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A high-performance and versatile technology for precision microbiome engineering

用于精密微生物组工程的高性能、多功能技术

基本信息

批准号：
10278809
负责人：
Samuel Henry Sternberg
金额：
$ 68.85万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-01 至 2025-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10278809
关键词：
Address Algorithm Design Area Automobile Driving Bacteria Bacterial Genes Bacterial Genome Basic Science Biological Biological Models CRISPR/Cas technology Candidate Disease Gene Catalogs Cells Clinical Clustered Regularly Interspaced Short Palindromic Repeats Collection Communities Complex Coupled Custom DNA DNA Double Strand Break Data Data Pooling Data Set Directed Molecular Evolution Disease Disease model Drug resistance Elements Engineering Environment Escherichia coli Exhibits Foundations Future Gastrointestinal tract structure Gene Silencing Genes Genetic Engineering Genome Genome engineering Gnotobiotic Guide RNA Health behavior Home Horizontal Gene Transfer Human Human Engineering Hydrolase Hyperactivity Immunity In Situ In Vitro Industrialization Insertional Mutagenesis Integrase Klebsiella oxytoca Klebsiella pneumoniae Knock-out Laboratories Mammalian Cell Metabolism Metagenomics Methods Microbe Modification Multidrug Resistance Gene Mus Mutation Names Nervous System Physiology Neuraxis Nucleotides Orthologous Gene Outcome Pathway interactions Performance Physiological Plasmids Play Pseudomonas putida RNA library Regulatory Element Research Role Scientist Specificity System Technology Therapeutic Time Transposase Variant Vibrio cholerae Vision Work bacterial community base bile acid metabolism bile salts carbapenemase data integration design dysbiosis exhaustion experimental study flexibility gain of function genetic approach genetic manipulation genetic payload genome-wide global health gut microbiome homologous recombination host microbiome in vivo innovation insight interest loss of function metabolic engineering metagenome microbial community microbiome microbiome composition microbiota microorganism mouse model next generation novel therapeutic intervention novel therapeutics pathogen pathogenic bacteria prevent programs repaired resistance gene reverse genetics screening site-specific integration synthetic biology tool transmission process vector

项目摘要

PROJECT SUMMARY The mammalian gastrointestinal tract is home to a complex and diverse collection of microorganisms that play crucial roles in metabolism, host immunity, and central nervous system function. Despite a growing appreciation for the importance of a balanced microbiome on human health and behavior, and the wide range of diseases that can result from dysbiosis, our ability to study and modify complex microbial communities in vivo remains severely limited. Sequencing efforts can exhaustively catalog bacterial diversity and abundance, but offer only observational information; gnotobiotic research in mice allows for tight control over colonization, but fails to represent natural host-microbiome interactions; and genetic engineering can be used to manipulate specific genes or pathways in select microbes, but not within native environments. To address these shortcomings, we propose to develop an innovative platform technology for precision microbiome engineering that will, for the first time, enable gene- and species-specific editing in vivo. Our approach centers around two recent breakthroughs made in our laboratories: a method for generating precise DNA insertions using CRISPR- transposon systems (INTEGRATE technology), and a method for mobilizing genetic payloads within the gut using broad-host-range conjugative vectors (MAGIC technology). By combining and expanding these tools, we will develop programmable, self-driving elements that disseminate broadly while retaining exquisite nucleotide- level specificity for target genomes. Our preliminary data provide strong evidence to substantiate the basis of our proposal and demonstrate feasibility. In a recent collaborative effort, we developed INTEGRATE for kilobase-scale bacterial genome engineering by systematically assessing genome-wide insertion specificity across a panel of guide RNAs, and demonstrating efficient activity in multiple clinically and industrially relevant bacterial species. In Aim 1, we will identify hyperactive INTEGRATE variants that function autonomously and proliferatively, and develop a comprehensive guide RNA design algorithm that incorporates empirical off-target data and large metagenome assembly information. In Aim 2, we will combinate MAGIC with INTEGRATE to enable mobile transmission and targeted integration within complex in vitro communities, as well as in a mouse model. Finally, in Aim 3, we will apply our tool for both gain-of-function and loss-of-function studies in vivo: 1) to deliver bile salt hydrolase genes in the murine gut and investigate their corresponding effects on microbiome composition and host metabolism, and 2) to inactivate multidrug resistance genes in a Klebsiella pneumoniae disease model. Collectively, our studies will advance powerful new synthetic biology tools that can be broadly and flexibly applied within any complex bacterial community of interest, for both basic research and eventual therapeutic applications.

项目总结