Development of screening approaches of highly specific bacteriophages based on bioinformatic analysis of CRISPR-Cas structures of Corynebacterium diphtheriae systems

This study aims to develop approaches for screening highly specific bacteriophages based on bio-informatic analysis of CRISPR-Cas structures of bacterial systems using the example of Corynebacterium diphtheriae. We proposed an algorithm for bioinformatic search and analysis of CRISPR-Cas structures of bacteria systems and phage screening through spacer sequences of CRISPR-cassette in genomes of Corynebacterium strains. 22 genome-wide sequences loaded from the GenBank database were selected as the target. 21 strains out of 22 had CRISPR-Cas systems. Using several search algorithms in CRISPR-Cas systems, one CRISPR-cassette was found in 23.8% of the tested strains and two in 76.2% of cases. Near the cassettes, a complete set of Cas-genes was identified, characteristic of two types of systems: Type-I Subtype-I-E and Type-II Subtype-II-C. The conducted analysis of the CRISPR-cassette spacer composition showed 3 to 42 spacers in the cassette. The cumulative total number of identified spacers amounted to 297, 64 spacers of which repeated in two or more CRISPR-cassettes, 159 spacers had no replicates. The three pairs of strains under study from this group had a complete match of spacer and consensus sequences, although they were isolated at different times and in multiple countries. A phylogenetic analysis was performed to confirm their common origin. Phages screening through the spacer sequences showed the highest compliance of the spacers with the phages protospacers, characteristic of the bacteria of the Mycobacteriaceae, Gordoniaceae, Streptomycetaceae, Corynebacteriaceae family belonging to the Actinobacteria type. One strain with multiple antibiotic resistance was identified, and its expected bacteriophage resistance was determined using this method. Thus, the developed bioinformatic analysis technology allowed the information on the expected resistance of the tested strains CRISPR-Cas system against the detected phages to be obtained, which in the long term enables the development of a platform of personalised bacteriophage treatment approaches.

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