Multiplexing CRISPR/Cas9-based Continuous Evolution for Improved Epigenome Editing

多重基于 CRISPR/Cas9 的连续进化以改进表观基因组编辑

• 批准号: 10331822
• 负责人: Niklaus Hoyt Evitt
• 金额: $ 4.41万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-15 至 2023-06-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10331822
• 关键词: Active Sites; Antibiotics; Benign; Binding; Bioethics; Biological; Biotechnology; CRISPR/Cas technology; Cell Line; Cells; Chemicals; Chimeric Proteins; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Custom; Cytosine; DNA; DNA Methylation; DNA Sequence; DNA-Directed DNA Polymerase; Diagnostic; Directed Molecular Evolution; Discipline of obstetrics; Disease; Distal; Dose; Engineering; Enzymes; Epigenetic Process; Escherichia coli; Evolution; Family; Flow Cytometry; Fluorescence; Future; Gene Expression; Generations; Genetic; Genome; Geometry; Guide RNA; Human Development; Hyperactivity; Hypermethylation; Immunoprecipitation; In Vitro; Industrialization; Leadership; Length; Maternal-fetal medicine; Mediating; Methods; Methylation; Modification; Molecular; Mutagenesis; Mutate; Mutation; Nature; Oncogenic; Oxides; Pathway interactions; Physicians; Plasmids; Play; Policy Maker; Polymerase; Process; Protein Region; Proteins; Publishing; RNA; Regulation; Reporter; Research; Role; Scientist; Site; Specific qualifier value; Structure; Structure of primordial sex cell; Students; System; Technology; Terminator Codon; Testing; Training; Translating; Variant; Work; base; bisulfite sequencing; catalyst; deep sequencing; demethylation; doctoral student; epigenome; epigenome editing; experimental study; fetal; gene therapy; genome editing; high risk; human disease; improved; improved functioning; in utero; in vivo; male; medical schools; mutant; novel; promoter; protein function; protein structure; repaired; response; screening; sperm cell; stem cells; therapeutic genome editing; tool; transcriptome sequencing; zygote

Cas9-guided fusion proteins target specific DNA sequences for custom chemical modification. Cas9-bound error- prone DNA polymerases (EvolvRs) diversify pre-specified DNA segments to facilitate user-defined mutagenesis and accelerate the pace of directed evolution. However, EvolvRs are limited by short target length and have yet to be applied to non-contiguous sites within a protein coding sequence, a common feature of enzyme active sites. Cas9-guided ten-eleven translocation (TET) enzymes promote targeted demethylation of modified cytosine bases to precisely alter gene expression. These Cas9-TET epigenome editors have the potential to elucidate the biological effects of specific epigenetic marks and provide therapies for numerous diseases. However, Cas9- TET epigenome editors often achieve incomplete demethylation, limiting their effect on gene expression as well as their technological and clinical promise. In Cas9-TET epigenome editing, dose-response relationships have been shown between effective TET concentration, demethylation, and gene expression. These correlations suggest that TET’s catalytic activity may limit the ultimate efficiency of Cas9-TET epigenome editing. Prior work has demonstrated that TET’s catalytic activity can be increased through mutation of active site residues and that simultaneous active site mutations can be synergistic. This study aims to multiplex EvolvR-based diversification to evolve non-contiguous protein regions comprising the TET active site and increase TET’s catalytic activity. The project will leverage nature’s array-based generation of Cas-targeting RNA molecules to parallelize EvolvR-based evolution of many DNA sequences. Multiplexed EvolvR’s function will be validated by rescue of fluorescence in GFP reporters and analysis with flow cytometry and deep sequencing. Multiplexed EvolvR will next be applied to increase TET activity through parallel mutagenesis of non-contiguous TET active site regions and enrichment of hyperactive TET variants through immunoprecipitation. Catalytic activity of hyperactive TET variants will be characterized in vitro. Subsequently, this study will apply known and novel TET variants to improve the efficiency of Cas9-TET epigenome editing. Hyperactive TET variants will be fused to catalytically inactive Cas9 and targeted to methylated promoters in reporter and endogenous systems. By way of bisulfite and RNA sequencing, changes in demethylation and gene expression will be assessed among Cas9-TET fusions with variable activity to determine whether TET activity limits efficiency of current epigenome editing. These experiments may yield engineered TET variants with improved activity and push epigenome editing technologies towards clinical utility. Combined with bioethics coursework, this research will train an MD/PhD student to become an independent physician-scientist who can clinically translate genome and epigenome editing technologies and guide policy makers in their responsible use. Through the Perelman School of Medicine’s global leadership in gene therapy and high-risk obstetrics, the student will prepare to become a maternal fetal medicine physician developing in utero genome editing therapies and fetal diagnostics.

Cas9引导的融合蛋白靶向特定的DNA序列进行定制化学修饰。Cas9结合错误- 倾向性DNA聚合酶（EvolvRs）使预先指定的DNA片段多样化以促进用户定义的诱变 并加快定向进化的步伐。然而，EvolvR受到短靶长度的限制， 将其应用于蛋白质编码序列内的非连续位点，这是酶活性的共同特征， 网站. Cas9引导的十-十一易位（泰特）酶促进修饰的胞嘧啶的靶向去甲基化 碱基来精确改变基因表达。这些Cas9-泰特表观基因组编辑器有潜力阐明 研究特定表观遗传标记的生物学效应，并为许多疾病提供治疗方法。然而，Cas9- 泰特表观基因组编辑器通常实现不完全去甲基化，也限制了它们对基因表达的影响 作为他们的技术和临床承诺。在Cas9-泰特表观基因组编辑中， 在有效的泰特浓度、去甲基化和基因表达之间。这些相关性 表明泰特催化活性可能限制Cas9-泰特表观基因组编辑的最终效率。先前工作 已经证明泰特的催化活性可以通过活性位点残基的突变而增加， 同时的活性位点突变可以是协同的。这项研究的目的是多路复用基于EvolvR 多样化以进化包含泰特活性位点的非连续蛋白质区域，并增加 泰特的催化活性。该项目将利用自然界基于阵列的Cas靶向RNA的生成 分子来并行化许多DNA序列的基于EvolvR的进化。多路复用的EvolvR的功能将是 通过拯救GFP报告基因中的荧光并用流式细胞术和深度测序进行分析来验证。 多重EvolvR接下来将用于通过非连续突变的平行诱变来增加泰特活性。 泰特活性位点区域和通过免疫沉淀富集高活性泰特变体。催化 高活性泰特变体的活性将在体外表征。随后，这项研究将适用于已知的 和新的泰特变体以提高Cas9-泰特表观基因组编辑的效率。过度活跃的泰特变体 将与无催化活性的Cas9融合，并靶向报告基因和内源基因中的甲基化启动子。 系统.通过亚硫酸氢盐和RNA测序的方式，去甲基化和基因表达的变化将被发现。 在具有可变活性的Cas9-泰特融合体中进行评估，以确定泰特活性是否限制了 当前的表观基因组编辑。这些实验可以产生具有改善的活性和生物活性的工程化的泰特变体。 将表观基因组编辑技术推向临床应用。结合生物伦理学课程，本研究 将培养一名MD/PhD学生成为一名独立的医生科学家，他们可以在临床上翻译基因组 和表观基因组编辑技术，并指导政策制定者负责任地使用这些技术。通过佩雷尔曼 医学院在基因治疗和高危产科领域的全球领导地位，学生将准备 成为一名母亲胎儿医学医生，在子宫内开发基因组编辑疗法和胎儿诊断。