Using the endogenous CRISPR-Cas system of Heliobacterium modesticaldum to delete the photochemical reaction center core subunit gene.

Appl Environ Microbiol. 2019 Sep 20;:

Authors: Baker PL, Orf GS, Kevershan K, Pyne ME, Bicer T, Redding KE

Abstract

In Heliobacterium modesticaldum, as in many Firmicutes, deleting genes by homologous recombination using standard techniques has been unsuccessful. 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 gene pshA produced colonies with multiple genotypes, rather than a clean gene replacement.Bacterial CRISPR-Cas systems have become powerful biotechnological tools for genome editing across all domains of life. In this study, we report the genetic structure of the Type I-A and I-E CRISPR-Cas systems from H. modesticaldum as well as methods to leverage the Type I-A system for genome editing. In silico analysis of the CRISPR spacers revealed a potential consensus protospacer adjacent motif (PAM) required for Cas3 recognition, which was then tested using an in vivo interference assay. Introduction of a homologous recombination plasmid that carried a miniature CRISPR array targeting sequences in pshA (downstream of a PAM sequence) produced non-phototrophic transformants with clean replacements of the pshA gene with ~80% efficiency. Mutants were confirmed by PCR, sequencing, optical spectroscopy, and growth characteristics. This methodology should be applicable to any genetic locus in the H. modesticaldum genome.IMPORTANCEThe heliobacteria are the only phototrophic members of the gram-positive phylum Firmicutes, which contains medically and industrially important members, such as Clostridium difficile and Clostridium 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 1980's, 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.

PMID: 31540988 [PubMed - as supplied by publisher]