，我们展示了如何 GQs 可以抑制靶标识别和 R 环形成，扭曲结构，并抑制 Cas9 介导的 DNA 切割。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 并可以解决实现瞬时和序列特异性转录的长期存在的问题 通过 PQS 进行监管。 PQS 已在重要癌基因和转录的启动子中被发现 在许多癌症中上调的因子。用小分子稳定这些癌基因启动子中的 GQ 分子导致其转录受到抑制，这使得这种方法成为潜在的抗 癌症治疗。然而，尽管它们具有结构特异性，这些小分子并没有序列 具体的。定点诱变也已用于修改 PQS 以控制基因表达；然而， 这种变化是永久性的。通过序列特异性靶向 PQS 并调节基因表达 暂时无法通过替代方法实现，我们建议可以通过 dCas9。我们假设通过靶向 PQS 或其附近区域可以上调或下调基因表达 与 dCas9。我们提供的酪氨酸羟化酶（TH）转录数据支持了这一假设。 该提案有两个具体目标：（1）确定目标链或非目标链中的 GQ 如何 DNA 影响 Cas9 的靶标识别、结合、构象状态、动力学和切割活性 （和 dCas9）使用体外单分子荧光，特别是 FRET 和集成方法。 (2) 至 通过 dCas9 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 KRAS 在许多癌症中上调，是一种癌基因，在信号传导和细胞中具有重要意义 增殖。 TH 是多巴胺生物合成中的限速酶。