Activity of zebrafish THAP9 transposase and zebrafish P element-like transposons.

Transposable elements are mobile DNA segments that are found ubiquitously across the three domains of life. One family of transposons, called P elements, were discovered in the fruit fly Drosophila melanogaster. Since their discovery, P element transposase-homologous genes (called THAP-domain containing 9 or THAP9) have been discovered in other animal genomes. Here, we show that the zebrafish (Danio rerio) genome contains both an active THAP9 transposase (zfTHAP9) and mobile P-like transposable elements (called Pdre). zfTHAP9 transposase can excise one of its own elements (Pdre2) and Drosophila P elements. Drosophila P element transposase (DmTNP) is also able to excise the zebrafish Pdre2 element, even though it's distinct from the Drosophila P element. However, zfTHAP9 cannot transpose Pdre2 or Drosophila P elements, indicating partial transposase activity. Characterization of the N-terminal THAP DNA binding domain of zfTHAP9 shows distinct DNA binding site preferences from DmTNP and mutation of the zfTHAP9, based on known mutations in DmTNP, generated a hyperactive protein,. These results define an active vertebrate THAP9 transposase that can act on the endogenous zebrafish Pdre and Drosophila P elements.

Highly efficient generation of isogenic pluripotent stem cell models using prime editing.

The recent development of prime editing (PE) genome engineering technologies has the potential to significantly simplify the generation of human pluripotent stem cell (hPSC)-based disease models. PE is a multicomponent editing system that uses a Cas9-nickase fused to a reverse transcriptase (nCas9-RT) and an extended PE guide RNA (pegRNA). Once reverse transcribed, the pegRNA extension functions as a repair template to introduce precise designer mutations at the target site. Here, we systematically compared the editing efficiencies of PE to conventional gene editing methods in hPSCs. This analysis revealed that PE is overall more efficient and precise than homology-directed repair of site-specific nuclease-induced double-strand breaks. Specifically, PE is more effective in generating heterozygous editing events to create autosomal dominant disease-associated mutations. By stably integrating the nCas9-RT into hPSCs we achieved editing efficiencies equal to those reported for cancer cells, suggesting that the expression of the PE components, rather than cell-intrinsic features, limit PE in hPSCs. To improve the efficiency of PE in hPSCs, we optimized the delivery modalities for the PE components. Delivery of the nCas9-RT as mRNA combined with synthetically generated, chemically-modified pegRNAs and nicking guide RNAs improved editing efficiencies up to 13-fold compared with transfecting the PE components as plasmids or ribonucleoprotein particles. Finally, we demonstrated that this mRNA-based delivery approach can be used repeatedly to yield editing efficiencies exceeding 60% and to correct or introduce familial mutations causing Parkinson's disease in hPSCs.

From muscles to nerves, our body is formed of many kinds of cells which can each respond slightly differently to the same harmful genetic changes. Understanding the exact relationship between mutations and cell-type specific function is essential to better grasp how conditions such as Parkinson's disease or amyotrophic lateral sclerosis progress and can be treated. Stem cells could be an important tool in that effort, as they can be directed to mature into many cell types in the laboratory. Yet it remains difficult to precisely introduce disease-relevant mutations in these cells. To remove this obstacle, Li et al. focused on prime editing, a cutting-edge 'search and replace' approach which can introduce new genetic information into a specific DNA sequence. However, it was unclear whether this technique could be used to efficiently create stem cell models of human diseases. A first set of experiments showed that prime editing is superior to conventional approaches when generating mutated genes in stem cells. Li et al. then further improved the efficiency and precision of the method by tweaking how prime editing components are delivered into the cells. The refined approach could be harnessed to quickly generate large numbers of stem cells carrying mutations associated with Parkinson's disease; crucially, prime editing could then also be used to revert a mutated gene back to its healthy form. The improved prime editing approach developed by Li et al. removes a major hurdle for scientists hoping to use stem cells to study genetic diseases. This could potentially help to unlock progress in how we understand and ultimately treat these conditions.

From conservation to structure, studies of magnetosome associated cation diffusion facilitators (CDF) proteins in Proteobacteria.

Magnetotactic bacteria (MTB) are prokaryotes that sense the geomagnetic field lines to geolocate and navigate in aquatic sediments. They are polyphyletically distributed in several bacterial divisions but are mainly represented in the Proteobacteria. In this phylum, magnetotactic Deltaproteobacteria represent the most ancestral class of MTB. Like all MTB, they synthesize membrane-enclosed magnetic nanoparticles, called magnetosomes, for magnetic sensing. Magnetosome biogenesis is a complex process involving a specific set of genes that are conserved across MTB. Two of the most conserved genes are mamB and mamM, that encode for the magnetosome-associated proteins and are homologous to the cation diffusion facilitator (CDF) protein family. In magnetotactic Alphaproteobacteria MTB species, MamB and MamM proteins have been well characterized and play a central role in iron-transport required for biomineralization. However, their structural conservation and their role in more ancestral groups of MTB like the Deltaproteobacteria have not been established. Here we studied magnetite cluster MamB and MamM cytosolic C-terminal domain (CTD) structures from a phylogenetically distant magnetotactic Deltaproteobacteria species represented by BW-1 strain, which has the unique ability to biomineralize magnetite and greigite. We characterized them in solution, analyzed their crystal structures and compared them to those characterized in Alphaproteobacteria MTB species. We showed that despite the high phylogenetic distance, MamBBW-1 and MamMBW-1 CTDs share high structural similarity with known CDF-CTDs and will probably share a common function with the Alphaproteobacteria MamB and MamM.

Specificity of protein-DNA interactions in hypersaline environment: structural studies on complexes of Halobacterium salinarum oxidative stress-dependent protein hsRosR.

Interactions between proteins and DNA are crucial for all biological systems. Many studies have shown the dependence of protein-DNA interactions on the surrounding salt concentration. How these interactions are maintained in the hypersaline environments that halophiles inhabit remains puzzling. Towards solving this enigma, we identified the DNA motif recognized by the Halobactrium salinarum ROS-dependent transcription factor (hsRosR), determined the structure of several hsRosR-DNA complexes and investigated the DNA-binding process under extreme high-salt conditions. The picture that emerges from this work contributes to our understanding of the principles underlying the interplay between electrostatic interactions and salt-mediated protein-DNA interactions in an ionic environment characterized by molar salt concentrations.

The 3-D structure of VNG0258H/RosR - A haloarchaeal DNA-binding protein in its ionic shell.

Protein-DNA interactions are highly dependent on salt concentration. To gain insight into how such interactions are maintained in the highly saline cytoplasm of halophilic archaea, we determined the 3-D structure of VNG0258H/RosR, the first haloarchaeal DNA-binding protein from the extreme halophilic archaeon Halobactrium salinarum. It is a dimeric winged-helix-turn-helix (wHTH) protein with unique features due to adaptation to the halophilic environment. As ions are major players in DNA binding processes, particularly in halophilic environments, we investigated the solution structure of the ionic envelope and located anions in the first shell around the protein in the crystal using anomalous scattering. Anions that were found to be tightly bound to residues in the positively charged DNA-binding site would probably be released upon DNA binding and will thus make significant contribution to the driving force of the binding process. Unexpectedly, ions were also found in a buried internal cavity connected to the external medium by a tunnel. Our structure lays a solid groundwork for future structural, computational and biochemical studies on complexes of the protein with cognate DNA sequences, with implications to protein-DNA interactions in hyper-saline environments.