# Novel genetic engineering tools for functional studies of the gut microbiome

> **NIH NIH F30** · COLUMBIA UNIVERSITY HEALTH SCIENCES · 2020 · $23,875

## Abstract

Project Summary
 In the past decade, advances in next-generation sequencing technologies have led to an explosion of
genomic studies of the human gut microbiome. Dysbiosis, or perturbation of the microbiome, has been linked to
diseases such as inflammatory bowel disease and obesity; genetically modifying the gut microbiome thus holds
promise as a method for treating these diseases. To achieve this goal, however, we must first develop a
mechanistic understanding of the complex ecological dynamics of the microbiome. This requires the ability to make
specific perturbations to the microbiome and observe the effects to determine causality, which we currently lack.
There are no tools available to genetically manipulate most gut microbes, including at least 30% of species that
are not cultivable in vitro. Engineering therapeutic functions into the microbiome also requires the ability to make
targeted genomic edits, which presents a further challenge. In this project, I propose an in situ genome engineering
system and a novel CRISPR-Transposon platform to address these key challenges.
 For in situ delivery of transposons into the gut microbiome, an E. coli donor strain will be orally gavaged
into a live mouse host. The donor strain will conjugate a replicative or integrative plasmid vector into native gut
microbes in situ to tag them with selectable markers such as GFP and antibiotic resistance genes. Using high-
throughput screening methods and metagenomic sequencing of 16S rRNA, I will identify genetically engineerable
microbes from fecal samples and determine the composition of reservoir populations for each plasmid vector. I will
also track these populations over time to make metagenomic measurements of the dynamics of horizontal gene
transfer (e.g. specific routes, time scales, and rates of transfer) in an animal-associated microbiome for the first
time, which will elucidate this important aspect of gut microbial ecology.
 In this in situ system, integrative plasmids utilize a randomly inserting transposon to add new genetic
functions into native microbes. Conversely, to enable targeted gene deletions and genetic knockout studies of
diverse microbes, I will develop the CRISPR-Transposon (CRISPR-Tn) platform for targeted transposon
mutagenesis. A fusion protein between a broad-host range transposase and a catalytically dead Cas9
endonuclease will be programmed to bind a specific genomic locus with by a synthetic guide RNA molecule.
Once tethered, the transposase will be forced to insert transposons site-specifically. This programmable, host-
independent system will enable targeted genome knockouts and operon insertions in a wide range of bacteria,
and combined with in situ conjugative delivery, will significantly expand our genetic engineering capabilities for
mechanistic studies of natural microbiomes.

## Key facts

- **NIH application ID:** 9870925
- **Project number:** 5F30DK111145-04
- **Recipient organization:** COLUMBIA UNIVERSITY HEALTH SCIENCES
- **Principal Investigator:** Sway Peng Chen
- **Activity code:** F30 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $23,875
- **Award type:** 5
- **Project period:** 2017-04-01 → 2020-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9870925

## Citation

> US National Institutes of Health, RePORTER application 9870925, Novel genetic engineering tools for functional studies of the gut microbiome (5F30DK111145-04). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9870925. Licensed CC0.

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