Using the Endogenous CRISPR-Cas System of Heliobacterium modesticaldum To Delete the Photochemical Reaction Center Core Subunit Gene
InHeliobacterium modesticaldum, as in manyFirmicutes, deleting genes by homologous recombination using standard techniques has been extremely difficult. The cells tend to integrate the introduced plasmid into the chromosome by a single recombination event rather than perform the double recombination required to replace the targeted locus. Transformation with a vector containing only a homologous recombination template for replacement of the photochemical reaction center genepshA produced colonies with multiple genotypes, rather than a clean gene replacement. To address this issue, we required an additional means of selection to force a clean gene replacement. In this study, we report the genetic structure of the type I-A and I-E CRISPR-Cas systems fromH. modesticaldum, as well as methods to leverage the type I-A system for genome editing.In silicoanalysis of the CRISPR spacers revealed a potential consensus protospacer adjacent motif (PAM) required for Cas3 recognition, which was then tested using anin vivointerference assay. Introduction of a homologous recombination plasmid that carried a miniature CRISPR array targeting sequences inpshA(downstream of a naturally occurring PAM sequence) produced nonphototrophic transformants with clean replacements of thepshAgene with ~80% efficiency. Mutants were confirmed by PCR, sequencing, optical spectroscopy, and growth characteristics. This methodology should be applicable to any genetic locus in theH. modesticaldumgenome. The heliobacteria are the only phototrophic members of the largely Gram-positive phylum Firmicutes, which contains medically and industrially important members, such asClostridium difficileandClostridium acetobutylicum. Heliobacteria are of interest in the study of photosynthesis because their photosynthetic system is unique and the simplest known. Since their discovery in the early 1980s, work on the heliobacteria has been hindered by the lack of a genetic transformation system. The problem of introducing foreign DNA into these bacteria has been recently rectified by our group; however, issues still remained for efficient genome editing. The significance of this work is that we have characterized the endogenous type I CRISPR-Cas system in the heliobacteria and leveraged it to assist in genome editing. Using the CRISPR-Cas system allowed us to isolate transformants with precise replacement of the pshA gene encoding the main subunit of the photochemical reaction center.
- Research Organization:
- Arizona State Univ., Tempe, AZ (United States)
- Sponsoring Organization:
- USDOE Office of Science (SC), Basic Energy Sciences (BES). Chemical Sciences, Geosciences, and Biosciences Division
- Grant/Contract Number:
- SC0010575
- OSTI ID:
- 1631821
- Journal Information:
- Applied and Environmental Microbiology, Vol. 85, Issue 23; ISSN 0099-2240
- Publisher:
- American Society for MicrobiologyCopyright Statement
- Country of Publication:
- United States
- Language:
- English
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