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

基本信息

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
项目类别：
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

项目摘要

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 引导的融合蛋白针对特定 DNA 序列进行自定义化学修饰。易于 DNA 聚合酶 (EvolvR) 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 受到目标长度较短的限制，尚未实现。应用于蛋白质编码序列内的非连续位点，这是酶活性的一个共同特征 Cas9 引导的 10-11 易位 (TET) 酶促进修饰胞嘧啶的靶向去甲基化。这些 Cas9-TET 表观基因组编辑器具有阐明基因表达的潜力。 Cas9- 可以识别特定表观遗传标记的生物学效应并为多种疾病提供治疗方法。 TET 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正如他们的技术和临床承诺一样，在 Cas9-TET 表观基因组编辑中，剂量反应关系已得到证实。有效 TET 浓度、去甲基化和基因表达之间存在这些相关性。表明 TET 的催化活性可能会限制 Cas9-TET 表观基因组编辑的最终效率。已经证明TET的催化活性可以通过活性位点残基的突变来提高，并且同时的活性位点突变可以具有协同作用。本研究旨在多重基于 EvolvR 的突变。多样化以进化包含 TET 活性位点的非连续蛋白质区域并增加 TET 的催化活性将利用基于阵列的 Cas 靶向 RNA 生成。并行化许多 DNA 序列的基于 EvolvR 的分子将是多重 EvolvR 的功能。通过流式细胞术和深度测序在 GFP 生产和分析中拯救荧光来验证。接下来将应用多重 EvolvR，通过非连续的平行诱变来增加 TET 活性。 TET 活性位点区域和通过免疫沉淀富集高活性 TET 变体。随后，该研究将应用已知的高活性 TET 变体的活性进行体外表征。和新颖的 TET 变体，以提高 Cas9-TET 表观基因组编辑的效率。将与催化失活的 Cas9 融合，并靶向报告基因和内源基因中的甲基化启动子通过亚硫酸氢盐和RNA测序，可以发现去甲基化和基因表达的变化。对具有可变活性的 Cas9-TET 融合体进行评估，以确定 TET 活性是否限制了目前的表观基因组编辑可能会产生具有改善的活性和活性的工程化 TET 变体。这项研究结合生物伦理学课程，将表观基因组编辑技术推向临床应用。将培训一名医学博士/博士生成为一名能够临床翻译基因组的独立医师科学家和表观基因组编辑技术，并通过佩雷尔曼指导政策制定者负责任地使用。医学院在基因治疗和高危产科领域处于全球领先地位，学生将准备成为一名在子宫基因组编辑疗法和胎儿诊断方面发展的母胎医学医师。