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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering Undergraduate Fire Talks

CRISPR-dCas9 Interference for In Vitro Inhibition of Disease-Associated Microbial Metabolites

Saturday, October 11, 2025

8:45 AM - 8:50 AM PDT

Location: Room 33B

Presenting Author(s)

Hania Timek

Undergraduate Researcher
University of Michigan College of Engineering, United States

Co-Author(s)

Hania Timek

Undergraduate Researcher
University of Michigan College of Engineering, United States
BH

Brian Hamp

PhD Candidate
University of Michigan College of Engineering, United States
AH

Amber Haley

PhD Pre-Candidate
University of Michigan College of Engineering, United States
CC

Collins I. Chimezie

PhD Pre-Candidate
University of Michigan College of Engineering
Ann Arbor, Michigan, United States

Last Author(s)

Jiahe Li, PhD

Assistant Professor
University of Michigan
Ann Arbor, Michigan, United States

Primary Investigator(s)

Jiahe Li, PhD

Assistant Professor
University of Michigan
Ann Arbor, Michigan, United States

Introduction: : The gut microbiome hosts thousands of microorganisms that play significant roles in influencing human health. Emerging evidence suggests that the onset of many common intestinal inflammatory diseases, such as inflammatory bowel disease (IBD), is heavily influenced by pathogenic bacteria outcompeting commensal bacteria in the gut via small-molecule metabolites (1, 2). Recently, the High Pathogenicity Island (HPI), a region of DNA abundantly found in pathogenic E. coli in gut microbiomes of IBD patients, has been identified to encode for some of these harmful small-molecule metabolites (3, 4). Most significantly, the HPI includes the gene irp1 that produces the iron siderophore yersiniabactin (Ybt), which pathogenic E. coli use in an iron deficient environment to acquire metal ions and gain a competitive advantage (5). In the gut, Ybt intracellularly sequesters zinc from macrophages and stabilizes macrophage hypoxia-inducible factors (HIF-α), amplifying bacterial colonization and promoting a pro-fibrotic genetic response. These effects ultimately lead to fibrosis and onset of IBD, qualifying Ybt as an important molecule to investigate. While recent studies have shown the knockout of the irp1 gene in HPI+ E. coli hindering Ybt biosynthesis, no probiotic bacteria have been developed to silence Ybt production in situ (4, 5). Thus, we engineered a non-pathogenic, therapeutic E. coli strain possessing tools to inhibit the biosynthesis of Ybt in HPI+ bacteria via CRISPR-dCas9-mediated, targeted transcriptional silencing of the irp1 gene.

Materials and

Methods: : Our main methods included 1) incorporating dCas9 machinery into eb-TP114, a self-transmissible and highly conjugative plasmid, and 2) quantifying the efficacy and specificity of our dCas9-mediated targeting of irp1 inhibition (Figure A). First, we used Atum Scoring Algorithms to design and optimize four single guided RNAs (sgRNAs) through which the dCas9 machinery could target four different regions of the irp1 gene. Subsequently, we cloned the sgRNAs individually into a plasmid that readily contains dCas9 and a known chloramphenicol spacer using single-fragment Gibson Assembly. The successful insertion of our sgRNAs was verified via gel electrophoresis, which showed the chloramphenicol spacer being replaced by our sgRNAs. Mediated by a plasmid containing BXB1 integrase, these dCas9-harboring plasmids were then conjugatively cloned into the backbone of eb-TP114, and transconjugates were verified via Kanamycin+ plates and sequencing. Lastly, transconjugates were conjugated into the chassis strain E. coli MG1655. In order to quantify the inhibition effect of our dCas9 machinery on irp1 expression for all four different sgRNAs, we performed quantitative PCR (qPCR) using HPI+ E. coli conjugated with our four different eB-TP114 plasmids carrying the dCas9 module. This would allow us to see transcription levels of the complementary mRNA strand of irp1 when targeted in various regions via our different sgRNAs. Additionally, we performed Liquid Chromatography Mass Spectrometry (LC-MS-QTOF) to quantify Ybt synthesized by HPI+ E. coli conjugated with one of our dCas9-harboring therapeutic plasmids in comparison to a scramble HPI+ control and an irp1 knockout strain.

Results, Conclusions, and Discussions:: Our dCas9-containing strains proved to be effective in suppressing irp1 expression and reducing Ybt synthesis; qPCR results showed all four dCas9-containing HPI+ strains exhibiting varying degrees of inhibition (Figure B). Most notably, our strain containing the most effective sgRNA (sgRNA_2) showed a 24-fold inhibition of irp1 expression when compared to a scramble HPI+ control. Additionally, Our targeted LC-MS results showed zero relative abundance of Ybt synthesized by the tested therapeutic strain when compared to a scramble HPI+ control and irp1 knockout strain (Figure C). Ultimately, the results suggest that we have successfully developed a therapeutic strain of bacteria that, when conjugated into HPI+ bacteria (as would be its tendency in the human gut) effectively hinders expression of irp1 and thus dramatically reduces synthesis of Ybt. This leads us to conclude that dCas9 machinery can successfully be transmitted via horizontal gene transfer to sterically prevent RNA polymerase in HPI+ bacteria from transcribing irp1, thereby reducing production of a harmful metabolite. Our study is significant because it advances scientific knowledge of the efficacy of bacterial-based biotherapeutics, CRISPR gene-editing technologies, and horizontal gene transfer manipulation. The engineered strains present an innovative solution to potentially minimize the effects of IBD, a disease that is clinically very challenging to treat, by reducing a problematic microbial metabolite produced by pathogenic bacteria found in the gut. Hence, our results are a valid contribution to the ongoing discussions in microbiome research surrounding living bacterial therapies and bridging the gap between synthetic biology and medicine. As there are many unknowns in the gut microbiome, our study suggests a way to expand scientific understanding of the role of pathogenic gut bacteria in inflammatory diseases. In the future, we aim to incorporate our therapeutic bacteria in a chronic colitis murine model to assess efficacy of our conjugative therapeutic strains in vivo.

Acknowledgements and/or References (Optional): : 1) Ni, J., Wu, G. D., Albenberg, L. & Tomov, V. T. Gut microbiota and IBD: causation or correlation? Nat Rev Gastroenterol Hepatol 14, 573-584 (2017). https://doi.org/10.1038/nrgastro.2017.88

2) Elmassry, M. M. et al. A meta-analysis of the gut microbiome in inflammatory bowel disease patients identifies disease-associated small molecules. Cell Host & Microbe 33, 218-234.e212 (2025). https://doi.org/10.1016/j.chom.2025.01.002

3) Hentschel, U. & Hacker, J. Pathogenicity islands: the tip of the iceberg. Microbes and Infection 3, 545-548 (2001). https://doi.org/https://doi.org/10.1016/S1286-4579(01)01410-1

4) Behnsen, J. et al. Siderophore-mediated zinc acquisition enhances enterobacterial colonization of the inflamed gut. Nature Communications 12, 7016 (2021). https://doi.org/10.1038/s41467-021-27297-2

5) Ahn, J. H. et al. Intestinal E. coli-produced yersiniabactin promotes profibrotic macrophages in Crohn's disease. Cell Host Microbe 33, 71-88.e79 (2025). https://doi.org/10.1016/j.chom.2024.11.012