The transposon-encoded protein TnpB processes its own mRNA into ωRNA for guided nuclease activity
Open accessAbstract
TnpB is a member of the Obligate Mobile Element Guided Activity (OMEGA) RNA-guided nuclease family, is harbored in transposons, and likely functions to maintain the transposon in genomes. Previously, it was shown that TnpB cleaves double- and single-stranded DNA substrates in an RNA-guided manner, but the biogenesis of the TnpB ribonucleoprotein (RNP) complex is unknown. Using in vitro purified apo TnpB, we demonstrate the ability of TnpB to generate guide omegaRNA (ωRNA) from its own mRNA through 5′ processing. We also uncover a potential cis -regulatory mechanism whereby a region of the TnpB mRNA inhibits DNA cleavage by the RNP complex. We further expand the characterization of TnpB by examining ωRNA processing and RNA-guided nuclease activity in 59 orthologs spanning the natural diversity of the TnpB family. This work reveals a new functionality, ωRNA biogenesis, of TnpB, and characterizes additional members of this biotechnologically useful family of programmable enzymes.
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Structural insights into the diversity and DNA cleavage mechanism of Fanzor
Xu, P., Saito, M., Faure, G., Maguire, S., Vo, C.-D.-T., Wilkinson, M. E., Kuang, H., Wang, B., Rice, W. J., Macrae, R. K., et al. (2024) · Cell · doi:10.1016/j.cell.2024.07.050 · Cited by 17 · see citing works
Feng, X., Xu, R., Liao, J., Zhao, J., Zhang, B., Xu, X., Zhao, P., Wang, X., Yao, J., Wang, P., et al. (2024) · Nature Communications · doi:10.1038/s41467-024-47697-4 · Cited by 15 · see citing works
“Our fi nding might be extended to diverse RNA-guided systems, including different TnpB nucleases 52,61,62 , Cas12 nucleases and recently identi fi ed eukaryotic Fanzor nucleases that are closely related to TnpB 31 – 33 , 63 .”
STAGE: A compact and versatile TnpB-based genome editing toolkit for Streptomyces
Luo, J., Chia, N., Qin, Y., Tan, P., Zhang, L., Yang, S., Yuan, Z., Liang, H., Lee, S. Y., & Tong, Y. (2025) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.2509146122 · Cited by 13 · see citing works
“Supporting this interpretation, previous in vitro studies have shown that the TnpB ortholog from Alicyclobacillus mac-rosporangiidus (AmaTnpB) possesses intrinsic RNA processing activity, enabling the generation of reRNA molecules required for target DNA cleavage ( 22 ).”
Koonin, E. V., & Krupovìč, M. (2023) · Current Opinion in Systems Biology · doi:10.1016/j.coisb.2023.100473 · Cited by 11 · see citing works
Antagonistic conflict between transposon-encoded introns and guide RNAs
Žedaveinytė, R., Meers, C., Le, H. C., Mortman, E. E., Tang, S., Lampe, G. D., Pesari, S. R., Gelsinger, D. R., Wiegand, T., & Sternberg, S. H. (2024) · Science · doi:10.1126/science.adm8189 · Cited by 9 · see citing works
RNA-guided genome engineering: paradigm shift towards transposons
Chang, C.-W., Truong, V. A., Pham, N. N., & Hu, Y. (2024) · Trends in biotechnology · doi:10.1016/j.tibtech.2024.02.006 · Cited by 9 · see citing works
CRISPR beyond: harnessing compact RNA-guided endonucleases for enhanced genome editing
Wang, F., Ma, S., Zhang, S., Ji, Q., & Hu, C. (2024) · Science China Life Sciences · doi:10.1007/s11427-023-2566-8 · Cited by 9 · see citing works
“…OMEGA systems share a foundational RNA-guided nucleic acid cleavage activity with their more widely recognized successors (Altae-Tran et al., 2021; Altae-Tran et al., 2023; Nakagawa et al., 2023; Nety et al., 2023; Saito et al., 2023; Sasnauskas et al., 2023; Xiang et al., 2024; Xu et al., 2023).”
Tang, H., Gao, J., Wang, H., Sun, M., Zhang, S., Song, C., & Li, Q. (2025) · Communications Biology · doi:10.1038/s42003-025-07521-1 · Cited by 8 · see citing works
“It has also been shown that TnpB can successfully optimize its mRNA into ω RNA 32 .”
Programmable RNA-guided endonucleases are widespread in eukaryotes and their virusesPreprint
Jiang, K., Lim, J., Sgrizzi, S. R., Trinh, M., Kayabölen, A., Yutin, N., Koonin, E. V., Abudayyeh, O. O., & Gootenberg, J. S. (2023) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2023.06.13.544871 · Cited by 6 · see citing works
“To demonstrate that these diverged HERMES actively process and associate with their cognate hRNAs, we focused on a family 2 HERMES nuclease from the unicellular green alga Chlamydomonas reinhardtii (CreHNuc) (Fig.”
InfluentialXu, Y. C., Yin, W., Cheng, Y., Zeng, W., Li, W., Chen, W., Wang, F., Peng, N., Ma, L., & Liu, T. (2024) · Communications Biology · doi:10.1038/s42003-024-07123-3 · Cited by 5 · see citing works
Fanzors, a family of eukaryotic RNA ‐guided DNA endonucleases
Jiang, K., Gootenberg, J. S., & Abudayyeh, O. O. (2025) · FEBS Letters · doi:10.1002/1873-3468.70038 · Cited by 4 · see citing works
“In contrast to the T-rich TAMs of TnpB and protospacer adjacent motifs of Cas12 family enzymes, the Fanzor TAM preference is diverse, with a G/C-rich preference observed for many viral Fanzor nucleases and A/T-rich preferences for the nuclear genome-encoded Fanzors [10 – 12].”
George, N. A., Zhou, Z., Anantharaman, K., & Hug, L. A. (2024) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2024.05.30.596742 · Cited by 4 · see citing works
Can Fusobacterium utilize nucleomodulins in the pathophysiology of CRC?
Anand, S., Korgaonkar, S., & Bose, C. (2025) · Frontiers in Cellular and Infection Microbiology · doi:10.3389/fcimb.2025.1644443 · Cited by 3 · see citing works
Transposons disrupt genomic stability and trigger cancers
Dong, W., Li, M., Li, P., Hou, X. S., He, F., & Ji, S. (2025) · Frontiers in Cellular and Infection Microbiology · doi:10.3389/fcimb.2025.1704405 · Cited by 3 · see citing works
TnpB: Transposon Retention Mechanisms as Potential Tools for Gene Editing
Shen, Q., Hao, Q., Xue, Y., & Zheng, Y. (2025) · Applied Biochemistry and Microbiology · doi:10.1134/s0003683825601088 · Cited by 2 · see citing works
George, N. A., Zhou, Z., Anantharaman, K., & Hug, L. A. (2025) · Virology Journal · doi:10.1186/s12985-025-02990-6 · Cited by 5 · see citing works
Fanzors: Mysterious TnpB-Like Bacterial Transposon-Related RNA-Guided DNA Nucleases of Eukaryotes
Karvelis, T., & Šikšnys, V. (2023) · The CRISPR Journal · doi:10.1089/crispr.2023.29164.tka · Cited by 2 · see citing works
Karmakar, S., Panda, D., Baig, M. J., & Molla, K. A. (2025) · Springer protocols handbooks/Springer protocols · doi:10.1007/978-1-0716-4358-7_20 · Cited by 1 · see citing works
Molecular basis of single-mismatch-induced nuclease-to-nickase conversion in TIGR–TasH
Zhou, R., Zhan, Y., Sun, Y., Wang, R., Wang, T., Liu, Z., Shan, Z., Li, X., Zhang, S., Sun, N., et al. (2026) · Nucleic Acids Research · doi:10.1093/nar/gkag743 · Cited by 1 · see citing works
“Compact effectors, including TnpB and Cas13an, the ancestors of Cas12 and Cas13 proteins, process pre-crRNAs and cleave target nucleic acids using a single active site [ 37 , 38 ], whereas larger effectors such as Cas12 and Cas13 employ separate active sites for the two activities.”
Tang, H., Gao, J., Sun, M., Zhang, S., & Li, Q. (2024) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2024.09.04.611128
“…editing in Pediococcus acidilactici and Sulfolobus islandicus 30 ; it was reported that TnpB could optimized its own mRNA into ωRNA successfully 31 ; recently, it is particularly noteworthy that precise genome editing and phenotypic rectification in a tyrosinaemia mouse model could be achieved…”
BackgroundRosalind Franklin Society Proudly Announces the 2023 Award Recipient for The CRISPR Journal
Nety, S. P. (2024) · The CRISPR Journal · doi:10.1089/crispr.2024.55675.rfs2023
Clark, D. P., Pazdernik, N. J., McGehee, M. R., & Rader, B. A. (2025) · Elsevier eBooks · doi:10.1016/b978-0-443-18484-0.00020-1
Brück, M., Daume, M., Randau, L., & Gomes‐Filho, J. V. (2025) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2025.07.28.667248
Archaea express circular isoforms of IS200/IS605-associated ωRNAs
Picinato, B. A., Franceschini-Santos, V. H., Zaramela, L. S., Vêncio, R. Z. N., & Koide, T. (2025) · Frontiers in Microbiology · doi:10.3389/fmicb.2025.1641342
Brück, M., Daume, M., Randau, L., & Gomes‐Filho, J. V. (2025) · mSystems · doi:10.1128/msystems.01485-25
A Unique Type V CRISPR-Cas System Encoded by a Group of Thermus VirusesPreprint
Trofimova, A. B., Demkina, A. O., Shmakov, S., Livenskyi, A., Serebryakova, M. V., Dmitriev, A. A., Rubinshteyn, I. D., Severinov, K., & Kolesnik, M. V. (2026) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.64898/2026.04.20.719267
“In many Type V CRISPR-Cas systems and RNA-guided TnpB nucleases, the maturation of guide RNAs relies on the nuclease activity of the RuvC domain of Cas12/TnpB proteins (64– 66).”
Zhang, W., Hu, J., Li, R., Sheng, D., Yue, X., She, Q., Zhang, Y., Li, Y., Li, Z., & Wu, C. (2026) · Nature Communications · doi:10.1038/s41467-026-74908-x
“The TnpB – reRNA complex recognizes a transposon-associated motif (TAM, typically 5 ’ -TTGAT-3 ’ ) situated directly beside the protospacer and then introduces a DSB precisely 3 nucleotides downstream of this sequence element 29,30 .”
Wang, R., Liu, H., Jia, S., Bai, S., Cao, X., Li, D., & Sun, Y. (2026) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.64898/2026.09.09.750255
“Although biochemical studies have demonstrated that TnpB can process its own transcript to generate reRNA molecules (Nety et al., 2023), this native overlapping configuration has not yet been shown to support efficient genome editing in vivo (Luo et al., 2025).”
- Authors
- Nety, S. P., Altae-Tran, H., Kannan, S., Demircioglu, F. E., Faure, G., Hirano, S., Mears, K., Zhang, Y., Macrae, R. K., & Zhang, F.
- Affiliations
- Broad Institute (US)
- Corresponding author
- Zhang, Feng
- ORCID
- Venue
- The CRISPR Journal
- Publisher
- SAGE Publications
- Published
- 2023-06-01
- Volume
- 6
- Issue
- 3
- Pages
- 232-242
- ISSN
- 2573-1599 (ISSN-L)
- 2573-1602
- PMC
- PMC10278001
- OpenAlex
- W4379377215
- Cited by
- 35
- References
- 53
- Citations by year
- 2026: 4 · 2025: 13 · 2024: 12 · 2023: 6
- FWCI
- 3.12
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- The CRISPR Journal (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Funders
- National Institutes of Health (5r01hg009761-06)
- National Human Genome Research Institute (R01-HG009761)
- National Institute of General Medical Sciences (T32GM007753, T32GM144273)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Keywords
- TnpB, transposable elements, ribonucleoprotein complexes, DNA cleavage inhibition, programmable nucleases
- MeSH terms
- Gene Editing, DNA Transposable Elements, RNA, RNA, Messenger, CRISPR-Cas Systems
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