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Abstract
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Clustered regularly interspaced short palindromic repeats (CRISPR) together with their
CRISPR-associated (Cas) genes are widely distributed in prokaryotes that provide an
adaptive defense mechanism against foreign invasive DNA. There is relatively little
knowledge about the CRISPR-Cas diversity and evolution in Lactobacillus brevis strains.
Therefore, in this study, a genome-mining approach was employed to investigate the
diversity and occurrence of the CRISPR-Cas system in 83 L. brevis strains. Moreover,
trans-activating CRISPR RNA (tracrRNA) and protospacer adjacent motif (PAM) as
pivotal elements for the successful targeting and inference of phages by the subtype
II CRISPR-Cas systems were surveyed. Finally, evolutionary paths of L. brevis strains
under selective pressure from foreign invasive DNA such as plasmids and phages of
studied strains were surveyed using acquisition and deletion events analysis of spacers.
A total of 127 confirmed CRISPRs were identified, which were distributed in 69 strains.
Among strains with confirmed CRISPRs, 35 strains only contained one CRISPR locus,
23 strains contained two CRISPR loci, and 12 strains contained three to six CRISPR loci.
L. brevis strains frequently harbor more than one CRISPR system. Analysis of confirmed
CRISPR arrays showed that 31 out of 127 confirmed CRISPRs included Cas genes
which were categorized as one of the II-A, II-C, and I-E subtypes. Analysis of subtype
II-A spacers reflected divergent evolution for 18 strains into 16 unique groups. Additional
analysis of spacer sequences also confirmed the implication of characterizing CRISPRCas
systems in targeting of phages and plasmids. The current study highlighted the
potential of utilizing CRISPR spacer polymorphism in genotyping lactobacillus strains.
Moreover, it provides deep insights into the occurrence, diversity, and functional impacts
of the CRISPR-Cas system in L. brevis strains.
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