Developing of a broad-host range platform for CRISPR/Cas9 genome editing: enhancing current generation in Shewanella oneidensis using CREATE
Abstract
Shewanella oneidensis remains an invaluable host for the discovery and engineering of pathways important for bioremediation of toxic and radioactive metals and understanding extracellular electron transfer. However, genetic manipulation of environmental bacteria is challenging due to the lack of genetic tools available. Transposon mutagenesis and targeted knockouts by suicide vectors have been extensively used in the last 20 years to elucidate cellular physiological traits in S. oneidensis. Nevertheless, these tools are typically used only in gene disruption applications and are insufficient to understand sequence-activity relationships at the amino acid level or to specifically enhance a desired activity. While CRISPR/Cas9 has been demonstrated to work in a wide range of organisms, its delivery to date has been focused on electroporation or natural competence. Shewanella and other environmental microorganisms are recalcitrant to transformation by these methods, instead requiring high-efficiency conjugation systems, typically using an E. coli donor strain. In this work, we will first develop and optimize a conjugation-based method to deliver the CRISPR/Cas9 genome-editing system in Shewanella. Our second aim is to utilize this system to implement the most recently developed genome editing technology, CRISPR EnAbled Trackable genome Engineering (CREATE), to enhance activity of the metal respiratory (Mtr) pathway under electrode reducing conditions. CREATE is a strategy which couples CRISPR/Cas9 with recombineering, microarray oligomer synthesis and next generation sequencing for genome-scale engineering. Over 50,000 mutants were generated in E. coli and used to map sequence-activity relationship for a range of applications. Average editing efficiency of 70% was achieved in E. coli demonstrating this powerful tool for efficiently generate and map a massive library of mutants.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Jul 07, 2017
- Source ID
- N000141712600
Entities
People
- Jeffery Gralnick
Organizations
- Office of Naval Research
- Regents of the University of Minnesota
- United States Navy