1. Orthologous CRISPR–Cas9 enzymes for combinatorial genetic screens.
Combinatorial genetic screening using CRISPR–Cas9 is a useful approach to uncover redundant genes and to explore complex gene networks. However, current methods suffer from interference between the single-guide RNAs (sgRNAs) and from limited gene targeting activity. To increase the efficiency of combinatorial screening, Fadi J Najm at Broad Institute of Harvard and MIT in Cambridge, Massachusetts, USA and his colleagues employ orthogonal Cas9 enzymes from Staphylococcus aureus and Streptococcus pyogenes. They used machine learning to establish S. aureus Cas9 sgRNA design rules and paired S. aureus Cas9 with S. pyogenes Cas9 to achieve dual targeting in a high fraction of cells. They also developed a lentiviral vector and cloning strategy to generate high-complexity pooled dual-knockout libraries to identify synthetic lethal and buffering gene pairs across multiple cell types, including MAPK pathway genes and apoptotic genes. Their orthologous approach also enabled a screen combining gene knockouts with transcriptional activation, which revealed genetic interactions with TP53. The “Big Papi” (paired aureus and pyogenes for interactions) approach described here will be widely applicable for the study of combinatorial phenotypes.
Read more, please click https://www.nature.com/articles/nbt.4048
2. Virus stamping for targeted single-cell infection in vitro and in vivo.
Genetic engineering by viral infection of single cells is useful to study complex systems such as the brain. However, available methods for infecting single cells have drawbacks that limit their applications. Here Rajib Schubert at ETH Zurich in Basel, Switzerland and his colleagues describe ‘virus stamping’, in which viruses are reversibly bound to a delivery vehicle—a functionalized glass pipette tip or magnetic nanoparticles in a pipette—that is brought into physical contact with the target cell on a surface or in tissue, using mechanical or magnetic forces. Different single cells in the same tissue can be infected with different viruses and an individual cell can be simultaneously infected with different viruses. They use rabies, lenti, herpes simplex, and adeno-associated viruses to drive expression of fluorescent markers or a calcium indicator in target cells in cell culture, mouse retina, human brain organoid, and the brains of live mice. Virus stamping provides a versatile solution for targeted single-cell infection of diverse cell types, both in vitro and in vivo.
Read more, please click https://www.nature.com/articles/nbt.4034
3. An extracellular network of Arabidopsis leucine-rich repeat receptor kinases.
The cells of multicellular organisms receive extracellular signals using surface receptors. The extracellular domains (ECDs) of cell surface receptors function as interaction platforms, and as regulatory modules of receptor activation. Understanding how interactions between ECDs produce signal-competent receptor complexes is challenging because of their low biochemical tractability. In plants, the discovery of ECD interactions is complicated by the massive expansion of receptor families, which creates tremendous potential for changeover in receptor interactions. The largest of these families in Arabidopsis thaliana consists of 225 evolutionarily related leucine-rich repeat receptor kinases (LRR-RKs), which function in the sensing of microorganisms, cell expansion, stomata development and stem-cell maintenance. Although the principles that govern LRR-RK signalling activation are emerging, the systems-level organization of this family of proteins is unknown. Here, to address this, Elwira Smakowska-Luzan at Vienna Biocenter (VBC) in Vienna, Austria and his colleagues investigated 40,000 potential ECD interactions using a sensitized high-throughput interaction assay, and produced an LRR-based cell surface interaction network (CSILRR) that consists of 567 interactions. To demonstrate the power of CSILRR for detecting biologically relevant interactions, they predicted and validated the functions of uncharacterized LRR-RKs in plant growth and immunity. In addition, they show that CSILRR operates as a unified regulatory network in which the LRR-RKs most crucial for its overall structure are required to prevent the aberrant signalling of receptors that are several network-steps away. Thus, plants have evolved LRR-RK networks to process extracellular signals into carefully balanced responses.
Read more, please click https://www.nature.com/articles/nature25184
4. Architecture of a channel-forming O-antigen polysaccharide ABC transporter.
O-antigens are cell surface polysaccharides of many Gram-negative pathogens that aid in escaping innate immune responses. A widespread O-antigen biosynthesis mechanism involves the synthesis of the lipid-anchored polymer on the cytosolic face of the inner membrane, followed by transport to the periplasmic side where it is ligated to the lipid A core to complete a lipopolysaccharide molecule. In this pathway, transport to the periplasm is mediated by an ATP-binding cassette (ABC) transporter, called Wzm–Wzt. Here Yunchen Bi at University of Virginia School of Medicine in Virginia, USA and her colleagues present the crystal structure of the Wzm–Wzt homologue from Aquifex aeolicus in an open conformation. The transporter forms a transmembrane channel that is sufficiently wide to accommodate a linear polysaccharide. Its nucleotide-binding domain and a periplasmic extension form ‘gate helices’ at the cytosolic and periplasmic membrane interfaces that probably serve as substrate entry and exit points. Site-directed mutagenesis of the gates impairs in vivo O-antigen secretion in the Escherichia coli prototype. Combined with a closed structure of the isolated nucleotide-binding domains, their structural and functional analyses suggest a processive O-antigen translocation mechanism, which stands in contrast to the classical alternating access mechanism of ABC transporters.
Read more, please click https://www.nature.com/articles/nature25190
5. Paternal chromosome loss and metabolic crisis contribute to hybrid inviability in Xenopus.
Hybridization of eggs and sperm from closely related species can give rise to genetic diversity, or can lead to embryo inviability owing to incompatibility. Although central to evolution, the cellular and molecular mechanisms underlying post-zygotic barriers that drive reproductive isolation and speciation remain largely unknown. Species of the African clawed frog Xenopus provide an ideal system to study hybridization and genome evolution. Xenopus laevis is an allotetraploid with 36 chromosomes that arose through interspecific hybridization of diploid progenitors, whereas Xenopus tropicalis is a diploid with 20 chromosomes that diverged from a common ancestor approximately 48 million years ago. Differences in genome size between the two species are accompanied by organism size differences, and size scaling of the egg and subcellular structures such as nuclei and spindles formed in egg extracts. Nevertheless, early development transcriptional programs, gene expression patterns, and protein sequences are generally conserved. Whereas the hybrid produced when X. laevis eggs are fertilized by X. tropicalis sperm is viable, the reverse hybrid dies before gastrulation. Here Romain Gibeaux at University of California in California, USA and his colleagues apply cell biological tools and high-throughput methods to study the mechanisms underlying hybrid inviability. They reveal that two specific X. laevis chromosomes are incompatible with the X. tropicalis cytoplasm and are mis-segregated during mitosis, leading to unbalanced gene expression at the maternal to zygotic transition, followed by cell-autonomous catastrophic embryo death. These results reveal a cellular mechanism underlying hybrid incompatibility that is driven by genome evolution and contributes to the process by which biological populations become distinct species.
Read more, please click https://www.nature.com/articles/nature25188