Abstract
The spirochete Borrelia burgdorferi, which causes Lyme disease, has the most segmented genome among known bacteria. In addition to a linear chromosome, the B. burgdorferi genome contains over 20 linear and circular endogenous plasmids. While many of these plasmids are dispensable under in vitro culture conditions, they are maintained during the natural life cycle of the pathogen. Plasmid-encoded functions are required for colonization of the tick vector, transmission to the vertebrate host, and evasion of host immune defenses. Different Borrelia strains can vary substantially in the type of plasmids they carry. The gene composition within the same type of plasmid can also differ from strain to strain, impeding the inference of plasmid function from one strain to another. To facilitate the investigation of the role of specific B. burgdorferi plasmids, we developed a Cas9-based approach that targets a plasmid for removal. As a proof-of-principle, we showed that targeting wild-type Cas9 to several loci on the endogenous plasmids lp25 or lp28-1 of the B. burgdorferi type strain B31 results in sgRNA-specific plasmid loss even when homologous sequences (i.e., potential sequence donors for DNA recombination) are present nearby. Cas9 nickase versions, Cas9D10A or Cas9H840A, also cause plasmid loss, though not as robustly. Thus, sgRNA-directed Cas9 DNA cleavage provides a highly efficient way to eliminate B. burgdorferi endogenous plasmids that are non-essential in axenic culture.
Funder
Howard Hughes Medical Institute
American Heart Association
National Institute of General Medical Sciences
Bay Area Lyme Foundation
Brandeis University
Publisher
Public Library of Science (PLoS)
Reference76 articles.
1. Estimating the frequency of Lyme disease diagnoses, United States, 2010–2018;KJ Kugeler;Emerg Infect Dis,2021
2. Epidemiology of Lyme disease.;PS Mead;Infect Dis Clin North Am,2015
3. Lyme borreliosis.;AC Steere;Nat Rev Dis Primers,2016
4. Of ticks, mice and men: understanding the dual-host lifestyle of Lyme disease spirochaetes;JD Radolf;Nat Rev Microbiol,2012
5. The divided bacterial genome: structure, function, and evolution;GC diCenzo;Microbiol Mol Biol Rev,2017
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