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Genetic manipulation is a fundamental procedure for the study of gene and operon functions and new characteristics acquisition. Modern CRISPR-Cas technology allows genome editing more precisely and increases the efficiency of transferring mutations in a variety of hard to manipulate organisms. Here, we describe new CRISPR-Cas vectors for genetic modifications in bacillary species. Our plasmids are single CRISPR-Cas plasmids comprising all components for genome editing and should be functional in a broad host range. They are highly efficient (up to 97%) and precise. The employment and delivery of these plasmids to bacillary strains can be easily achieved by conjugation from Escherichia coli. During our research we also demonstrated the absence of compatibility between CRISPR-Cas system and non-homologous end joining in Bacillus subtilis.

New CRISPR-Cas9 vectors for genetic modifications ofBacillusspecies