Cas4/1 dual nuclease activities enable prespacer maturation and directional integration in a type I-G CRISPR-Cas system

Abstract

CRISPR-Cas adaptive immune systems uptake short ‘spacer’ sequences from foreign DNA and 8 incorporate them into the host genome to serve as templates for crRNAs that guide interference 9 against future infections. Adaptation in CRISPR systems is mediated by Cas1-Cas2 complexes 10 that catalyze integration of prespacer substrates into the CRISPR array. Many DNA targeting 11 systems also require Cas4 endonucleases for functional spacer acquisition. Cas4 selects 12 prespacers containing a protospacer adjacent motif (PAM) and removes the PAM prior to 13 integration, both of which are required to ensure host immunization. Cas1 has also been shown 14 to function as a nuclease in some systems, but a role for this nuclease activity in adaptation has 15 not been demonstrated. We identified a type I-G Cas4/1 fusion with a nucleolytically active Cas1 16 domain that can directly participate in prespacer processing.