Biology subjects

Ronda, C.

Publications and source records attributed to Ronda, C..

5 recordsLinked to original sources

bioRxiv2026

Half-match recombination drives bridge RNA-guided excision and off-target insertion

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.

molecular biology↗

bioRxiv2025

Reconstructing the emergence of the human chorion via HIPPO-mediated trophoblast induction

The first lineage decision in the mammalian blastocyst commits outer cells to the trophectoderm and initiates the trajectory that gives rise to the placental chorion. The molecular sequence that unfolds downstream of HIPPO pathway inactivation, linking human trophectoderm specification to the early organization of the chorion, has remained unknown. Here, we establish a developmentally informed model that leverages HIPPO pathway modulation to induce the native trophectoderm trajectory in the absence of exogenous BMP or WNT signaling. We first transiently reset primed human pluripotent stem cells into a trophectoderm-competent ground state, followed by LATS kinase inhibition to set the trajectory in motion. To benchmark fidelity, we built an embryo-chorion single-cell reference integrating published early human and placental transcriptomes and applied a computational stage-matching tool to align our cultures to natural development. Stage matching revealed an ordered progression along the trophectoderm trajectory from early TE to post-implantation trophoblast. With extended culture, all major cell types of the nascent chorion emerged, encompassing both trophoblast and chorionic mesoderm lineages. Within the trophoblast, we identified proliferative and non-cycling villous cytotrophoblast, a columnar population connecting villous and extravillous domains, as well as syncytia and extravillous subtypes. When cultured in suspension, these lineages self-organized into three-dimensional organoids that recapitulated the stromal-epithelial architecture and proliferative-syncytial polarity of the emergent chorion. We identified CLDN6 as a defining surface marker of columnar trophoblast, the population that bridges villous and extravillous compartments. Prospective isolation of living CLDN6+ trophoblast revealed their capacity to reacquire a proliferative villous state and, under directed cues, generate both syncytial and extravillous fates, confirming their proposed dual developmental potential within the chorion. Together, these findings establish a developmentally informed framework that connects human trophectoderm specification to the emergent chorion and provides a dynamic platform for investigating the earliest steps of placental specification and the origins of implantation disorders.

cell biology↗

bioRxiv2023

Bacterial genome engineering using CRISPR RNA-guided transposases

CRISPR-associated transposons (CASTs) have the potential to transform the technology landscape for kilobase-scale genome engineering, by virtue of their ability to integrate large genetic payloads with high accuracy, easy programmability, and no requirement for homologous recombination machinery. These transposons encode efficient, CRISPR RNA-guided transposases that execute genomic insertions in E. coli at efficiencies approaching [~]100%, generate multiplexed edits when programmed with multiple guides, and function robustly in diverse Gram-negative bacterial species. Here we present a detailed protocol for engineering bacterial genomes using CAST systems, including guidelines on the available homologs and vectors, customization of guide RNAs and DNA payloads, selection of common delivery methods, and genotypic analysis of integration events. We further describe a computational crRNA design algorithm to avoid potential off-targets and CRISPR array cloning pipeline for DNA insertion multiplexing. Starting from available plasmid constructs, the isolation of clonal strains containing a novel genomic integration event-of-interest can be achieved in 1 week using standard molecular biology techniques.

bioengineering↗

bioRxiv2020

CRISPR RNA-guided integrases for high-efficiency and multiplexed bacterial genome engineering

Tn7-like transposons are pervasive mobile genetic elements in bacteria that mobilize using heteromeric transposase complexes comprising distinct targeting modules. We recently described a Tn7-like transposon from Vibrio cholerae that employs a Type I-F CRISPR-Cas system for RNA-guided transposition, in which Cascade directly recruits transposition proteins to integrate donor DNA downstream of genomic target sites complementary to CRISPR RNA. However, the requirement for multiple expression vectors and low overall integration efficiencies, particularly for large genetic payloads, hindered the practical utility of the transposon. Here, we present a significantly improved INTEGRATE (insertion of transposable elements by guide RNA-assisted targeting) system for targeted, multiplexed, and marker-free DNA integration of up to 10 kilobases at ~100% efficiency. Using multi-spacer CRISPR arrays, we achieved simultaneous multiplex insertions in three genomic loci, and facile multi-loci deletions when combining orthogonal integrases and recombinases. Finally, we demonstrated robust function in other biomedically- and industrially-relevant bacteria, and developed an accessible computational algorithm for guide RNA design. This work establishes INTEGRATE as a versatile and portable tool that enables multiplex and kilobase-scale genome engineering.

molecular biology↗

bioRxiv2018

Multiplex transcriptional characterizations across diverse and hybrid bacterial cell-free expression systems

Cell-free expression systems enable rapid prototyping of genetic programs in vitro. However, current throughput of cell-free measurements is often limited by the use of single-channel reporter assays. Here, we describe DNA Regulatory element Analysis by cell-Free Transcription and Sequencing (DRAFTS), a rapid and robust in vitro approach for multiplexed measurement of transcriptional activities from thousands of regulatory sequences in a single reaction. We employed this method in active cell lysates developed from ten diverse bacterial species. Interspecies analysis of transcriptional profiles from >1,000 diverse regulatory sequences revealed functional differences in gene expression that could be predictively modeled. Finally, we constructed and examined the transcriptional capacities of dual-species \"hybrid\" cell lysates that can simultaneously harness gene expression properties of multiple organisms. We expect that this cell-free multiplex transcriptional measurement approach will improve genetic circuit prototyping in new bacterial chassis for synthetic biology.

synthetic biology↗