CRISPR-delivery particles targeting nuclear receptor-interacting protein 1 (Nrip1) in adipose cells to enhance energy expenditure.

RNA-guided, engineered nucleases derived from the prokaryotic adaptive immune system CRISPR-Cas represent a powerful platform for gene deletion and editing. When used as a therapeutic approach, direct delivery of Cas9 protein and single-guide RNA (sgRNA) could circumvent the safety issues associated with plasmid delivery and therefore represents an attractive tool for precision genome engineering. Gene deletion or editing in adipose tissue to enhance its energy expenditure, fatty acid oxidation, and secretion of bioactive factors through a "browning" process presents a potential therapeutic strategy to alleviate metabolic disease. Here, we developed "CRISPR-delivery particles," denoted CriPs, composed of nano-size complexes of Cas9 protein and sgRNA that are coated with an amphipathic peptide called Endo-Porter that mediates entry into cells. Efficient CRISPR-Cas9-mediated gene deletion of ectopically expressed GFP by CriPs was achieved in multiple cell types, including a macrophage cell line, primary macrophages, and primary pre-adipocytes. Significant GFP loss was also observed in peritoneal exudate cells with minimum systemic toxicity in GFP-expressing mice following intraperitoneal injection of CriPs containing Gfp-targeting sgRNA. Furthermore, disruption of a nuclear co-repressor of catabolism, the Nrip1 gene, in white adipocytes by CriPs enhanced adipocyte browning with a marked increase of uncoupling protein 1 (UCP1) expression. Of note, the CriP-mediated Nrip1 deletion did not produce detectable off-target effects. We conclude that CriPs offer an effective Cas9 and sgRNA delivery system for ablating targeted gene products in cultured cells and in vivo, providing a potential therapeutic strategy for metabolic disease.

Fugu ESTs: new resources for transcription analysis and genome annotation.

The draft Fugu rubripes genome was released in 2002, at which time relatively few cDNAs were available to aid in the annotation of genes. The data presented here describe the sequencing and analysis of 24,398 expressed sequence tags (ESTs) generated from 15 different adult and juvenile Fugu tissues, 74% of which matched protein database entries. Analysis of the EST data compared with the Fugu genome data predicts that approximately 10,116 gene tags have been generated, covering almost one-third of Fugu predicted genes. This represents a remarkable economy of effort. Comparison with the Washington University zebrafish EST assemblies indicates strong conservation within fish species, but significant differences remain. This potentially represents divergence of sequence in the 5' terminal exons and UTRs between these two fish species, although clearly, complete EST data sets are not available for either species. This project provides new Fugu resources, and the analysis adds significant weight to the argument that EST programs remain an essential resource for genome exploitation and annotation. This is particularly timely with the increasing availability of draft genome sequence from different organisms and the mounting emphasis on gene function and regulation.