Anti-CRISPR-mediated Acylation and Bioreversible Esterification for Precision Genome Editing

用于精准基因组编辑的抗 CRISPR 介导的酰化和生物可逆酯化

• 批准号: 10657417
• 负责人: Axel O Vera
• 金额: $ 2.27万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10657417
• 关键词: Acetyl Coenzyme A; Acetylation; Acylation; Address; Bacteria; Bacteriophages; Basic Science; Binding; Cell Culture Techniques; Cells; Charge; Chromosomal translocation; Clustered Regularly Interspaced Short Palindromic Repeats; Coenzyme A; Computer Analysis; Confocal Microscopy; Cytosol; DNA; Development; Electroporation; Enzymes; Esterification; Exposure to; Fellowship; Fluorescence Microscopy; Genome; Goals; Guide RNA; Human; Hydrolysis; Immunity; Learning; Liquid Chromatography; Lysine; Mammalian Cell; Masks; Mass Spectrum Analysis; Mediating; Medicine; Methods; Outcome; Phosphines; Proteins; Research; Ribonucleoproteins; Site; Specificity; System; Testing; Therapeutic; Time; United States National Institutes of Health; Viral; detection assay; diazo compound; endonuclease; esterase; exposed human population; genome editing; genotoxicity; in vivo; inhibitor; next generation sequencing; novel; novel strategies; prevent; response; small molecule; spatiotemporal; tandem mass spectrometry; vector

PROJECT SUMMARY/ABSTRACT Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems use RNA-guided proteins (e.g., Cas9, Cas12a) to cleave DNA. Cas9 and Cas12a share many similarities and have been repurposed for genome editing in human cells because of their programmability, simplicity, and efficiency, with applications in basic research and medicine. Cas12a is generally more efficient and specific than Cas9. However, since the concentration of Cas protein is much higher than that of the DNA target in genome editing, prolonged Cas12a activity also leads to off-target DNA cleavage, chromosomal translocations, and genotoxicity. Equipping Cas12a with an “on-off” switch can overcome these challenges by shortening Cas12a’s window of exposure and increasing its specificity—the on-target to off-target ratio of DNA cleavage. We developed a novel strategy that repurposes a CRISPR-Cas inhibitor protein (i.e., an anti-CRISPR) to selectively acylate or “cage” Cas12a with a small-molecule-removable group. We propose to deliver small molecules and anti- CRISPR proteins into human cells via fast, bioreversible, and in vivo-compatible means to activate and inactivate Cas12a, respectively, and increase its specificity. With the help of experts at the outstanding MIT and Broad Institute facilities, I will learn how to use liquid chromatography–tandem mass spectrometry (LC–MS/MS) to assess the selectivity of our novel Cas12a acylation strategy, and mammalian cell culture, high throughput confocal microscopy, next-generation sequencing, and computational analysis to assess anti-CRISPR delivery and Cas12a genome-editing specificity. Overall, this research will introduce the first approach for the site-selective and reversible acylation of Cas12a lysine residues and a new bioreversible esterification strategy for the traceless delivery of anti-CRISPR proteins that together will increase genome- editing specificity.

项目概要/摘要 成簇规则间隔短回文重复序列 (CRISPR)/CRISPR 相关 (Cas) 系统的使用 用于切割 DNA 的 RNA 引导蛋白（例如 Cas9、Cas12a）具有许多相似之处，并且具有许多相似之处。 由于其可编程性、简单性和可靠性，已被重新用于人类细胞的基因组编辑 效率，在基础研究和医学中的应用通常更有效和特异性。 然而，由于Cas蛋白的浓度远高于DNA靶标的浓度。 基因组编辑、Cas12a 活性延长也会导致脱靶 DNA 切割、染色体易位、 和遗传毒性。 为 Cas12a 配备“开关”开关可以通过缩短 Cas12a 的窗口来克服这些挑战。 暴露并提高其特异性——DNA 切割的正靶与脱靶比率。 重新利用 CRISPR-Cas 抑制剂蛋白（即抗 CRISPR）来选择性酰化或 带有小分子可去除基团的“笼”Cas12a 我们建议提供小分子和抗-。 通过快速、生物可逆和体内兼容的方式将 CRISPR 蛋白引入人体细胞，以激活和 在麻省理工学院杰出专家的帮助下，分别灭活Cas12a，并增加其特异性。 和布罗德研究所的设施，我将学习如何使用液相色谱-串联质谱法 (LC-MS/MS) 来评估我们的新型 Cas12a 酰化策略和哺乳动物细胞培养的选择性，高 通过共聚焦显微镜、下一代测序和计算分析来评估 总体而言，本研究将引入第一个抗 CRISPR 传递和 Cas12a 基因组编辑特异性。 Cas12a赖氨酸残基的位点选择性和可逆酰化方法以及一种新的生物可逆方法 用于无痕传递抗 CRISPR 蛋白的酯化策略，这些蛋白一起将增加基因组- 编辑特异性。