One CRISPR array supplies guide RNAs to two CRISPR–Cas subtypes
image: Cas6a and Cas6b use distinct recognition strategies to independently process a shared pre-crRNA, generating interchangeable crRNAs that guide both type I-A and type I-B CRISPR–Cas3 interference complexes and enable their functional coexistence.
Credit: HIGHER EDUCATION PRESS
CRISPR–Cas systems protect bacteria and archaea against invading genetic elements by using CRISPR RNAs, or crRNAs, to guide Cas proteins to matching foreign DNA. Many organisms encode more than one CRISPR–Cas subtype, but it remains unclear how different subtypes coordinate their use of CRISPR arrays and guide RNAs within the same cell.
In this study, we investigated the frequent coexistence of type I-A and type I-B CRISPR–Cas systems. Comparative genomic analysis revealed that nearly one-third of type I-A CRISPR–Cas3 loci are located next to a type I-B locus. In many cases, the two subtypes appear to rely on a single shared CRISPR array rather than maintaining separate sets of guide sequences.
Using the archaeon Thermococcus siculi RG-20 as a model, we showed that the crRNA-processing enzymes Cas6a and Cas6b can independently recognize and cleave the same precursor crRNA. Interestingly, the two enzymes use distinct recognition strategies: Cas6b mainly interacts with the 5′-proximal region of the CRISPR repeat, whereas Cas6a primarily recognizes a more central region. Despite these differences, both enzymes efficiently generate mature crRNAs from the shared precursor.
Most importantly, the crRNAs produced by either Cas6a or Cas6b can be incorporated into both type I-A and type I-B interference complexes. These shared guide RNAs can therefore direct either CRISPR subtype to recognize and destroy complementary target DNA. Structural modelling and mutational analyses further identified RNA-binding residues that are essential for crRNA processing and subsequent interference activity.
Together, our findings reveal a flexible guide-RNA-sharing mechanism in which one CRISPR array can supply functional crRNAs to two coexisting type I subtypes. This shared-array arrangement may explain how CRISPR interference modules lacking their own arrays remain active and may provide a more resilient defence strategy when one CRISPR pathway is inhibited by viral anti-CRISPR proteins.