Half-match recombination drives bridge RNA-guided excision and off-target insertion
- 1UC Berkeley
Abstract
IS110-family bridge recombinases are a recently identified class of compact, RNA-guided editors in which a bridge RNA (bRNA) directs the recombination of a donor DNA into a target site. In the current model, the bRNA engages fully complementary donor and target sequences within a single synaptic complex to drive double-stranded recombination, implying that the transposon is cut from its donor site rather than copied, yet neither the strandedness of the excised intermediate nor the requirement for full complementarity has been tested directly. Here we reconstituted IS621 recombination in a cell-free transcription-translation system, building representative arrangements of the excision and insertion reactions and characterizing the outcomes. We find that IS621 predominantly excises a single strand, releasing a single-stranded circle and leaving the donor site intact, consistent with copy-and-paste transposition. By introducing mismatches into the bRNA target sequences, we further find that excision proceeds independently of target-site complementarity, relying strictly on donor-arm recognition; we term this "half-match" recombination, because a substrate matching only half of the bRNA is sufficient. We also find half-match activity during insertion, both in vitro and in a published genome-editing experiment, where it accounts for approximately half of non-target insertion reads. Half-match recombination provides both a mechanistic explanation for off-target insertion and a framework for the rational design of high-fidelity bridge recombinases.
Related
- Agent-guided epitope excision enables economical de novo binder design
- CRISPRi-mediated silencing of racR reveals molecular strategies for surviving lethal stress coupled to Rac prophage excision
- CRISPR-mediated gap-based replacement enables site-specific DNA lesion insertion into long DNA and nucleosomes
- Nucleotide excision repair in tumor response to DNA-damaging anticancer therapies: a comprehensive review.
- Production of membrane-embedded Bcl-2 proteins - Use of cell-free synthesis in continuous exchange for co-translational insertion of Bcl-2 proteins in lipid bilayer nanodiscs
BibTeX
@misc{xie2026halfmatch,
title = {{Half-match recombination drives bridge RNA-guided excision and off-target insertion}},
author = {B. Xie and K. Hu and H. Yang and J. E. Patel and C. Ronda and B. E. Rubin},
howpublished = {bioRxiv},
year = {2026},
month = aug,
doi = {10.64898/2026.08.31.748434},
url = {https://www.biorxiv.org/content/10.64898/2026.08.31.748434v1},
note = {CC BY-NC},
}
RIS
TY - UNPB
TI - Half-match recombination drives bridge RNA-guided excision and off-target insertion
AU - B. Xie
AU - K. Hu
AU - H. Yang
AU - J. E. Patel
AU - C. Ronda
AU - B. E. Rubin
PY - 2026
DA - 2026/08/31
PB - bioRxiv
DO - 10.64898/2026.08.31.748434
UR - https://www.biorxiv.org/content/10.64898/2026.08.31.748434v1
AB - IS110-family bridge recombinases are a recently identified class of compact, RNA-guided editors in which a bridge RNA (bRNA) directs the recombination of a donor DNA into a target site. In the current model, the bRNA engages fully complementary donor and target sequences within a single synaptic complex to drive double-stranded recombination, implying that the transposon is cut from its donor site rather than copied, yet neither the strandedness of the excised intermediate nor the requirement for full complementarity has been tested directly. Here we reconstituted IS621 recombination in a cell-free transcription-translation system, building representative arrangements of the excision and insertion reactions and characterizing the outcomes. We find that IS621 predominantly excises a single strand, releasing a single-stranded circle and leaving the donor site intact, consistent with copy-and-paste transposition. By introducing mismatches into the bRNA target sequences, we further find that excision proceeds independently of target-site complementarity, relying strictly on donor-arm recognition; we term this "half-match" recombination, because a substrate matching only half of the bRNA is sufficient. We also find half-match activity during insertion, both in vitro and in a published genome-editing experiment, where it accounts for approximately half of non-target insertion reads. Half-match recombination provides both a mechanistic explanation for off-target insertion and a framework for the rational design of high-fidelity bridge recombinases.
ER -