Developing Retrons into Genetic Engineering Tools for Non-Canonical Host Microbes
Abstract
There is an ongoing need to develop efficient genome modification tools that produce dynamic and scar-free mutations in a variety of microbial hosts. Retrons are unique retroelements present in a wide variety of bacteria and consist of three primary elements, (1)a guide sequence (#msr#), (2) a targeting sequence (#msd#), and (3) a unique reverse transcriptase. Following transcription of the retron cassette, the reverse transcriptase is cleaved for translation after which it produces a unique hybrid DNA-RNA complex (#msDNA#) that contains the guide RNA and reverse transcribed targeting (single stranded) DNA. The targeting DNA from the msDNA complex can then introduce edits into homologous DNA sequences. Relative to other methods of genome modification, such as CRISPR/Cas9, retronshave several potential advantages. First, retrons can introduce multiple simultaneous changes at different loci, enabling potentially dramatic shifts in phenotype. Second, retrons allow for transcriptional control over mutation rate by introducing error-prone RNApolymerases that affect transcription of the non-coding RNA. Finally, retrons allow for continuous evolution because the reverse transcribed DNA is homologous to and can theoretically edit its own parent sequence. Despite this potential, retrons have received relatively little attention as genomic engineering tools. Addressing this challenge, the overall goal of this proposal is to develop retrons into efficient and modular genomic engineering tools for use a in a variety of microbial hosts relevant to the ONR mission.To accomplish this goal, we will pursue three complementary aims. First, we will optimize retron-based editing in the model electroactive bacteria Shewanella oneidensis. Second, we will use retrons to engineer critical electron transport proteins in this organism. Finally, we will discover new retrons from a variety of bacterial species and leverage them to further improve editing efficiency. Thepresence of reversetranscriptases and the ability of retrons to produce single stranded DNA could enable several emerging technologies relevant to the DoD including long-term biological sensing and recording of environmental conditions, rapid changes in phenotype, continuous evolution of diverse protein targets, and genetic transformations of microbial consortia. Given our initial engineeringtarget of S. oneidensis, we envision potential retron applications in improving biological sensing, superior interfacing with bioelectronics, enabling rapid changes to biomaterial synthesis, and engineering of microbial consortia relevant to deep sea power generation.Approved for Public Release
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Nov 09, 2024
- Source ID
- N000142412637
Entities
People
- Benjamin K Keitz
Organizations
- Office of Naval Research
- United States Navy
- University of Texas at Austin