CRISPR-Cas Spacer Integration in a type III-A system

Adaptive immunity is an important asset to compete in the universal arms race of evolution. Prokaryotes possess an adaptive immune system in the form of clustered regularly interspaced short palindromic repeats (CRISPR) and their CRISPR-associated (Cas) proteins. New immunological memories are saved in the form of spacers in between repeats of the CRISPR array. The key components to acquire new spacers include a Cas1-2 complex, a CRISPR array comprising a leader sequence with at least one repeat and often auxiliar proteins like IHF. In the type I-E system of Escherichia coli, IHF binds within the leader junction and bends the CRISPR array in a sharp U-Turn. This interaction specifies correct spacer integration. Thermotoga profunda encodes a III-A Cas1-2 complex and HU, a protein similar to IHF. Both proteins belong to the type II DNA-binding protein family and have a highly conserved structure. IHF and HU-like proteins are typically 10 kDa in size, share an α helical core with two positively charged β-ribbon arms and form a dimer. We demonstrate specific integration of new spacers only in the presence of the complex and TpHU. Additionally, TpCas1-2 has low intrinsic specificity and TpHU is necessary to avoid ectopic integration. Our results enhance the current state of knowledge as HU proteins were previously not associated with CRISPR-Cas spacer integration.