ACTIVITY OF CRISPR-CAS SYSTEMS IN LACTICASEIBACILLUS PARACASEI

SIMONUTTI, A.; MERCANTI, D.; MOINEAU, S.; GENEVIÈVE M. ROUSSEAU; QUIBERONI ANDREA; PUJATO SILVINA ALICIA

Congreso; 32 CONGRESO BRASILERO DE MICROBIOLOGÍA; 2023

The fermentative dairy industry is fundamentally based on the activity of lactic bacteria. During the manufacturing processes, the activity of the starter bacteria can be delayed by bacteriophages. The attack from these bacteriophages causes the destruction of bacterial cells, hindering the normal growth and development of acidity by the ferments.CRISPR systems confer "acquired", sequence-specific, and heritable immunity against phages and other genetic elements that are foreign and invasive to the bacterial cell, such as plasmids. A CRISPR locus is composed of short, highly conserved repeat DNA sequences within the cluster, separated by variable sequences known as spacers. The immunization process is based on the incorporation of new spacers into the CRISPR locus, from phage sequences or plasmids. Subsequently, CRISPRs are transcribed into small RNAs (crRNAs) that guide a protein complex to cut, invading, genetic material. Despite the popularity of CRISPR-Cas9-based genomic editing, few endogenous CRISPR systems have been characterized to date.The objective of this work was to select bacteria with probiotic potential that contain active CRISPR-Cas systems and explore the possibility of taking advantage of this natural immunization technology to provide the industry with a 'food-grade' tool for the development of cultures improved in their phagoresistance. For this, the presence of CRISPR-Cas systems in 49 Lacticaseibacillus strains belonging to the INLAIN collection was analyzed, and 14 CRISPR-Cas systems were sequenced. To evaluate the functionality of the CRISPR-Cas system, interference tests were performed using the pNZ123 plasmid with an insert containing the first spacer of the strain and the PAM sequence (Protospacer Adjacent Motif) corresponding to the CRISPR system analyzed.52% of the Lacticaseibacillus strains analyzed presented CRISPR-Cas systems in their genome. Out of the 14 CRISPR-Cas systems sequenced, 13 were type II-A CRISPR-Cas systems, showing between 21 and 49 spacers. 5% of the spacers were similar to phage genomes or plasmids. Only 1 strain presented the type I-E CRISPR system, with 79 spacers not previously reported. 23% of these showed 100% homology to phages sequences of Lb. paracasei.The interference tests showed a high functionality of the CRISPR-Cas system in the Lacticaseibacillus strains studied. The CRISPR type II-A system showed higher system activity compared to the I-E system. These results are promising, as the demonstrated functionality of the CRISPR system would allow obtaining probiotic strains of Lacticaseibacillus with improved phage resistance.