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：成簇的规则间隔回文重复(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 以特定序列为目标。Cas9及其工程突变的内切酶死亡Cas9(DCas9)，用途 CRISPR-RNA(CrRNA)作为靶向20个核苷酸的长DNA序列的指南。一个活动的CRISPR 复合体需要crRNA和靶DNA之间近乎完美的互补和R环的形成。 尽管它经常在不同的应用程序中使用，并且对它有广泛的了解，但它的功能和 Cas9在靶向易于折叠成二级结构的序列方面的局限性尚不清楚。 二级结构，如G-四链(GQs)，在人类基因组中含量丰富，并且 在生理和医学上都具有重要意义。在最近的两份出版物中，我们展示了如何 GQS可以抑制靶识别和R环的形成，扭曲结构，抑制Cas9介导的 DNA裂解。我们建议进行系统的单分子和整体生物物理研究，以 确定位于Cas9目标站点附近的GQ如何影响其功能的关键方面。 针对潜在的GQ形成序列(PQ)也为CRISPR提供了新的应用场所 并可以解决在实现瞬时和序列特异性转录方面的长期问题 通过PQ进行监管。已经在显著癌基因和转录的启动子中发现了PQs 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 分子导致它们的转录被抑制，这使得这种方法成为一种潜在的反 癌症治疗。然而，尽管这些小分子具有结构的特殊性，但它们并不是序列 具体的。定点突变也被用于修饰PQs以控制基因表达；然而， 这样的变化是永久性的。序列特异性靶向PQs调控基因表达 暂时不可能使用替代方法，我们建议可以通过 DCas9。我们假设，通过靶向PQ或其附近，基因表达可以上调或下调 使用dCas9。我们提供了关于酪氨酸羟化酶(TH)转录的数据，支持这一假说。 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和系综方法。(2)至 用dCas9靶向c-myc、KRAS和TH基因启动子中的PQs，以确定它们的mRNA和 蛋白质的表达可以在相关的细胞系中进行调节。而c-myc编码转录因子 这在许多癌症中上调，KRAS是一种癌基因，在信号和细胞中具有重要意义 扩散。TH是多巴胺生物合成中的限速酶。