Shared CRISPR arrays underpin type I-A/I-B coexistence.

Abstract

CRISPR-Cas systems provide adaptive immunity in prokaryotes, yet how multiple CRISPR-Cas subtypes coexist and coordinate within a single genome remains unclear. Comparative genomic analysis revealed that nearly one-third of type I-A CRISPR-Cas3 systems are adjacent to a type I-B system, often sharing a single CRISPR array. Using Thermococcus siculi RG-20 (Tsi) as a model, we show that purified TsiCas6a and TsiCas6b independently recognize and cleave the shared pre-crRNA, producing mature crRNAs with comparable efficiency. Plasmid interference assays further demonstrated that crRNAs produced by either Cas6a or Cas6b enzyme could guide both type I-A and type I-B interference complexes. This interchangeability shows that crRNAs generated by either Cas6a or Cas6b can be loaded into, and function with, both type I-A and type I-B interference complexes. Structural modelling revealed distinct but complementary recognition strategies for Cas6a and Cas6b, and mutational analysis of their RNA-binding residues impaired pre-crRNA cleavage and abolished interference activity. Together, these results uncover a shared-array logic in which Cas6a/Cas6b-compatible processing routes a single pre-crRNA to multiple type I effectors-providing a potential mechanism for subtype co-existence, a plausible explanation for array-less (or "orphan") interference modules, and an evolutionary bet-hedging strategy that prevents Acrs from shutting down immunity wholesale.