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引导的融合蛋白靶向特定的DNA序列进行自定义化学修饰。 cas9结合错误 - 俯卧的DNA聚合酶（EVOLVRS）多样化预先指定的DNA片段，以促进用户定义的诱变并加速了定向进化的速度。但是，Evolvr受到目标长度短的限制，尚未要应用于蛋白质编码序列中的非连续位点，这是活性酶的共同特征站点。 CAS9引导的十个时期易位（TET）酶促进了修饰的胞嘧啶的靶向脱甲基化碱基恰好改变基因表达。这些Cas9-Tet表观基因组编辑有可能阐明特定表观遗传标记的生物学作用并为众多疾病提供疗法。但是，cas9- TET表观基因组编辑通常达到不完全的脱甲基化，也限制了其对基因表达的影响作为他们的技术和临床承诺。在Cas9-Tet表观基因组编辑中，剂量反应关系具有它们在有效的TET浓度，脱甲基化和基因表达之间显示。这些相关性表明TET的催化活性可能会限制Cas9-Tet表观组编辑的最终效率。先前的工作已经证明，可以通过保持活跃部位保留的突变来增加TET的催化活性，并且单纯形活动位点突变可能是协同的。这项研究旨在基于EVOLVR多重多元化以进化出完成TET活性位点并增加的非连续蛋白区域 TET的催化活性。该项目将利用大自然的基于阵列的cas靶向RNA的生成分子以平行许多DNA序列的基于EVOLVR的演变。多路复用Evolvr的功能将是通过在GFP记者中挽救荧光并通过流式细胞仪和深度测序进行分析来验证。接下来，多路复用Evolvr将通过无连接的平行诱变来增加TET活性 TET活跃部位区域和通过免疫沉淀富集了多动性TET变体。催化多动TET变体的活性将在体外表征。随后，本研究将应用已知和新型TET变体以提高CAS9-TET表观基因组编辑的效率。多动性TET变体将融合到催化活跃的CAS9中，并针对记者和内生中的甲基化启动子系统。通过亚硫酸盐和RNA测序，脱甲基化和基因表达的变化将是评估具有可变活性的Cas9-TET融合量，以确定TET活动是否限制当前剧集编辑。这些实验可能会产生具有改善活性的工程TET变体，并且将表观基因组编辑技术推向临床实用程序。结合生物伦理学课程，这项研究将培训一名MD/PhD学生，成为一名独立的身体科学家，他们可以在临床上翻译基因组以及表观基因组编辑技术和指导决策者负责使用。通过Perelman 医学院在基因疗法和高风险产科领域的全球领导力，学生将准备成为子宫基因组编辑疗法和胎儿诊断的主要胎儿医学。