Publications
Methods in EnzymologyFeb 2025 DOI:
10.1016/bs.mie.2025.01.039

Determining the biochemical function of type IV CRISPR ribonucleoprotein complexes and accessory proteins

Williams, Andrew A.; Redman, Olivine; Domgaard, Hannah; Armbrust, Matthew J.; Jackson, Ryan N.
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Genes
Abstract
Type IV CRISPR systems are phylogenetically diverse and poorly understood. However, recently, major strides have been made toward understanding type IV-A systems. In type IV-A systems, a multi-subunit ribonucleoprotein complex, called the Csf complex, uses a CRISPR-derived guide to bind double-stranded DNA, forming an R-loop to which a helicase called CRISPR-associated DinG (CasDinG) is recruited. It is proposed that the ATP-dependent helicase activity of CasDinG then unwinds duplex DNA near the targeting site, impairing RNA transcription, and gene expression. Here we describe methods used to investigate the type IV-A system from Pseudomonas aeruginosa strain 83 including a plasmid clearance assay, expression and purification of type IV ribonucleoprotein complexes and proteins, nucleic acid binding assays, and CasDinG helicase assays. These methods provide a foundation for future work aimed at understanding these enigmatic systems.
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Genes

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