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Abstract

Type IV CRISPR-Cas modules belong to class 1 prokaryotic adaptive immune
systems, which are defined by the presence of multisubunit effector
complexes. They usually lack the known Cas proteins involved in
adaptation and target cleavage, and their function has not been
experimentally addressed. To investigate RNA and protein components of
this CRISPR-Cas type, we located a complete type IV cas gene locus and
an adjacent CRISPR array on a megaplasmid of Aromatoleum aromaticum
EbN1, which contains an additional type I-C system on its chromosome.
RNA sequencing analyses verified CRISPR RNA (crRNA) production and
maturation for both systems. Type IV crRNAs were shown to harbour
unusually short 7 nucleotide 5'-repeat tags and stable 3' hairpin
structures. A unique Cas6 variant (Csf5) was identified that generates
crRNAs that are specifically incorporated into type IV
CRISPR-ribonucleoprotein (crRNP) complexes. Structures of RNA-bound Csf5
were obtained. Recombinant production and purification of the type IV
Cas proteins, together with electron microscopy, revealed that Csf2 acts
as a helical backbone for type IV crRNPs that include Csf5, Csf3 and a
large subunit (Csf1). Mass spectrometry analyses identified
protein-protein and protein-RNA contact sites. These results highlight
evolutionary connections between type IV and type I CRISPR-Cas systems
and demonstrate that type IV CRISPR-Cas systems employ crRNA-guided
effector complexes.