Abstract
Cas9 can process poly(T) single-stranded DNA molecules upon activation in an RNA-guided manner. Here, we uncover key determinants underlying this function. First, we show that unflanked R-loops in the RNA 5′ side favor trans-cleavage activity, which occur when targeting short double-stranded DNA molecules. Second, we show that elongated guide RNA spacers beyond the canonical 20 bases, even by a few bases, severely impair this collateral activity. Third, although trans-cleavage is mediated by the RuvC domain, we show that a catalytically active HNH domain contributes to an efficient process. Analysis of structural models provides tentative mechanistic insights. Together, these findings illustrate that fine modulation of Cas9 function can be achieved.
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References
Barrangou, R. & Marraffini, L. A. CRISPR-Cas systems: prokaryotes upgrade to adaptive immunity. Mol. Cell 54, 234–244 (2014).
Pickar-Oliver, A. & Gersbach, C. A. The next generation of CRISPR-Cas technologies and applications. Nat. Rev. Mol. Cell Biol. 20, 490–507 (2019).
Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).
Wu, Y. et al. Highly efficient therapeutic gene editing of human hematopoietic stem cells. Nat. Med. 25, 776–783 (2019).
Zalatan, J. G. et al. Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds. Cell 160, 339–350 (2015).
Xu, X. & Qi, L. S. A CRISPR-dCas toolbox for genetic engineering and synthetic biology. J. Mol. Biol. 431, 34–47 (2019).
Chen, J. S. et al. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science 360, 436–439 (2018).
Gootenberg, J. S. et al. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science 356, 438–442 (2017).
Chen, J. et al. Trans-nuclease activity of Cas9 is activated by DNA or RNA target binding. Nat. Biotechnol. 43, 558–568 (2025).
Jinek, M. et al. Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. Science 343, 1247997 (2014).
Mekler, V., Minakhin, L. & Severinov, K. Mechanism of duplex DNA destabilization by RNA-guided Cas9 nuclease during target interrogation. Proc. Natl. Acad. Sci. USA 114, 5443–5448 (2017).
Jiao, C. et al. TracrRNA reprogramming enables direct PAM-independent detection of RNA with diverse DNA-targeting Cas12 nucleases. Nat. Commun. 15, 5909 (2024).
Ma, E., Harrington, L. B., O’Connell, M. R., Zhou, K. & Doudna, J. A. Single-stranded DNA cleavage by divergent CRISPR-Cas9 enzymes. Mol. Cell 60, 398–407 (2015).
Kirillov, B. et al. Uncertainty-aware and interpretable evaluation of Cas9-grna and Cas12a-grna specificity for fully matched and partially mismatched targets with deep kernel learning. Nucleic Acids Res. 50, e11 (2022).
Marino, N. D., Pinilla-Redondo, R. & Bondy-Denomy, J. CRISPR-Cas12a targeting of ssDNA plays no detectable role in immunity. Nucleic Acids Res. 50, 6414–6422 (2022).
Allawi, H. T. & SantaLucia, J. Thermodynamics and NMR of internal G·T mismatches in DNA. Biochemistry 36, 10581–10594 (1997).
Marquez-Costa, R. et al. Multiplexable and biocomputational virus detection by CRISPR-Cas9-mediated strand displacement. Anal. Chem. 95, 9564–9574 (2023).
Mullally, G. et al. 5’ modifications to CRISPR-Cas9 gRNA can change the dynamics and size of R-loops and inhibit DNA cleavage. Nucleic Acids Res. 48, 6811–6823 (2020).
Kim, D. et al. The architecture of the SARS-CoV-2 transcriptome. Cell 181, 914–921 (2020).
Piepenburg, O., Williams, C. H., Stemple, D. L. & Armes, N. A. DNA detection using recombination proteins. PLoS Biol. 4, e204 (2006).
Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).
Sternberg, S. H., LaFrance, B., Kaplan, M. & Doudna, J. A. Conformational control of DNA target cleavage by CRISPR-Cas9. Nature 527, 110–113 (2015).
Jiang, F. et al. Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage. Science 351, 867–871 (2016).
Huai, C. et al. Structural insights into DNA cleavage activation of the CRISPR-Cas9 system. Nat. Commun. 8, 1375 (2017).
Pacesa, M. et al. R-loop formation and conformational activation mechanisms of Cas9. Nature 609, 191–196 (2022).
Bravo, J. P. K. et al. Structural basis for mismatch surveillance by CRISPR-Cas9. Nature 603, 343–347 (2022).
DeLano, W. L. The case for open-source software in drug discovery. Drug Discov. Today 10, 213–217 (2005).
Acknowledgments
Work supported by the Spanish Ministry of Science, Innovation, and Universities and AEI/10.13039/501100011033 (PDC2022-133941-I00 and PID2021-127671NB-I00, co-financed by the European Union NextGenerationEU/PRTR and European Regional Development Fund) and the Valencia Regional Government (CIPROM/2022/21). S.B. acknowledges a Juan de la Cierva contract from the Spanish Ministry of Science, Innovation, and Universities (JDC2023-052427-I) and R.D.-M. a predoctoral contract from the Valencia Regional Government (CIACIF/2023/119).
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G.R. designed the research. R.M.-M. performed the experiments under the supervision of G.R. and supported by R.R., S.B., and R.D.-M. All authors analyzed the data. G.R. wrote the manuscript. All authors revised the final manuscript.
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Montagud-Martínez, R., Ruiz, R., Baldanta, S. et al. CRISPR-Cas9 trans-cleavage is hindered by a flanked R-loop, an elongated spacer, and an inactive HNH domain. Nat Commun (2026). https://doi.org/10.1038/s41467-026-68789-3
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DOI: https://doi.org/10.1038/s41467-026-68789-3
