In situ genetic perturbation of gut bacteria with engineered phage vectors and CRISPR

Project Information

crInSitu

Grant agreement ID: 101044479

DOI

10.3030/101044479

EC signature date 22 April 2022

Start date 1 September 2022

End date 31 August 2027

Funded under

• European Research Council (ERC)

Total cost

€ 1 999 780,00

EU contribution

€ 1 999 780,00

Coordinated by

INSTITUT PASTEUR
France

Periodic Reporting for period 1 - crInSitu (In situ genetic perturbation of gut bacteria with engineered phage vectors and CRISPR)

Reporting period: 2022-09-01 to 2025-02-28

Summary of the context and overall objectives of the project

Humans live in a symbiotic relationship with trillions of microorganisms that inhabit our bodies and play an essential role in digestion, the maturation of our immune response, protection against pathogens, production of vitamins, brain–gut axis and more. More and more causal relationships are being reported between the presence of specific communities, specific bacteria, or specific genes with disease onset or aggravation. Bacterial antigens are involved in autoimmune disease, bacterial toxins can cause a range of acute and chronic diseases including cancer, bacteria can modify drugs used in the treatment of a broad range of disease potentially impacting therapeutic outcomes, bacteria have also been shown to impact the success of immunotherapies. Because of these diverse effects of bacteria on a vast range of diseases, there is a growing interest in manipulating the microbiome to improve health. Yet, there is a huge knowledge and technological gap in our ability to carry out precise interventions in the microbiome.

The ability to genetically manipulate bacteria in situ to either bring novel function, or modify existing pathways would be a game changer both as a tool to better understand the microbiome and its effects on health and disease, and to develop innovative therapies targeting the microbiome. The main goal of this proposal will be to bridge this gap by developing the next generation of synthetic biology tools to study and manipulate the microbiome based on phage delivery vectors and CRISPR-Cas systems.

CRISPR-Cas systems have recently been repurposed to modify genomes, control gene expression and selectively remove target bacterial species, offering powerful novel strategies to investigate the microbiome. We propose here to develop CRISPR tools for the manipulation of several abundant species of the microbiome. We will further engineer bacteriophages as DNA delivery vectors that will enable us to introduce CRISPR-Cas systems in bacteria of interest directly in the animal gut. Preliminary results indicate that DNA delivery by phages to the gut is efficient enough to enable genome-wide pooled genetic screens with CRISPR-Cas. Using this strategy we will probe the fitness effect of thousands of microbial genes in key species, providing phenotypes for some of the estimated 5 million bacterial genes in the microbiome, most of which of unknown function.This will shed light on the genetic requirements for growth in the gut and on the niche occupied by different members of the microbiome. Altogether, the knowledge and technologies developed in this project will be instrumental both to further our understanding of the gut microbiome and how it contributes to health and disease. These technologies also have the potential to be used in the development of future microbiome targeted therapies.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

During the first part of the project we have focused our efforts on establishing tools and methods to enable in situ genetic perturbations of bacteria in the gut environment. These efforts have been organized around three main aims, with the first aim being the establishment of tools to enable robust genetic manipulation of bacteria and bypass their defenses against horizontal gene transfer. An important bottleneck to solve when modifying wild bacterial isolates is to overcome restriction modification systems. We have made interesting progress in the establishment of a novel method we call Rand-seq. In this approach a stretch of random DNA is introduced by a minimal vector into different strains of interest. Sequencing of the successful transformants and comparison with the original pool of sequences enables to identify depleted sequences that have been restricted. We have developed a computational algorithm that enables the identification of restricted motifs in the depleted sequences, and we have now shown that the method can identify previously unknown restricted sequences in a small panel of E. coli strains. This aim also benefits from the characterization of bacterial defense systems beyond restriction modification systems. We have worked on the PARIS immune system and initiated the characterization of additional novel defense systems.

A second aim is to engineer bacteriophage vectors for DNA delivery. We have performed work on the engineering of chimeric phages using phage lambda as a scaffold with the goal of extending its host range. In addition we have made progress on the use of Diversity Generating Retroelements to enable the directed evolution of phage tail fiber genes, which will enable the selection of phage variants able to recognize novel receptors.

Finally, a third aim is to perform in situ genetic perturbation in an animal model to probe gene function in high-throughput. We have improved CRISPRi methodologies and established a method enabling us to perform CRISPRi screens in the mouse gut environment. Experiments have been performed with mice under different diets, using different bacterial strains, and using a model of gut inflammation. The results obtained enable to shed light on the genetic requirements for growth, and at the same time provide a detailed picture of the mouse gut environment under these different conditions.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Our work on bacterial defenses has led us to perform a detailed characterization of the PARIS bacterial immune system. This work published in Nature, revealed how this immune system senses proteins deployed by bacteriophages to block restriction modification systems. We described the structure and molecular mechanism of this system, which acts by degrading tRNAs of the host and how bacteriophages can overcome this system by carrying their own tRNAs (Burman, Nature, 2024). We are now extending our work to the characterization of other defenses, with a particular focus on defenses susceptible of blocking DNA delivery to target bacteria.

Work performed with Eligo Bioscience has led to the demonstration of two key methodologies of the project in another study published in Nature (Brödel, Nature, 2024). First, the engineering of chimeric phage particles based on phage lambda enabled the delivery of DNA to various bacterial strains of E. coli and K. pneumoniae, second, a base editor delivered using these engineered phage vectors enable to efficiently introduce a mutation in a target bacterial population in the gut of mice. This work provides compelling evidence that it is indeed possible to genetically modify a whole target bacterial population directly in the gut environment. This is an important breakthrough which further motivates our work on expending this strategy to additional bacteria of the gut microbiome.

Finally, we have improved CRISPRi methodologies by deciphered a toxicity mechanism of dCas9 that is important to take into account for future use of the tool (Rostain, NAR, 2023), and we have further worked on the design of optimized dCas9 proteins using machine learning methods (Malbranke, PLoS. Comp. Biol., 2023).