Characterizing function of type IV-C1 CRISPR-Cas systems from Thermococcus onnurineus and Pyrococcus abyssi: RNA sequencing of small RNAs bound by type IV-C1 CRISPR-Cas complex and long read sequencing of targeted plasmids.
Type IV-C CRISPR-Cas systems have not been characterized until now. This sequencing dataset was part of a study aimed at understanding how these systems function. To determine whether the type IV-C1 CRISPR-Cas interference complex (crRNP) associates with CRISPR guide RNAs (crRNAs), complexes were his-tagged, purified, and bound small RNAs were sequenced. This was done in both a native host organism (Thermococcus onnurineus) and in a heterologous expression system (Pyrococcus abyssi type IV-C1 system expressed in E. coli). Resulting data showed that type IV-C1 CRISPR-Cas systems can bind host-expressed crRNAs. To study the nature of DNA breaks caused by type IV-C1 systems, in vitro assays were conducted with purified IV-C1 crRNP and plasmids bearing target sequences. Following incubation, plasmids were sequenced (Oxford Nanopore full-length sequencing) to determine where type IV-C1 complexes created DNA breaks.