This week’s theme is genes to function for drug screens…with a macabre theme of zombies! As more genes are discovered through GWAS and large-scale sequencing in humans, there is a pressing need to understand function. There are at least two steps: (1) fine-mapping the most likely causal genes and causal variants; and (2) functional interrogation of causal genes and causal variants to move towards a better understanding of causal human biology for drug screens (“from genes to screens”).
Genome-editing represents one very powerful tool, and the latest article from the laboratory of Feng Zhang at the Broad Institute takes genome-editing to a new level (see Genetic Engineering & Biotechnology News commentary here). They engineer the dead!
Genome-scale gene activation by an engineered CRISPR-Cas9 complex, Nature (December 2014).
Since its introduction in late 2012, the CRISPR-Cas9 gene-editing technology has revolutionized the ways scientists can apply to interrogate gene functions. Using a catalytically inactive Cas9 protein (dead Cas9, dCas9) tethered to an engineered single-guide RNA (sgRNA) molecule, the authors demonstrated the ability to conduct robust gain-of-function genetic screens through programmable, targeted gene activation.
Earlier this year, the laboratories of Stanley Qi, Jonathan Weissman and others \ reported the use of dCas9 conjugated with a transcriptional activator for gene activation (see Cell paper here). However, there were limitations of early versions of dCas9-based transcription activators, including effectiveness of gene activation and scalability (both of which are critical for establishing pooled, genome-scale gain-of-function screens).
Based on their previous crystallographic work on the atomic structure of the Cas9–sgRNA–target DNA tertiary complex, the Zhang lab took a structure-guided approach to design a much better dCas9-based transcription activation system: they re-positioned the dCas9-VP64 (a transcription activator) conjugation for more optimal functional output and, importantly, engineered the sgRNA to incorporate multiple protein-binding RNA aptamers. Through these smart engineering steps, they assembled a synthetic transcription activation complex consisting of multiple distinct effector domains modeled after natural transcription activation processes and showed that a single engineered sgRNA can facilitate robust, targeted gene activation. They further showed that this synergistic activation mediator (or “SAM”) system can facilitate genome-scale gain-of-function genetic screens when combined with a compact pooled sgRNA library.
[Thanks to Richard Chen for contributing this article and write-up…and the clever title: Rise of the dead!]
Other papers of interest related to functional interrogation of variants, genes and pathways:
Meta-analysis of the TNFAIP3 region in psoriasis reveals a risk haplotype that is distinct from other autoimmune diseases, Genes & Immunity (December 2014). This gene locus is of great personal interest to me, as it was one of the first GWAS hits in rheumatoid arthritis (RA) that I helped identify (here). At the time in 2007, we had strong statistical evidence for at least two independent signals contributing to risk of RA. Shortly thereafter, we and others determined that variants in the 6q23/TNFAIP3 locus contributed to risk of other autoimmune diseases, include SLE and psoriasis. However, disentangling the underlying biology has hampered by fine-mapping of the locus and additional functional interrogation of the most likely causal gene (TNFAIP3) and associated variants. In the current study published in Genes & Immunity, researchers at UCSF and elsewhere fine-mapped this region in psoriasis. Using bioinformatics tools and functional annotation of the top variants, they suggest that altered gene function might be due to long noncoding RNAs. They conclude: “Overall, our findings identify novel candidate causal variants of TNFAIP3 in psoriasis and highlight the complex genetic architecture of this locus in autoimmune susceptibility.”
White-to-brown metabolic conversion of human adipocytes by JAK inhibition, Nature Cell Biology (December 2014). Adipogenesis has been a major focus in obesity research for its direct association with diabetes, hypertension, hyperlipidemia, and cancer. Adipose tissue consists of two distinct fat cells: white and brown. White fat is the storage bin for triglycerides, brown fat dissipates chemical energy in the form of heat. Adipogenic maturation via trans differentiation or de-differentiation between the two types of fat cells can be exploited for therapeutic interventions, as these cells performs distinct lipid metabolic functions primarily due to the gene expression pattern. In this paper the authors have generated a stem cell based in vitro tool to screen compounds. They identified potential small molecules that may turn white (bad) fat into brown (good) fat. [Thanks to Vilas Wagh and Sergey Lezhnin for contributing this article.]
Happy Holidays to everyone! See you in 2015.