# A high-performance and versatile technology for precision microbiome engineering

> **NIH NIH R01** · COLUMBIA UNIVERSITY HEALTH SCIENCES · 2022 · $676,224

## Abstract

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.

## Key facts

- **NIH application ID:** 10475816
- **Project number:** 5R01EB031935-02
- **Recipient organization:** COLUMBIA UNIVERSITY HEALTH SCIENCES
- **Principal Investigator:** Samuel Henry Sternberg
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $676,224
- **Award type:** 5
- **Project period:** 2021-09-01 → 2025-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10475816

## Citation

> US National Institutes of Health, RePORTER application 10475816, A high-performance and versatile technology for precision microbiome engineering (5R01EB031935-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10475816. Licensed CC0.

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