Genomic Science Program
U.S. Department of Energy | Office of Science | Biological and Environmental Research Program

Leveraging Type I-F CRISPR-Associated Transposase Regulators to Improve Editing Efficiency

Authors:

Leo Song2* (LeoSong@lbl.gov), Amanda T. Alker2, Sophia E. Swartz2, Jigyasa Arora2, Sara Smith2, Rachel Rovinsky2, Abby Wang2, Agnès Oromí-Bosch2, Jonathan Martinson2, Robin Herbert1, Adam M. Deutschbauer1, Brady F. Cress2, Benjamin E. Rubin2, Jennifer A. Doudna2, Trent R. Northen1

Institutions:

1Lawrence Berkeley National Laboratory; 2 University of California–Berkeley

URLs:

Goals

The goal of this program is to understand the interactions, localization, and dynamics of grass rhizosphere microbial communities at the molecular level (genes, proteins, metabolites) to enable accurate predictions and interventions to effectively manage and harness microbes to achieve DOE missions in sustainable energy and carbon cycling.

Abstract

Functional understanding of microbial gene functions is largely based on genetic interrogation of isolated organisms, providing limited insights into the importance of genes within microbial communities, including the rhizosphere, which is the focus of the program. To address these knowledge gaps, recently researchers have created a generalizable toolset for targeted genome editing of individual organisms within complex microbial communities that uses type I-F CRISPR-associated transposons (CASTs) to make targeted genetic edits to complex microbial communities (Rubin et al. 2022). CASTs are broadly dispersed across bacteria and capable of integrating large genomic payloads. However, little is known about the host molecular factors which regulate CAST integration, and their widespread utilization is limited by low editing efficiency across diverse, non-model bacteria. To expand the range and applicability of Type I-F CASTs as editing tools, the group employed a genome wide mutant screening approach to identify putative regulators of CAST transposition in established model systems in which CAST is known to integrate. Candidate regulator hits were individually validated, with a particular focus on well-characterized genes involved in known mechanisms. Next, the team conducted a bioinformatic survey for the conservation of the candidate regulator hits across broad bacterial phyla. Finally, the team leveraged its findings by constructing vectors that incorporate these key regulators to increase editing efficiency. These results will shed light on the molecular mechanisms underlying CAST integration and enable more efficient editing in diverse non-model microorganisms. This information will enable the team to extend the application of community editing to better understand the molecular mechanisms governing assembly and interactions in the rhizosphere.

References

Rubin, B. E., et al. 2022. “Species- and Site-Specific Genome Editing in Complex Bacterial Communities,” Nature Microbiology 7, 34–47. DOI:10.1038/s41564-021-01014-7.

Funding Information

This material by m-CAFEs Microbial Community Analysis and Functional Evaluation in Soils, (m-CAFEs@lbl.gov), a Science Focus Area led by Lawrence Berkeley National Laboratory, is based upon work supported by the U.S. DOE, Office of Science, BER program under contract number DE-AC02-05CH11231.