Nature biotechnology最新文献

While natural proteins are incredibly diverse biomacromolecules, they present only a subset of the sequence and structural space that is chemically possible¹. The field of protein design has the potential to illuminate the unexplored regions of this space, to deepen our understanding of sequence–structure–function relationships, and to enable the development of novel proteins that could be used to address societal challenges.

Protein design has evolved dramatically over the past 40 years from rational design to contemporary data-driven approaches. The field has now reached milestones such as the design of small-molecule² and protein³ binders, entirely novel folds^4,5,6, and has been recognized with a Nobel Prize. However, it can be difficult to identify current challenges and opportunities within the field as no easily searchable resource provides an overview of proteins that have been designed to date. Such a resource would improve our ability to learn from previous efforts and guide the development of future design methods.

Cell-type-specific regulatory elements such as enhancers can direct expression of recombinant adeno-associated viruses (AAVs) to specific cell types, but this approach is limited by the relatively small packaging capacity of AAVs. In this study, we used spatial genomics to show that transcriptional crosstalk between individual AAV genomes provides a general method for cell-type-specific expression of large cargo by separating distally acting regulatory elements into a second AAV genome. We identified and profiled transcriptional crosstalk in AAV genomes carrying 11 different enhancers active in mouse brain. We developed spatial genomics methods to identify and localize AAV genomes and their concatemeric forms in cultured cells and in tissue, and we demonstrate here that transcriptional crosstalk is dependent upon concatemer formation. Finally, we leveraged transcriptional crosstalk to drive expression of a 3.2-kb Cas9 cargo in a cell-type-specific manner with systemically administered engineered AAVs, and we demonstrate AAV-delivered, minimally invasive, cell-type-specific gene editing in wild-type mice that recapitulates known disease phenotypes.

Gene transfer can be studied using genetically encoded reporters or metagenomic sequencing but these methods are limited by sensitivity when used to monitor the mobile DNA host range in microbial communities. To record information about gene transfer across a wastewater microbiome, a synthetic catalytic RNA was used to barcode a highly conserved segment of ribosomal RNA (rRNA). By writing information into rRNA using a ribozyme and reading out native and modified rRNA using amplicon sequencing, we find that microbial community members from 20 taxonomic orders participate in plasmid conjugation with an Escherichia coli donor strain and observe differences in 16S rRNA barcode signal across amplicon sequence variants. Multiplexed rRNA barcoding using plasmids with pBBR1 or ColE1 origins of replication reveals differences in host range. This autonomous RNA-addressable modification provides information about gene transfer without requiring translation and will enable microbiome engineering across diverse ecological settings and studies of environmental controls on gene transfer and cellular uptake of extracellular materials.