Biology subjects

Paix, A.

Publications and source records attributed to Paix, A..

8 recordsLinked to original sources

bioRxiv2026

Endogenous gene tagging in the model brown alga Ectocarpus using a simplified CRISPR/Cas method

Brown algae represent one of the few eukaryotic lineages to have independently evolved complex multicellularity, providing a powerful comparative system for investigating the molecular and evolutionary principles underlying multicellular development. Ectocarpus has emerged as the principal model for this lineage, supported by extensive genomic and transcriptomic resources. However, mechanistic and functional studies have remained limited by the available reverse-genetic tools. While recent CRISPR-Cas developments have enabled targeted gene knock-outs, the lack of knock-in (KI) approaches for endogenous protein tagging and precise genomic insertion remains a major experimental bottleneck. Here, we establish a comprehensive CRISPR-Cas genome-engineering framework for Ectocarpus that enables both targeted gene disruption and precise genomic insertion. We demonstrate efficient knock-in of multiple peptide tags at endogenous loci, enabling direct analysis of native proteins. By combining robust gene knock-out with endogenous protein tagging, this framework substantially expands the experimental possibilities for brown algal research and establishes Ectocarpus as a genetically tractable system for functional genomics, providing a foundation for genome engineering across stramenopiles.

developmental biology↗

bioRxiv2026

Efficient Endogenous Tagging in the Sea Urchin, Lytechinus pictus, Using CRISPR/Cas9-mediated Split-Fluorescent Protein Knock-In

Precise knock-in of fluorescent reporters is a powerful tool for studying the dynamic cellular and molecular processes of embryogenesis. However, conventional CRISPR-Cas9 knock-in of large inserts, such as full-length fluorescent proteins, is inefficient. This has limited its application in many emerging model systems, including sea urchins. Here, we overcome this barrier using a transgenic Lytechinus pictus line that constitutively and ubiquitously expresses a large fragment of mNeonGreen (mNG3K1-10). In this line, fluorescence is only reconstituted when CRISPR-mediated knock-in delivers mNG211, the 11th beta strand of the fluorescent protein, to complement the constitutively expressed fragment. Because this strategy requires integrating only the short 11th-strand, together with short homology arms ([~]130 nt total), by homology directed repair, it circumvents the size constraints that limit conventional full-length reporter knock-ins using CRISPR. Using this approach, we achieved integration efficiencies of 14-22%, roughly an order of magnitude higher than those obtained with full-length fluorescent protein knock-ins. This provides a streamlined, scalable method for endogenous protein visualization in echinoderm embryos and a valuable resource for studying gene function, morphogenesis, and toxicant response in this classic developmental model.

developmental biology↗

bioRxiv2025

Mesoglea biogenesis reveals a cryptic aboral valve for pressure regulation in cnidarian morphogenesis

Cnidarians are classically defined by a single oral opening, a hallmark of the "blind gut" model in early animal evolution. Here, we identify a pressure-sensitive aboral valve in Nematostella vectensis that operates independently of digestion. This valve dissipates elevated hydraulic pressure during morphogenesis, by expelling fluid through transient epidermal ruptures triggered by muscular ring opening. This unexpected function emerged from a comprehensive analysis of mesogleal basement membrane biogenesis. We show that the global dynamics of this extracellular matrix transduce muscular hydraulics to drive tissue rearrangement and stabilize shape, while localized FGFRb-dependent matrix remodeling establishes the aboral valve. By positioning the mesoglea as an integrator of biomechanics, tissue remodeling, and aboral valve function, these findings expand non-bilaterian openings beyond the digestive paradigm as a hydraulic regulator.

developmental biology↗

bioRxiv2025

Selection-free CRISPR-Cas9 editing protocol for distant Dictyostelid species

Dictyostelids are a species-rich clade of cellular slime molds that are widely found in soils and have been studied for over a century. Most research focusses on Dictyostelium discoideum, which - due to its ease of culturing and genetic tractability - has been adopted as a model species in the fields of developmental biology, cell biology and microbiology. Over decades, genome editing methods in D. discoideum have steadily improved but remain relatively time-consuming and limited in scope, effective in a few species only. Here, we introduce a CRISPR-Cas9 editing protocol that is cloning-free, selection-free, highly-efficient, and effective across Dictyostelid species. After optimizing our protocol in D. discoideum, we obtained knock-out efficiencies of [~]80% and knock-in efficiencies of [~]30% without antibiotic selection. Efficiencies depend on template concentrations, insertion sizes, homology arms and target sites. Since our protocol is selection-free, we can isolate mutants as soon as one day post-transfection, vastly expediting the generation of knock-outs, fusion proteins and expression reporters. Our protocol also makes it possible to generate several knock-in mutations simultaneously in the same cells. Boosted by cell-sorting and fluorescent microscopy, we could readily apply our CRISPR-Cas9 editing protocol to phylogenetically distant Dictyostelid species, which diverged hundreds of millions of years ago and have never been genome edited before. Our protocol therefore opens the door to performing broad-scale genetic interrogations across Dicyostelids.

genetics↗

bioRxiv2024

Systemic coordination of whole-body tissue remodeling in sea anemone local regeneration

The complexity of regeneration extends beyond local wound responses (1,2), eliciting systemic processes that engage the entire organism. However, their functional relevance, and the spatial and temporal orchestration of the underlying molecular processes distant from the injury site remain unknown. Here, we demonstrate that local regeneration in the cnidarian Nematostella vectensis involves a systemic homeostatic response, leading to coordinated whole-body remodeling. Leveraging spatial transcriptomics, endogenous protein tagging, and live imaging, we comprehensively dissect this systemic response at the organismal scale. We identify proteolysis as a critical process driven by both local and systemic upregulation of metalloproteases. We show that metalloproteinase expression levels and activity scale with the extent of tissue loss, leading to proportional long-range movement of tissue and its associated extracellular matrix. Our findings illuminate the adaptive nature of the systematic response in regeneration. We propose that this integrated regenerative mechanism, shifting the system from a steady to a dynamic homeostatic state, allows the organism to cope with a wide range of injuries.

developmental biology↗

bioRxiv2022

An oblique plane microscope for mesoscopic imaging of freely moving organisms with cellular resolution

Several important questions in biology require non-invasive and three-dimensional imaging techniques with appropriate spatiotemporal resolution that permit live organisms to move in an unconstrained fashion over an extended field-of-view. While selective-plane illumination microscopy (SPIM) has emerged as a powerful method to observe live biological specimens at high spatio-temporal resolution, typical implementations often necessitate constraining sample mounting or lack the required volumetric speed. Here, we report on an open-top, dual-objective oblique plane microscope (OPM) capable of observing millimeter sized, freely moving animals at cellular resolution. We demonstrate the capabilities of our mesoscopic OPM (MesOPM) by imaging the behavioural dynamics of the sea anemone Nematostella vectensis over 1.56 x 1.56 x 0.25 mm at 1.5 x 2.8 x 5.3{micro}m resolution and 0.5Hz volume rate.

bioengineering↗

bioRxiv2021

Endogenous protein tagging in medaka using a simplified CRISPR/Cas9 knock-in approach

The CRISPR/Cas9 system has been used to generate fluorescently labelled fusion proteins by homology directed repair in a variety of species. Despite its revolutionary success, there remains an urgent need for increased simplicity and efficiency of genome editing in research organisms. Here, we establish a simplified, highly efficient and precise strategy for CRISPR/Cas9 mediated endogenous protein tagging in medaka (Oryzias latipes). We use a cloning-free approach that relies on PCR amplified donor fragments containing the fluorescent reporter sequences flanked by short homology arms (30-40bp), a synthetic sgRNA and streptavidin tagged Cas9. We generate six novel knock-in lines with high efficiency of F0 targeting and germline transmission. Whole Genome Sequencing (WGS) results reveal single-copy integration events only at the targeted loci. We provide an initial characterization of these fusion-protein lines, significantly expanding the repertoire of genetic tools available in medaka. In particular, we show that the mScarlet-pcna knock-in line has the potential to serve as an organismal-wide label for proliferative zones and an endogenous cell cycle reporter.

developmental biology↗

bioRxiv2017

Precision genome editing using synthesis-dependent repair of Cas9-induced DNA breaks

The RNA-guided DNA endonuclease Cas9 has emerged as a powerful new tool for genome engineering. Cas9 creates targeted double-strand breaks (DSBs) in the genome. Knock-in of specific mutations (precision genome editing) requires homology-directed repair (HDR) of the DSB by synthetic donor DNAs containing the desired edits, but HDR has been reported to be variably efficient. Here, we report that linear DNAs (single and double-stranded) engage in a high-efficiency HDR mechanism that requires only [~]35 nucleotides of homology with the targeted locus to introduce edits ranging from 1 to 1000 nucleotides. We demonstrate the utility of linear donors by introducing fluorescent protein tags in human cells and mouse embryos using PCR fragments. We find that repair is local, polarity-sensitive, and prone to template switching, characteristics that are consistent with gene conversion by synthesis-dependent strand-annealing (SDSA). Our findings enable rational design of synthetic donor DNAs for efficient genome editing.\n\nSignificanceGenome editing, the introduction of precise changes in the genome, is revolutionizing our ability to decode the genome. Here we describe a simple method for genome editing that takes advantage of an efficient mechanism for DNA repair called synthesis-dependent strand annealing. We demonstrate that synthetic linear DNAs (ssODNs and PCR fragments) with [~]35bp homology arms function as efficient donors for SDSA repair of Cas9-induced double-strand breaks. Edits from 1 to 1000 base pairs can be introduced in the genome without cloning or selection.

synthetic biology↗