The rearrangement and diversification of the genome have endowed numerous enzymes through evolution. The emergence of new genes and their functional diversification, immune development, virus dissemination, and mobile genetic elements (MGEs) occur during this process. Genomic rearrangements involving mutational changes such as insertion, deletion, and inversion contribute to genetic diversity. These rearrangements often occur through homologous recombination or transposition by foreign genetic material, such as viruses and mobile genetic components.
Hsu and colleagues have discovered a structural non-coding RNA (ncRNA) expressed by insertion sequence (IS) elements from the IS110 family. This ncRNA, named bridge RNA, specifically binds to the recombinase enzyme encoded by IS110 and contains two internal loops that base-pair with the target DNA sequence and donor DNA sequence. IS elements are abundant in bacteria and archaea and belong to the least autonomous MGEs. MGEs are everywhere and typically move through transposases, integrases, homing endonucleases, or recombinases. Members of the IS110 family are cut-and-paste MGEs that create a circular form as part of the transposition process, leaving no trace in the genome. The target and donor binding loops of Bridge RNA can be separately programmed. This capability allows for sequence-specific recombination between two DNA molecules. The modular design enables the addition of DNA to specific genomic targets and the programmable removal and inversion of DNA segments.
Both computational and experimental analyses demonstrate how bridge RNA directs IS110 recombinase to recognize and bind to target DNA and donor DNA. Strong nucleotide covariance signals show evolutionarily conserved base-pairing interactions with the target and donor sequences. Additionally, experimental reports in E. coli elucidate how the Bridge RNA system can be programmable through high-efficiency screening techniques and genome-wide insertion experiments. The IS110 bridge recombination system represents a new class of programmable DNA editing tools that complement existing technologies like CRISPR.
Bridge RNAs contain a highly compact sequence of approximately 150–250 nucleotides and encode their single effector recombinases (approximately 300-460 amino acids). IS110 targeting is achieved using internal binding loops reminiscent of tRNA hairpin loops or snoRNA internal loops, distinct from the terminal binding sequences of CRISPR-Cas or Argonaute guide RNAs. Unlike the known single-strand base-pairing mechanisms of RNA guide systems, each RNA loop encodes segments that base-pair with staggered regions of the top and bottom strand of each cognate DNA binding partner. RNA guidance of the donor form of the IS110 element also demonstrates a previously unseen mechanism of DNA movement. Initial studies of various IS110 orthologs reveal significant mechanistic diversity between both the IS110 and IS1111 subfamilies.
The IS110 bridge system provides a necessary and sufficient RNA-guided recombinase for direct DNA recombination. Modular reprogramming of target and donor recognition by bispecific Bridge RNA uniquely enables the three fundamental DNA rearrangements known for manipulating large DNA sequences and overall genome organization. The results of this study propose that the IS110 bridge recombination system enhances the diversity of nucleic acid-guided systems and offers a mechanism beyond CRISPR and RNA interference for the essential DNA rearrangements required for genome design. Insights into the evolutionary origins and molecular mechanisms of IS110 transposition provide new opportunities for engineering nucleic acid-guided systems for various biotechnological and therapeutic applications. The bridge recombination mechanism is poised to establish a new frontier in genome design, beyond RNA interference and CRISPR-based mechanisms.
Author: Fatma Duran
Editor: Elif Duymaz
References: Durrant, M.G., Perry, N.T., Pai, J.J. et al. Bridge RNAs direct programmable recombination of target and donor DNA. Nature 630, 984–993 (2024). https://doi.org/10.1038/s41586-024-07552-4
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