RNA Polymerase Backtracking Promotes Type III-A CRISPR DNA Targeting

Abstract

Type III CRISPR-Cas systems provide transcription-dependent immunity against mobile genetic elements, yet the mechanism underlying their DNA targeting remains unclear. Although the type III effector Cas10-Csm functions as a single-stranded DNase in vitro, type III systems eliminate double-stranded DNA targets in vivo, suggesting that transcription generates critical DNA substrates. Here, we show that conserved RNA polymerase anti-backtracking factors, such as GreA, selectively inhibit DNA-targeting of type III immunity. GreA overexpression abolishes protection against both phages and conjugative plasmids, whereas mutations that disrupt GreA anti-backtracking activity eliminate this effect. In contrast, immunity mediated by the type II CRISPR-Cas system or the auxiliary type III RNase Csm6 remain unaffected, demonstrating that GreA specifically suppresses Cas10-dependent DNA interference. These findings identify RNA polymerase backtracking as a key determinant of Cas10-Csm activity and support a model in which arrested transcription complexes generate substrates for DNA degradation, opening up new anti-CRISPR strategies against type III.