RNA Polymerase Backtracking Promotes Type III-A CRISPR DNA Targeting
- 1University of Wisconsin-Madison
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.
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BibTeX
@misc{viera2026polymerase,
title = {{RNA Polymerase Backtracking Promotes Type III-A CRISPR DNA Targeting}},
author = {A. J. Hernandez Viera and N. R. Gross and P. Q. Tran and O. Shahin and J. C. Kosmopoulos and K. Anantharaman and C. Y. Mo},
howpublished = {bioRxiv},
year = {2026},
month = nov,
doi = {10.64898/2026.11.04.764256},
url = {https://www.biorxiv.org/content/10.64898/2026.11.04.764256v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - RNA Polymerase Backtracking Promotes Type III-A CRISPR DNA Targeting
AU - A. J. Hernandez Viera
AU - N. R. Gross
AU - P. Q. Tran
AU - O. Shahin
AU - J. C. Kosmopoulos
AU - K. Anantharaman
AU - C. Y. Mo
PY - 2026
DA - 2026/11/04
PB - bioRxiv
DO - 10.64898/2026.11.04.764256
UR - https://www.biorxiv.org/content/10.64898/2026.11.04.764256v1
AB - 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.
ER -