The widespread IS200/IS605 transposon family encodes diverse programmable RNA-guided endonucleases
Open accessAbstract
Tracing the origin of CRISPR-Cas: CRISPR-Cas systems have transformed genome editing and other biotechnologies; however, the broader origins and diversity of RNA-guided nucleases have largely remained unexplored. Altae-Tran et al . show that three distinct transposon-encoded proteins, IscB, IsrB, and TnpB, are naturally occurring, reprogrammable RNA-guided DNA nucleases (see the Perspective by Rousset and Sorek). In addition to identifying diverse guide-encoding mechanisms, the authors elucidate the evolutionary relationship between IsrB, IscB, and CRISPR-Cas9. Overall, these newly characterized systems, called OMEGA (for obligate mobile element–guided activity) systems, are found in all domains of life and may be harnessed for biotechnology development. —DJ
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The IS200/IS605 family spreads through peel-and-paste transposition catalyzed by homodimeric TnpA interacting with terminal hairpins at the left and right ends of the element.
“The IS200/IS605 family of MGEs spreads through “peel-and-paste” transposition catalyzed by the homodimeric transposase TnpA interacting with terminal hairpins at the left end (LE) and right end (RE) of the element.”
From Sequence modeling and design from molecular to genome scale with Evo (2024) · cites this paper as [68]
The 'Evolution and diversity of IscB systems' section says that TnpA mobilizes transposon ends, and the Discussion refers to TnpA-catalyzed transposition. Our copy does not describe that process as 'peel-and-paste' or say that TnpA is homodimeric and interacts with terminal hairpins at both ends (pages unavailable). (LLM, conf=0.84)
Experimental — accuracy not yet measuredIS605 elements contain an RNA-guided TnpB nuclease and a cognate ωRNA that bias selfish inheritance of the transposable element.
“IS605 elements additionally contain an RNA-guided TnpB nuclease and a cognate ωRNA that bias the selfish inheritance of the transposable element (Fig. 4A) (66–69).”
From Sequence modeling and design from molecular to genome scale with Evo (2024) · cites this paper as [68]
The section ‘IS200/IS605 elements encode diverse RNA-guided nucleases’ (page unavailable) identifies transposon-encoded TnpB, its associated ωRNA, and RNA-guided cleavage activity. The Discussion (page unavailable) says the biological functions of these systems remain unknown; securing maintenance of IS200/IS605 insertions is a hypothesis, not an established effect on selfish inheritance. (LLM, conf=0.88)
Experimental — accuracy not yet measuredThe discovery of TnpB's RNA-guided nuclease activity expands the toolkit available for genome editing, particularly in organisms where traditional CRISPR systems may not be as effective.
“The discovery of TnpB’s RNA-guided nuclease activity expands the toolkit available for genome editing, particularly in organisms where traditional CRISPR systems may not be as effective.”
From Emerging applications of gene editing technologies for the development of climate-resilient crops (2025) · cites this paper as Altae-Tran et al., 2021
The ‘IS200/IS605 elements encode diverse RNA-guided nucleases’ section reports reprogrammable RNA-guided DNA cleavage by TnpB, and the Discussion describes TnpB as part of a diverse family of programmable nucleases. The copy does not establish that TnpB expands genome-editing capability specifically in organisms where traditional CRISPR systems are less effective; the genome-editing experiment reported here used IscB, not TnpB. No page numbers are available in the indexed passages. (LLM, conf=0.86)
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“Nature | Vol 616 | 13 April 2023 | 397 variants (Cas12m to Cas12a) may have evolved from a condensed and duplicated version of the ωRNA ancestor to allow the formation of a CRISPR array (with palindromic repeats), and (2) the tracrRNA–crRNA pair present in some Cas12 systems (Cas12f to Cas12b) may have evolved from a…”
BackgroundSelf-citationThe miniature CRISPR-Cas12m effector binds DNA to block transcription
Wu, W. Y., Mohanraju, P., Liao, C., Adiego-Pérez, B., Creutzburg, S. C. A., Makarova, K. S., Keessen, K., Lindeboom, T. A., Khan, T. S., Prinsen, S. H. P., et al. (2022) · Molecular Cell · doi:10.1016/j.molcel.2022.11.003 · Cited by 100 · see citing works
Self-citationXiang, G., Li, Y., Sun, J., Huo, Y., Cao, S., Cao, Y., Guo, Y., Yang, L., Cai, Y., Zhang, Y. E., et al. (2023) · Nature Biotechnology · doi:10.1038/s41587-023-01857-x · Cited by 102 · see citing works
“As for even smaller Cas12fs and IscBs (400–700 aa), they generally show lower activity (median: 10% editing efficiency) 12–16 , 18 .”
InfluentialAn engineered hypercompact CRISPR-Cas12f system with boosted gene-editing activity
Wu, T., Liu, C., Zou, S., Lyu, R., Yang, B., Yan, H., Zhao, M., & Tang, W. (2023) · Nature Chemical Biology · doi:10.1038/s41589-023-01380-9 · Cited by 101 · see citing works
GMOs or non-GMOs? The CRISPR Conundrum
Ahmad, A., Jamil, A., & Munawar, N. (2023) · Frontiers in Plant Science · doi:10.3389/fpls.2023.1232938 · Cited by 106 · see citing works
Structural basis for RNA-guided DNA cleavage by IscB-ωRNA and mechanistic comparison with Cas9
Schuler, G. A., Hu, C., & Ke, A. (2022) · Science · doi:10.1126/science.abq7220 · Cited by 95 · see citing works
Discovery of Diverse CRISPR-Cas Systems and Expansion of the Genome Engineering Toolbox
Koonin, E. V., Gootenberg, J. S., & Abudayyeh, O. O. (2023) · Biochemistry · doi:10.1021/acs.biochem.3c00159 · Cited by 92 · see citing works
“51 The putative II-D loci are a minimal type II version that consists only of a small Cas9 protein ( ∼ 700 amino acids) and a CRISPR array (Figure 2).”
BackgroundResultInfluentialSelf-citationDevelopment of miniature base editors using engineered IscB nickase
Han, D., Xiao, Q., Wang, Y., Zhang, H., Dong, X., Li, G., Kong, X., Wang, S., Song, J., Zhang, W., et al. (2023) · Nature Methods · doi:10.1038/s41592-023-01898-9 · Cited by 92 · see citing works
CRISPR/Cas9 Landscape: Current State and Future Perspectives
Tyumentsev, A. I., Tyumentsev, A. I., & Акимкин, В. Г. (2023) · International Journal of Molecular Sciences · doi:10.3390/ijms242216077 · Cited by 95 · see citing works
“Like Cas12, Fanzor (Fz) proteins are believed to be putative descendants of the recently discovered OMEGA effector TnpB (Transposase B, Transposon-associated protein) [379,380].”
BackgroundEvolutionary plasticity and functional versatility of CRISPR systems
Koonin, E. V., & Makarova, K. S. (2022) · PLoS Biology · doi:10.1371/journal.pbio.3001481 · Cited by 89 · see citing works
“In a phylogenetic tree rooted by the recently discovered IsrB proteins, homologs of IscB and Cas9 that lack the HNH domain, the Cas9 branch, is lodged with IscB, supporting this ancestral relationship [49].”
BackgroundSelf-citationViral delivery of an RNA-guided genome editor for transgene-free germline editing in Arabidopsis
Weiss, T. J., Kamalu, M., Shi, H., Li, Z., Amerasekera, J., Zhong, Z., Adler, B. A., Song, M. M., Vohra, K., Wirnowski, G., et al. (2025) · Nature Plants · doi:10.1038/s41477-025-01989-9 · Cited by 86 · see citing works
Mammalian genome innovation through transposon domestication
Modzelewski, A. J., Chong, J. G., Wang, T., & He, L. (2022) · Nature Cell Biology · doi:10.1038/s41556-022-00970-4 · Cited by 82 · see citing works
Lopes, R., & Prasad, M. K. (2024) · Frontiers in Bioengineering and Biotechnology · doi:10.3389/fbioe.2023.1339189 · Cited by 82 · see citing works
Click editing enables programmable genome writing using DNA polymerases and HUH endonucleases
da Silva, J. F., Tou, C. J., King, E. M., Eller, M. L., Rufino‐Ramos, D., Ma, L., Cromwell, C. R., Metović, J., Benning, F. M. C., Chao, L. H., et al. (2024) · Nature Biotechnology · doi:10.1038/s41587-024-02324-x · Cited by 83 · see citing works
“…functionality of the CE architecture with SaCas9 indicates that it could also be compatible with other miniature RNA-guided nickases (for example, Cas9 orthologs 94 or IscB 95 ), in which a smaller coding sequence would facilitate improved component manufacturing and delivery owing to reduced size.”
Structural mechanism of bridge RNA-guided recombination
Hiraizumi, M., Perry, N. T., Durrant, M. G., Soma, T., Nagahata, N., Okazaki, S., Athukoralage, J. S., Isayama, Y., Pai, J. J., Pawluk, A., et al. (2024) · Nature · doi:10.1038/s41586-024-07570-2 · Cited by 84 · see citing works
Chen, W., Ma, J., Wu, Z., Wang, Z., Zhang, H., Fu, W., Pan, D., Jin, S. K., & Ji, Q. (2023) · Molecular Cell · doi:10.1016/j.molcel.2023.06.014 · Cited by 79 · see citing works
Diversity, evolution, and classification of the RNA-guided nucleases TnpB and Cas12
Altae-Tran, H., Shmakov, S., Makarova, K. S., Wolf, Y. I., Kannan, S., Zhang, F., & Koonin, E. V. (2023) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.2308224120 · Cited by 74 · see citing works
“Although the RNA - guided DNA cleavage by OMEGA systems has been characterized in mechanistic detail, the biological role of these RNA - guided nucleases in the transposon life cycle remains uncertain. ω RNAs are highly structured RNAs with multiple hairpins and an additional, typically variable region that serves as…”
InfluentialSelf-citationRecent application of CRISPR-Cas12 and OMEGA system for genome editing
Badon, I. W., Oh, Y., Kim, H., & Lee, S. H. (2023) · Molecular Therapy · doi:10.1016/j.ymthe.2023.11.013 · Cited by 73 · see citing works
“24 The results of treating ISDra2 TnpB and SpuFanzor effectors in human-derived cell lines showed a typical indel pattern very similar to that of Cas12 endonucleases.”
Cheng, P., Mao, C., Tang, J., Yang, S., Cheng, Y., Wang, W., Gu, Q., Han, W., Chen, H., Li, S., et al. (2024) · Cell Research · doi:10.1038/s41422-024-00989-2 · Cited by 76 · see citing works
“46 We fi rst focused on the transposase-related RNA-guided nuclease TnpB, 31,47 whose relatively low editing activity in mammalian cells limits its wide applications.”
Li, Z., Wang, J., Xu, J.-P., Yang, X. F., & Yang, X. F. (2023) · Military Medical Research · doi:10.1186/s40779-023-00447-x · Cited by 68 · see citing works
“Being only two-fifths the size of Cas9, IscB and TnpB can mediate double-strand DNA cleavage at the target sites with a 3′ or 5′ transposon-associated motif (TAM), and both have been adopted for genome editing in human cells [27, 28].”
BackgroundInfluentialPudžiuvelytė, I., Olechnovič, K., Godliauskaitė, E., Sermokas, K., Urbaitis, T., Gasiūnas, G., & Kazlauskas, D. (2024) · Bioinformatics · doi:10.1093/bioinformatics/btae157 · Cited by 65 · see citing works
“Two groups of TnpBs (namely, TnpB2 and TnpB-Kra, which contains TnpB from Ktedonobacter racemifer (Altae-Tran et al. 2021)) showed higher predicted thermal stability compared to a group represented by TnpB from Deinococcus radiodur-ans ISDra2.”
Xu, W., Zhang, S., Qin, H., & Yao, K. (2024) · Journal of Translational Medicine · doi:10.1186/s12967-024-05957-3 · Cited by 67 · see citing works
The compact Casπ (Cas12l) ‘bracelet’ provides a unique structural platform for DNA manipulation
(2023) · doi:10.1038/s41422-022-00771-2 · Cited by 64 · see citing works
“(Schuler et al., 2022) Then starting from the IscB or other ancestors like TnpB for type V effectors,(Altae-Tran et al., 2021; Karvelis et al., 2021; Koonin et al., 2017a) this trend may suggest an RNA-protein co-evolution path underlying the CRISPR effectors (Figure S11a, b) (Cech, 2009; Poole et…”
BackgroundStructure-guided discovery of ancestral CRISPR-Cas13 ribonucleases
Yoon, P. H., Zhang, Z. T., Loi, K. J., Adler, B. A., Lahiri, A., Vohra, K., Shi, H., Rabelo, D. B., Trinidad, M. I., Boger, R. S., et al. (2024) · Science · doi:10.1126/science.adq0553 · Cited by 59 · see citing works
Transposon-encoded nucleases use guide RNAs to promote their selfish spread
Meers, C., Le, H. C., Pesari, S. R., Hoffmann, F. T., Walker, M. W. G., Gezelle, J., Tang, S., & Sternberg, S. H. (2023) · Nature · doi:10.1038/s41586-023-06597-1 · Cited by 60 · see citing works
“The following active site mutations were introduced to inactivate the endonuclease domains of the respective 3×FLAG-tagged proteins: Gst IscB (D87A, H238A, H239A); Gst TnpB (D196A); Spy Cas9 (D10A, H840A); As Cas12a (D908A).”
BackgroundInfluentialProgrammable RNA-guided DNA endonucleases are widespread in eukaryotes and their viruses
Jiang, K., Lim, J., Sgrizzi, S., Trinh, M., Kayabolen, A., Yutin, N., Bao, W., Kato, K., Koonin, E. V., Gootenberg, J. S., et al. (2023) · Science Advances · doi:10.1126/sciadv.adk0171 · Cited by 60 · see citing works
“This relationship is supported by phylogenetic analysis of the RuvC-like domains, which indicates independent origins of Cas12s of different type V subtypes from distinct groups of TnpBs ( 6 , 7 ).”
InfluentialSelf-citationTIGR-Tas: A family of modular RNA-guided DNA-targeting systems in prokaryotes and their viruses
Faure, G., Saito, M., Wilkinson, M. E., Quinones‐Olvera, N., Xu, P., Flam-Shepherd, D., Kim, S. S., Reddy, N. R., Zhu, S., Evgeniou, L., et al. (2025) · Science · doi:10.1126/science.adv9789 · Cited by 62 · see citing works
“Given that RNA-guided endonucleases belonging to CRISPR and OMEGA systems require a protospacer-adjacent motif (PAM) or target-adjacent motif (TAM) for efficient target DNA cleavage (12), we tested if Tas nucleases have a similar requirement.”
Self-citationDe novo gene synthesis by an antiviral reverse transcriptase
Tang, S., Conte, V., Zhang, D. J., Žedaveinytė, R., Lampe, G. D., Wiegand, T., Tang, L. C., Wang, M., Walker, M. W. G., George, J. T., et al. (2024) · Science · doi:10.1126/science.adq0876 · Cited by 58 · see citing works
Advances in gene editing without residual transgenes in plants
He, Y., Mudgett, M., & Zhao, Y. (2021) · PLANT PHYSIOLOGY · doi:10.1093/plphys/kiab574 · Cited by 57 · see citing works
Emerging trends in prime editing for precision genome editing
Lee, J., Kweon, J., & Kim, Y. (2025) · Experimental & Molecular Medicine · doi:10.1038/s12276-025-01463-8 · Cited by 60 · see citing works
Non-GM Genome Editing Approaches in Crops
Gong, Z., Cheng, M., & Botella, J. R. (2021) · Frontiers in Genome Editing · doi:10.3389/fgeed.2021.817279 · Cited by 51 · see citing works
“Obligated mobile element-guided activity (OMEGA) is another class of transposon-encoded RGENs with a reduced size (∼400 amino acids) (Altae-Tran et al., 2021).”
BackgroundHypercompact adenine base editors based on a Cas12f variant guided by engineered RNA
Kim, D. Y., Chung, Y., Lee, Y., Jeong, D., Park, K., Chin, H. J., Lee, J. M., Park, S., Ko, S., Ko, J., et al. (2022) · Nature Chemical Biology · doi:10.1038/s41589-022-01077-5 · Cited by 52 · see citing works
Biology and applications of CRISPR–Cas12 and transposon-associated homologs
Wu, W. Y., Adiego-Pérez, B., & John van der, O. O. S. T. (2024) · Nature Biotechnology · doi:10.1038/s41587-024-02485-9 · Cited by 54 · see citing works
“For DNA-targeting Cas12 nucleases, the first step of target recognition is generally based on a specific interaction between the bases/base pairs of the PAM/TAM nucleotides (mostly located in the non-target strand) and specific amino acids at one end of the positively charged target binding groove of the Cas12 protein…”
InfluentialProkaryotic Argonaute Proteins as a Tool for Biotechnology
Kropocheva, E., Lisitskaya, L., Agapov, A., Musabirov, A. A., Kulbachinskiy, A. V., & Esyunina, D. (2022) · Molecular Biology · doi:10.1134/s0026893322060103 · Cited by 52 · see citing works
“The possibility of introducing targeted changes into the cell genome using these nucleases was shown [57, 58].”
BackgroundA naturally DNase-free CRISPR-Cas12c enzyme silences gene expression
Huang, C. J., Adler, B. A., & Doudna, J. A. (2022) · Molecular Cell · doi:10.1016/j.molcel.2022.04.020 · Cited by 51 · see citing works
Compendium of Plant-Specific CRISPR Vectors and Their Technical Advantages
Alok, A., Chauhan, H., Upadhyay, S. K., Pandey, A. K., Kumar, J., & Singh, K. (2021) · Life · doi:10.3390/life11101021 · Cited by 50 · see citing works
Structure of the IscB–ωRNA ribonucleoprotein complex, the likely ancestor of CRISPR-Cas9
Kato, K., Okazaki, S., Kannan, S., Altae-Tran, H., Demircioglu, F. E., Isayama, Y., Ishikawa, J., Fukuda, M., Macrae, R. K., Nishizawa, T., et al. (2022) · Nature Communications · doi:10.1038/s41467-022-34378-3 · Cited by 49 · see citing works
Self-citationThe expanded CRISPR toolbox for constructing microbial cell factories
Teng, Y., Jiang, T., & Yan, Y. (2023) · Trends in biotechnology · doi:10.1016/j.tibtech.2023.06.012 · Cited by 50 · see citing works
Karmakar, S., Panda, D., Panda, S., Dash, M., Saha, R., Das, P., Avinash, S. P., Shih, J., Yang, Y., Nayak, A. K., et al. (2024) · Plant Biotechnology Journal · doi:10.1111/pbi.14416 · Cited by 50 · see citing works
CRISPR/Cas systems accelerating the development of aptasensors
Zhu, C., Zhang, F., Li, H., Chen, Z., Yan, M., Li, L., & Qu, F. (2022) · TrAC Trends in Analytical Chemistry · doi:10.1016/j.trac.2022.116775 · Cited by 48 · see citing works
Self-citationA programmable seekRNA guides target selection by IS1111 and IS110 type insertion sequences
Siddiquee, R., Pong, C. H., Hall, R. M., & Ataide, S. F. (2024) · Nature Communications · doi:10.1038/s41467-024-49474-9 · Cited by 48 · see citing works
“…and shortest lengths of cargo insert that can be ef fi ciently moved, a clear advantage of the systems reported here over others studied to date 35 – 38 is the fact that only a single protein of modest size (Tnp are about 340 aa for IS 1111 s and 315 – 450 for IS 110 s) plus a short seekRNA is…”
Going viral: The role of mobile genetic elements in bacterial immunity
Beamud, B., Benz, F., & Bikard, D. (2024) · Cell Host & Microbe · doi:10.1016/j.chom.2024.05.017 · Cited by 47 · see citing works
Structure of the OMEGA nickase IsrB in complex with ωRNA and target DNA
Hirano, S., Kappel, K., Altae-Tran, H., Faure, G., Wilkinson, M. E., Kannan, S., Demircioglu, F. E., Yan, R., Shiozaki, M., Yu, Z., et al. (2022) · Nature · doi:10.1038/s41586-022-05324-6 · Cited by 45 · see citing works
Self-citationLi, Z., Guo, R., Sun, X.-Z., Li, G., Shao, Z., Huo, X., Yang, R., Liu, X., Cao, X., Zhang, H., et al. (2024) · Nature Communications · doi:10.1038/s41467-024-45197-z · Cited by 46 · see citing works
Emerging applications of gene editing technologies for the development of climate-resilient crops
Chavhan, R. L., Jaybhaye, S. G., Hinge, V. R., Deshmukh, A. S., Shaikh, U. S., Jadhav, P. K., Kadam, U. S., & Hong, J. C. (2025) · Frontiers in Genome Editing · doi:10.3389/fgeed.2025.1524767 · Cited by 43 · see citing works
Reprogrammable RNA-targeting CRISPR systems evolved from RNA toxin-antitoxins
Zilberzwige‐Tal, S., Altae-Tran, H., Kannan, S., Wilkinson, M. E., Vo, C.-D.-T., Strebinger, D., Edmonds, K. K., Yao, C.-C. J., Mears, K. S., Shmakov, S., et al. (2025) · Cell · doi:10.1016/j.cell.2025.01.034 · Cited by 42 · see citing works
“6 We sought to determine if the likely ancestors of Cas13, c13c1, AbiF, and F13a1/2 are also associated with ncRNAs, and if so, what their biological function is.”
Self-citationA strategy for Cas13 miniaturization based on the structure and AlphaFold
Zhao, F., Zhang, T., Sun, X., Zhang, X., Chen, L., Wang, H., Li, J., Peng, F., Lai, L., Sui, T., et al. (2023) · Nature Communications · doi:10.1038/s41467-023-41320-8 · Cited by 43 · see citing works
“Cas13bt, EsCas13d, RspCas13d and PspCas13b were obtained from Addgene (#176316, #108303, #108305, and #103862, respectively) 7,16,23 .”
Prado, G. S., Rocha, D. C., dos Santos, L. N., Contiliani, D. F., Nóbile, P. M., Martinati, J. C., Padilha, L., Maluf, M. P., Lubini, G., Pereira, T. C., et al. (2024) · Frontiers in Plant Science · doi:10.3389/fpls.2023.1331258 · Cited by 41 · see citing works
Exploiting viral vectors to deliver genome editing reagents in plants
Shen, Y., Tao, Y., Li, Z., Kimutai, T. H., Song, H., Dong, O. X., & Wan, J. (2024) · aBIOTECH · doi:10.1007/s42994-024-00147-7 · Cited by 41 · see citing works
“Meanwhile, more compact sequence-specific nucleases, such as Cas12f, IscB, and TnpB, are strong candidates to be delivered using virus-based systems (Han Altae-Tran et al. 2021; Karvelis et al. 2021; Siksnys et al. 2020).”
BackgroundEukaryotic RNA-guided endonucleases evolved from a unique clade of bacterial enzymes
Yoon, P. H., Skopintsev, P., Shi, H., Chen, L.-X., Adler, B. A., Al-Shimary, M. J., Craig, R. J., Loi, K. J., DeTurk, E. C., Li, Z., et al. (2023) · Nucleic Acids Research · doi:10.1093/nar/gkad1053 · Cited by 36 · see citing works
Engineering IscB to develop highly efficient miniature editing tools in mammalian cells and embryos
Xue, N., Hong, D., Zhang, D., Wang, Q., Zhang, S., Yang, L., Chen, X., Li, Y., Han, H., Hu, C., et al. (2024) · Molecular Cell · doi:10.1016/j.molcel.2024.07.007 · Cited by 41 · see citing works
The Bibliometric Landscape of Gene Editing Innovation and Regulation in the Worldwide
Wei, X., Pu, A., Liu, Q., Hou, Q., Zhang, Y., An, X., Long, Y., Jiang, Y., Dong, Z., Wu, S., et al. (2022) · Cells · doi:10.3390/cells11172682 · Cited by 39 · see citing works
Engineered IscB–ωRNA system with expanded target range for base editing
Xiao, Q., Li, G., Han, D., Wang, H., Yao, M., Ma, T., Zhou, J., Zhang, Y., Zhang, X., He, B., et al. (2024) · Nature Chemical Biology · doi:10.1038/s41589-024-01706-1 · Cited by 39 · see citing works
“This is similar to a previous study showing that OgeuIscB exhibited indel activity with a guide containing a spacer length of 14–26 nt 17 .”
Developments and prospects for doubled haploid wheat
Eliby, S., Беккужина, С. С., Kishchenko, O., Iskakova, G., Kylyshbayeva, G., Jatayev, S., Soole, K. L., Langridge, P., Borisjuk, N. V., & Shavrukov, Y. N. (2022) · Biotechnology Advances · doi:10.1016/j.biotechadv.2022.108007 · Cited by 38 · see citing works
Molecular basis and engineering of miniature Cas12f with C-rich PAM specificity
Su, M., Li, F., Wang, Y., Gao, Y., Lan, W., Shao, Z., Zhu, C., Tang, N., Gan, J., Wu, Z., et al. (2023) · Nature Chemical Biology · doi:10.1038/s41589-023-01420-4 · Cited by 38 · see citing works
“However, most of these compact nucleases exhibit only low or minimal genome editing capacity in mammalian cells in their natural forms 11,23,27,28,31 .”
TnpB homologues exapted from transposons are RNA-guided transcription factors
Wiegand, T., Hoffmann, F. T., Walker, M. W. G., Tang, S., Richard, E., Le, H. C., Meers, C., & Sternberg, S. H. (2024) · Nature · doi:10.1038/s41586-024-07598-4 · Cited by 36 · see citing works
The transposon-encoded protein TnpB processes its own mRNA into ωRNA for guided nuclease activity
Nety, S. P., Altae-Tran, H., Kannan, S., Demircioglu, F. E., Faure, G., Hirano, S., Mears, K., Zhang, Y., Macrae, R. K., & Zhang, F. (2023) · The CRISPR Journal · doi:10.1089/crispr.2023.0015 · Cited by 35 · see citing works
“The limited TAM diversity of TnpBs may be explained by the co-evolution of TnpA and TnpB, as TnpA also utilizes the TAM to recognize the transposon ssDNA for excision and insertion.(1,2,6,43,52) Although it is not yet clear how, if at all, TnpB interacts with TnpA and the transposon DNA, the fact that both proteins po…”
BackgroundMethodInfluentialThe type V effectors for CRISPR/Cas-mediated genome engineering in plants
Zhang, R., Chai, N., Liu, T., Zheng, Z., Lin, Q., Xie, X., Wen, J., Yang, Z. F., Liu, Y., & Zhu, Q. (2024) · Biotechnology Advances · doi:10.1016/j.biotechadv.2024.108382 · Cited by 36 · see citing works
TadA reprogramming to generate potent miniature base editors with high precision
Zhang, S., Song, L., Yuan, B., Zhang, C., Cao, J., Chen, J., Qiu, J., Tai, Y., Chen, J., Qiu, Z., et al. (2023) · Nature Communications · doi:10.1038/s41467-023-36004-2 · Cited by 36 · see citing works
CRISPR-assisted transcription activation by phase-separation proteins
Liu, J., Chen, Y., Nong, B., Luo, X., Cui, K., Li, Z., Zhang, P., Tan, W., Yang, Y., Ma, W., et al. (2023) · Protein & Cell · doi:10.1093/procel/pwad013 · Cited by 36 · see citing works
“It is conceivable that if SpCas9 is further replaced with smaller Cas proteins such as IscB (Kapitonov et al., 2015; Altae-Tran et al., 2021; Schuler et al., 2022) and Cas12f (Xu et al., 2021b), the resulting fusion proteins along with the gRNA may be packaged into a single vector (e.g.,…”
BackgroundEngineering miniature IscB nickase for robust base editing with broad targeting range
Han, L., Hu, Y., Mo, Q., Yang, H., Gu, F., Bai, F., Sun, Y., & Ma, H. (2024) · Nature Chemical Biology · doi:10.1038/s41589-024-01670-w · Cited by 38 · see citing works
“The TAM of OgeuIscB was characterized to be NWRRNA (W represents A or T, and R represents A or G) by a biochemical TAM screen assay 22,24 .”
Assessing and engineering the IscB–ωRNA system for programmed genome editing
Yan, H., Tan, X., Zou, S., Sun, Y., Ke, A., & Tang, W. (2024) · Nature Chemical Biology · doi:10.1038/s41589-024-01669-3 · Cited by 36 · see citing works
“Identified in the human gut metagenome, OgeuIscB–ωRNA has been reported to generate double-stranded breaks (DSBs) in mammalian cells 2 .”
InfluentialApplication of CRISPR/Cas-based gene-editing for developing better banana
Tripathi, L., Ntui, V. O., & Tripathi, J. N. (2024) · Frontiers in Bioengineering and Biotechnology · doi:10.3389/fbioe.2024.1395772 · Cited by 35 · see citing works
Effective genome editing with an enhanced ISDra2 TnpB system and deep learning-predicted ωRNAs
Marquart, K. F., Mathis, N., Mollaysa, A., Müller, S., Kissling, L., Rothgangl, T., Schmidheini, L., Kulcsár, P. I., Allam, A., Kaufmann, M. M., et al. (2024) · Nature Methods · doi:10.1038/s41592-024-02418-z · Cited by 41 · see citing works
“IscB and TnpB proteins are typically more compact than Cas9 and Cas12 endonucleases 4 and are guided by RNA species derived from the transposon right-end (RE) element to bind and cleave substrate DNA 4,5 .”
Coral holobionts and biotechnology: from Blue Economy to coral reef conservation
van de Water, J. A. J. M., Tignat‐Perrier, R., Allemand, D., & Ferrier‐Pagès, C. (2021) · Current Opinion in Biotechnology · doi:10.1016/j.copbio.2021.10.013 · Cited by 36 · see citing works
Evolution-guided protein design of IscB for persistent epigenome editing in vivo
Kannan, S., Altae-Tran, H., Zhu, S., Xu, P., Strebinger, D., Oshiro, R., Faure, G., Moeller, L., Pham, J., Mears, K. S., et al. (2025) · Nature Biotechnology · doi:10.1038/s41587-025-02655-3 · Cited by 36 · see citing works
“OMEGA–IscB, the ancestor of Cas9 14 , is a particularly compelling candidate for genome and epigenome editing due to its small size (~300–550 amino acids (aa)), which renders it more conducive to delivery via adeno-associated viruses (AAVs); its dual nuclease composition, which enables control over…”
InfluentialSelf-citationRecent advances in CRISPR-based single-nucleotide fidelity diagnostics
Kohabir, K. A. V., Sistermans, E. A., & Wolthuis, R. M. F. (2025) · Communications Medicine · doi:10.1038/s43856-025-00933-4 · Cited by 39 · see citing works
Language Modeling Materializes a World Model of Protein BiologyPreprint
Candido, S., Hayes, T., Derry, A., Rao, R., Lin, Z., Verkuil, R., Wu, B. Z., Lee, J. S., Bruguera, E. S., Keval, J. A., et al. (2026) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.64898/2026.06.03.729735 · Cited by 65 · see citing works
Compact Cas9d and HEARO enzymes for genome editing discovered from uncultivated microbes
Goltsman, D. S. A., Alexander, L. M., Lin, J., Ocampo, R. F., Freeman, B., Lamothe, R. C., Rivas, A. P., Temoche-Diaz, M. M., Chadha, S., Nordenfelt, N. S., et al. (2022) · Nature Communications · doi:10.1038/s41467-022-35257-7 · Cited by 33 · see citing works
Mechanistic and evolutionary insights into a type V-M CRISPR–Cas effector enzyme
Omura, S. N., Nakagawa, R., Südfeld, C., Warren, R. V., Wu, W. Y., Hirano, H., Laffeber, C., Kusakizako, T., Kise, Y., Lebbink, J. H. G., et al. (2023) · Nature Structural & Molecular Biology · doi:10.1038/s41594-023-01042-3 · Cited by 33 · see citing works
Advances in the application of CRISPR-Cas technology in rapid detection of pathogen nucleic acid
Li, X., Zhong, J., Li, H., Qiao, Y., Mao, X., Fan, H., Zhong, Y., İmani, S., Zheng, S., & Li, J. (2023) · Frontiers in Molecular Biosciences · doi:10.3389/fmolb.2023.1260883 · Cited by 33 · see citing works
“It won the fi rst of Nature ’ s fi ve most in fl uential scienti fi c events in the past 10 years and the Nobel Prize in Chemistry in 2020 (Makarova et al., 2020; Altae-Tran et al., 2021).”
Harnessing artificial intelligence to advance CRISPR-based genome editing technologies
Thomson, T., Li, G. L., Strilchuk, A. W., Cui, H., Wang, B., & Li, B. (2025) · Nature Reviews Genetics · doi:10.1038/s41576-025-00907-1 · Cited by 32 · see citing works
“Recently discovered ancestral Cas-related proteins, such as the transposon-encoded TnpB and IscB nucleases, which use an associated ωRNA for target recognition, are emerging as programmable genome editing tools 28–30 .”
Structure and engineering of miniature Acidibacillus sulfuroxidans Cas12f1
Wu, Z., Liu, D., Pan, D. Y., Yu, H., Shi, J. F., Ma, J., Fu, W., Wang, Z., Zheng, Z., Qu, Y., et al. (2023) · Nature Catalysis · doi:10.1038/s41929-023-00995-4 · Cited by 33 · see citing works
CRISPR-Cas Technology: Emerging Applications in Clinical Microbiology and Infectious Diseases
Serajian, S., Ahmadpour, E., Oliveira, S. M. R., Pereira, M. D. L., & Heidarzadeh, S. (2021) · Pharmaceuticals · doi:10.3390/ph14111171 · Cited by 34 · see citing works
“[147] -CRISPR enzyme’s ancestors reported in microbes [148,149]”
BackgroundPromoter editing for the genetic improvement of crops
Shi, L., Su, J., Cho, M., Song, H., Dong, O. X., Liang, Y., & Zhang, Z. (2023) · Journal of Experimental Botany · doi:10.1093/jxb/erad175 · Cited by 33 · see citing works
Modularity and diversity of target selectors in Tn7 transposons
Faure, G., Saito, M., Benler, S., Peng, I., Wolf, Y. I., Strecker, J. G., Altae-Tran, H., Neumann, E. N., Li, D., Makarova, K. S., et al. (2023) · Molecular Cell · doi:10.1016/j.molcel.2023.05.013 · Cited by 30 · see citing works
Self-citationDNA on the move: mechanisms, functions and applications of transposable elements
Schmitz, M., & Querques, I. (2023) · FEBS Open Bio · doi:10.1002/2211-5463.13743 · Cited by 31 · see citing works
“Strikingly, adaptive immunity relying on CRISPR-Cas systems in prokaryotes and the V(D)J recombination pathway in vertebrates, have arisen independently via domestication of recombinases encoded by unrelated TEs [26 – 28].”
BackgroundFoley, N. M., Harris, A. J., Bredemeyer, K. R., Ruedi, M., Puechmaille, S. J., Teeling, E. C., Criscitiello, M. F., & Murphy, W. J. (2024) · Cell Genomics · doi:10.1016/j.xgen.2023.100482 · Cited by 31 · see citing works
Bio–Nano Toolbox for Precision Alzheimer's Disease Gene Therapy
Liu, Y., Xia, X., Zheng, M., & Shi, B. (2024) · Advanced Materials · doi:10.1002/adma.202314354 · Cited by 30 · see citing works
Genome editing in plants using the TnpB transposase system
Li, Q., Wang, Y., Hou, Z., Zong, H., Wang, X., Zhang, Y. E., Wang, H., Chen, H., Wang, W., & Duan, K. (2024) · aBIOTECH · doi:10.1007/s42994-024-00172-6 · Cited by 29 · see citing works
“The class 2 CRISPR effectors Cas9 and Cas12 may have evolved, independently, from an ancestral TnpB-like nuclease, which is still commonly found in insertion sequences (ISs) of the IS200/IS605 and IS607 families of transposons (Altae-Tran et al. 2021; Kapitonov et al. 2016).”
BackgroundAragonés, V., Aliaga, F., Pasin, F., & Daròs, J. (2022) · Biotechnology Journal · doi:10.1002/biot.202100504 · Cited by 29 · see citing works
CRISPR/Cas-mediated germplasm improvement and new strategies for crop protection
Dong, G., & Fan, Z. (2024) · Crop Health · doi:10.1007/s44297-023-00020-x · Cited by 28 · see citing works
“The OMEGA effector TnpB is the putative ancestor of Cas12, suggesting that TnpB may also be the ancestor of the Fanzor (Fz) protein encoded by eukaryotic trans-posons [43].”
Engineering a New Generation of Gene Editors: Integrating Synthetic Biology and AI Innovations
Chia, B. S., Seah, S., Wang, B., Shen, K., Srivastava, D., & Chew, W. L. (2025) · ACS Synthetic Biology · doi:10.1021/acssynbio.4c00686 · Cited by 29 · see citing works
Recruitment of Mobile Genetic Elements for Diverse Cellular Functions in Prokaryotes
Benler, S., & Koonin, E. V. (2022) · Frontiers in Molecular Biosciences · doi:10.3389/fmolb.2022.821197 · Cited by 28 · see citing works
“…while Cas9 was derived from a transposon-encoded nuclease IscB in a single evolutionary event (Kapitonov et al., 2016; Altae-Tran et al., 2021), Cas12 apparently evolved from transposon nucleases of the TnpB family on multiple, independent occasions (Faure et al., 2019; Altae-Tran et al., 2021).”
BackgroundInfluentialSelf-citationMini-PE, a prime editor with compact Cas9 and truncated reverse transcriptase
Lan, T., Chen, H., Tang, C., Wei, Y., Liu, Y., Zhou, J., Zhuang, Z., Zhang, Q., Chen, M., Zhou, X., et al. (2023) · Molecular Therapy — Nucleic Acids · doi:10.1016/j.omtn.2023.08.018 · Cited by 28 · see citing works
Miniature CRISPR-Cas12 Systems: Mechanisms, Engineering, and Genome Editing Applications
Tang, N., & Ji, Q. (2024) · ACS Chemical Biology · doi:10.1021/acschembio.4c00247 · Cited by 26 · see citing works
CRISPR-Cas, Argonaute proteins and the emerging landscape of amplification-free diagnostics
Santiago‐Frangos, A., Nemudryi, A. A., Nemudraia, A., Wiegand, T., Nichols, J. E., Krishna, P., Scherffius, A. M., Zahl, T., Wilkinson, R. A., & Wiedenheft, B. (2022) · Methods · doi:10.1016/j.ymeth.2022.06.002 · Cited by 25 · see citing works
“Recently, TnpB proteins, the evolutionary precursors to Cas12, have been shown to be RNAguided DNA-binding proteins with collateral nuclease activity [47,48].”
- Authors
- Altae-Tran, H., Kannan, S., Demircioglu, F. E., Oshiro, R., Nety, S. P., McKay, L. J., Dlakić, M., Inskeep, W. P., Makarova, K. S., Macrae, R. K., Koonin, E. V., & Zhang, F.
- Affiliations
- Broad Institute (US)
- Montana State University (US)
- United States National Library of Medicine (US)
- Corresponding author
- Altae-Tran, Han
- Kannan, Soumya
- Zhang, Feng
- ORCID
- Venue
- Science
- Published
- 2021-10
- Volume
- 374
- Issue
- 6563
- Pages
- 57-65
- ISSN
- 0036-8075 (ISSN-L)
- 1095-9203
- PMC
- PMC8929163
- OpenAlex
- W3198344223
- Cited by
- 407
- References
- 65
- Citations by year
- 2026: 78 · 2025: 92 · 2024: 102 · 2023: 77 · 2022: 42 · 2021: 16
- FWCI
- 18.66
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Science (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion)
- Montana State University ScholarWorks (Montana State University) (submittedVersion)
- Funders
- National Science Foundation (1950770)
- U.S. Department of Energy
- Edward Mallinckrodt, Jr. Foundation
- Open Philanthropy Project
- Joint Genome Institute
- National Institutes of Health (5r01hg009761-06, HG009761, 1R01- HG009761)
- National Park Service
- National Heart, Lung, and Blood Institute (DP1HL141201)
- National Human Genome Research Institute (R01-HG009761)
- U.S. National Library of Medicine
- Division of Environmental Biology
- 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, RNA-guided endonuclease, genome editing, transposable elements, RNA-guided DNA cleavage
- MeSH terms
- CRISPR-Associated Protein 9, RNA, Guide, CRISPR-Cas Systems, Bacterial Proteins, DNA Transposable Elements, Endodeoxyribonucleases, Genetic Code, Genetic Variation, Conserved Sequence, Evolution, Molecular, RNA, Untranslated, CRISPR-Cas Systems, CRISPR-Associated Proteins
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