Biology subjects

Bourgeois, B.

Publications and source records attributed to Bourgeois, B..

3 recordsLinked to original sources

bioRxiv2024

Systematic Development of Reprogrammed Modular Integrases Enables Precise Genomic Integration of Large DNA Sequences

Despite recent progress in the ability to manipulate the genomes of eukaryotic cells1-3, there is still no effective and practical method to precisely integrate large synthetic DNA constructs into desired chromosomal sites using a programmable integrase. Serine integrases can perform the necessary molecular steps4, but only if their natural target site is first installed into the recipient genome by other methods. A more elegant approach would be to directly reprogram the serine integrase itself to target a desired genomic site that is different from the natural recognition site of the integrase5. Here, we describe the development of a Modular Integrase (MINT) platform, a versatile protein-guided genome editing tool that can facilitate site-directed targeted integration (TI) of synthetic DNA into chromosomal sites. Through a combination of structural modeling, directed evolution, and screening in human cells we have reprogrammed the specificity of the serine integrase Bxb1. We demonstrate the therapeutic potential of the MINT platform by retargeting Bxb1 to the human AAVS1 and TRAC loci where wild-type Bxb1 has no detectable activity. By combining the MINT platform with known activity-increasing Bxb1 mutants, we achieved 14% TI at the AAVS1 locus, and by additionally fusing zinc finger DNA binding domains to engineered Bxb1 variants, we achieved 35% TI at the TRAC locus in human K562 cells. To further demonstrate clinical potential, we achieved stable 15% TI of a functional donor construct in human T cells.

synthetic biology↗

bioRxiv2024

Vinegar injections can be used safely to control outbreaks of crown-of-thorns starfish (Acanthaster solaris) during the peak spawning season

Concerns are mounting over the effects of COTS outbreaks, prompting the need for integrated management strategies. Although direct control methods are short-term and localized, they remain one of the few operational tools that can be easily implemented by local actors. Vinegar injections have recently emerged as highly effective method; however their impact on reproductive behavior remains untested. Here, we investigated the short-term spawning response of mature COTS to double injections of household vinegar. First, COTS abundances and reproductive status were monitored during a massive outbreak affecting New Caledonias reefs. In-situ and laboratory experiments were then conducted to determine whether injected COTS would eventually release their eggs and trigger synchronized spawning among mature individuals in close proximity. Our results indicated that injections had no significant effect on spawning behavior, even in densely populated aggregations (>4000 COTS.ha-1). In the field, starfish exhibited ripe gonads with high gamete content (up to 35% of body weight) three days after conspecifics were injected. In the laboratory, mature COTS kept with injected, decaying individuals in a confined volume did not expel their gametes after two days. This suggests that vinegar injections could be used at any time, even during peak spawning, without risking synchronized spawning in the affected areas.

ecology↗

bioRxiv2023

Hexokinase 1 forms rings that constrict mitochondria during energy stress

Metabolic enzymes can adapt during energy stress, but the precise mechanisms and consequences of these adaptations remain understudied. Here, we discovered that hexokinase 1 (HK1), a key glycolytic enzyme, clusters into ring-like structures around mitochondria during energy stress. These HK1-rings constrict mitochondria at contact sites with the endoplasmic reticulum (ER) and prevent mitochondrial fission by displacing the dynamin-related protein 1 (Drp1) from mitochondrial constriction sites. Mechanistically, we identified that the lack of ATP and glucose-6-phosphate (G6P) promotes the clustering of HK1. Moreover, we found several mutations that are critical for the formation of HK1-rings. Utilizing these mutations, we could show that HK1-rings keep mitochondria connected and rewire cellular metabolism during energy stress. Our findings highlight that HK1 is a robust energy stress sensor that regulates the shape, connectivity and metabolic activity of mitochondria. Thus, the formation of HK1-rings may affect mitochondrial function in energy stress-related pathologies.

cell biology↗