CRISPR-Cas systems assure adaptive immunity against invading mobile genetic elements in bacteria and archaea. Among them, type IV-A CRISPR-Cas systems stand out due to their distinct mechanism. Type IV-A systems lack the typical DNA/RNA nuclease activity, instead, these multisubunit systems employ a helicase DinG, which unwinds the DNA rather than cleaving it, thus causing transcriptional interference [1][2]. While subtypes IV-A1 and IV-A3 have been thoroughly characterized through structural and biochemical studies, subtype IV-A2 remains largely unexplored. This is mainly because type IV-A2 systems diverge from other subtypes with the absence of a Cas8 homologous protein, which in type IV-A1 and A3 systems, together with Cas5, is responsible for protospacer adjacent motif (PAM) recognition [1][3][4]. However, in our study using a bioinformatic guilt-by-association approach, it was discovered that CRISPR-Cas type IV-A2 systems co-occur with a small, conserved gene near the cas operon, which is transcribed in the opposite direction. Investigating the function of the protein encoded by this gene will not only deepen the understanding of type IV-A2 mechanisms but also lead to their potential applications in gene editing in the future. Here, we present the preparatory studies laying the groundwork for the structural and functional characterization of one of type IV-A2 systems.
STUDY OF TYPE IV-A2 CRISPR-CAS SYSTEMS
Gintarė Žvejytė1, Rimvydė Čepaitė1, Patrick Pausch1
1 Life Sciences Center - European Molecular Biology Laboratory (LSC-EMBL) Partnership Institute for Genome Editing Technologies, Vilnius University, Vilnius, Lithuania
[1] R. Pinilla-Redondo et al., “Type IV CRISPR–Cas systems are highly diverse and involved in competition between plasmids”, Nucleic Acids Res., vol. 48, no. 4, pp. 2000–2012, Dec. 2019. Accessed: Feb. 16, 2025. [Online]. Available: https://doi.org/10.1093/nar/gkz1197
[2] V. M. Crowley et al., “A Type IV-A CRISPR-Cas System in Pseudomonas aeruginosa Mediates RNA-Guided Plasmid Interference In Vivo”, CRISPR J., vol. 2, no. 6, pp. 434–440, Dec. 2019. Accessed: Feb. 16, 2025. [Online]. Available: https://doi.org/10.1089/crispr.2019.0048
[3] N. Cui et al., “Type IV-A CRISPR-Csf complex: Assembly, dsDNA targeting, and CasDinG recruitment”, Mol. Cell, Jun. 2023. Accessed: Feb. 16, 2025. [Online]. Available: https://doi.org/10.1016/j.molcel.2023.05.036
[4] R. Čepaitė et al., “Structural variation of types IV-A1- and IV-A3-mediated CRISPR interference”, Nature Commun., vol. 15, no. 1, Oct. 2024. Accessed: Feb. 16, 2025. [Online]. Available: https://doi.org/10.1038/s41467-024-53778-1