资源环境科技发展态势分析平台

Reverse genetics has been an indispensable tool to gain insights into viral pathogenesis and vaccine development. The genomes of large RNA viruses, such as those from coronaviruses, are cumbersome to clone and manipulate inEscherichia coliowing to the size and occasional instability of the genome(1-3). Therefore, an alternative rapid and robust reverse-genetics platform for RNA viruses would benefit the research community. Here we show the full functionality of a yeast-based synthetic genomics platform to genetically reconstruct diverse RNA viruses, including members of theCoronaviridae,FlaviviridaeandPneumoviridaefamilies. Viral subgenomic fragments were generated using viral isolates, cloned viral DNA, clinical samples or synthetic DNA, and these fragments were then reassembled in one step inSaccharomyces cerevisiaeusing transformation-associated recombination cloning to maintain the genome as a yeast artificial chromosome. T7 RNA polymerase was then used to generate infectious RNA to rescue viable virus. Using this platform, we were able to engineer and generate chemically synthesized clones of the virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)(4), which has caused the recent pandemic of coronavirus disease (COVID-19), in only a week after receipt of the synthetic DNA fragments. The technical advance that we describe here facilitates rapid responses to emerging viruses as it enables the real-time generation and functional characterization of evolving RNA virus variants during an outbreak.

CRISPR-Cas immunity protects prokaryotes against invading genetic elements(1). It uses the highly conserved Cas1-Cas2 complex to establish inheritable memory (spacers)(2-5). How Cas1-Cas2 acquires spacers from foreign DNA fragments (prespacers) and integrates them into the CRISPR locus in the correct orientation is unclear(6,7). Here, using the high spatiotemporal resolution of single-molecule fluorescence, we show that Cas1-Cas2 selects precursors of prespacers from DNA in various forms-including single-stranded DNA and partial duplexes-in a manner that depends on both the length of the DNA strand and the presence of a protospacer adjacent motif (PAM) sequence. We also identify DnaQ exonucleases as enzymes that process the Cas1-Cas2-loaded prespacer precursors into mature prespacers of a suitable size for integration. Cas1-Cas2 protects the PAM sequence from maturation, which results in the production of asymmetrically trimmed prespacers and the subsequent integration of spacers in the correct orientation. Our results demonstrate the kinetic coordination of prespacer precursor selection and PAM trimming, providing insight into the mechanisms that underlie the integration of functional spacers in the CRISPR loci.

Cas1-Cas2 selects precursor prespacers from DNA fragments in a length- and PAM-sequence-dependent manner, and these precursors are trimmed by DnaQ exonucleases to enable integration into the CRISPR locus in the correct orientation.

Structures of the orphan G-protein-coupled receptor GPR52 in ligand-free, G-protein-coupled and ligand-bound states reveal that extracellular loop 2 occupies the orthosteric binding pocket and functions as a built-in agonist to activate the receptor.