A Unique Type V CRISPR-Cas System Encoded by a Group of Thermus Viruses

Trofimova, A. B.; Demkina, A. O.; Shmakov, S. A.; Livenskyi, A. D.; Serebryakova, M. V.; Dmitriev, A. A.; Rubinshteyn, I. D.; Severinov, K. V.; Kolesnik, M. V.

doi:10.64898/2026.04.20.719267

bioRxiv · 10.64898/2026.04.20.719267

A Unique Type V CRISPR-Cas System Encoded by a Group of Thermus Viruses

Trofimova, A. B.·Demkina, A. O.·Shmakov, S. A.·Livenskyi, A. D.·Serebryakova, M. V.·Dmitriev, A. A.·Rubinshteyn, I. D.·Severinov, K. V.·Kolesnik, M. V.

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Abstract

CRISPR-Cas are widespread adaptive immune systems that protect bacteria and archaea from mobile genetic elements such as bacteriophages. Metagenomic sequencing identified CRISPR-Cas systems in phage genomes; however, their functions remain largely unknown. Here, we present Cas12r-CRISPR, a novel type V CRISPR-Cas system encoded by Lalka phages infecting thermophilic Thermus bacteria. We determined Cas12r-CRISPR PAM consensus sequence and crRNA structure and showed, that when provided with appropriate spacers and expressed in Thermus thermophilus, Cas12r-CRISPR efficiently interferes with plasmid transformation as well as infection by diverse Thermus phages. In the course of Lalka phage infection, the Cas12r-CRISPR locus is expressed with middle phage genes and its transcripts are among the most abundant phage RNAs. Notably, most Cas12r-CRISPR spacers target integrative mobile elements widespread in Thermus genomes. Both Lalka phages and targeted integrative mobile elements use host tRNA genes as attachment sites. We therefore propose that Cas12r-CRISPR participates in an inter-MGEs conflict. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/719267v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@f90c35org.highwire.dtl.DTLVardef@90a063org.highwire.dtl.DTLVardef@111f936org.highwire.dtl.DTLVardef@1008132_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Trofimova, A. B., Demkina, A. O., Shmakov, S. A., Livenskyi, A. D., Serebryakova, M. V., Dmitriev, A. A., Rubinshteyn, I. D., Severinov, K. V., Kolesnik, M. V.. 2026-04-21. A Unique Type V CRISPR-Cas System Encoded by a Group of Thermus Viruses. https://doi.org/10.64898/2026.04.20.719267

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