Postdoctoral Scholar, University of California San Francisco
Universal platform technology to develop therapeutics for dosage sensitive diseases
Haploinsufficiency, having only one functional copy of a gene, leads to a wide range of human disease and has been associated with over 300 genes. Large-scale exome sequencing studies estimated that there could be around 3000 genes that can potentially contribute to disease upon heterozygous loss of function. Here, we tested if CRISPR activation (CRISPRa) could rescue haploinsufficiency in vivo. We targeted the promoter or enhancer of an existing functional copy of haploinsufficient gene using single guide RNA and a nuclease deficient dCas9 fused with an activator domain VP64 in mice. Haploinsufficiency of SIM1, a transcription factor expressed in the hypothalamus that is involved in the regulation of food intake through the leptin pathway, results in severe obesity in humans and mice. CRISPRa targeting of either the Sim1 promoter or its ~270kb distant hypothalamic enhancer using transgenic mice, rescued the obesity phenotype in Sim1 heterozygous mice. Despite using a ubiquitous promoter for CRISPRa, Sim1 was upregulated only in tissues where the promoter or enhancer are active, suggesting that cis-regulatory elements can determine CRISPRa tissue-specificity. To further translate CRISPRa into a potential post-natal therapeutic strategy, we delivered dCas9-VP64 and sgRNA targeting either the Sim1 promoter or its enhancer using adeno-associated virus (AAV) to the hypothalamus. Transcriptional upregulation of Sim1 in the hypothalamus after AAV stereotaxic injections led to reversal of the weight gain phenotype of Sim1 heterozygous mice in a long lasting manner for both promoter and enhancer targeted CRISPRa. Our results show that CRISPRa could be used to exploit the inherent tissue specificity of cis-regulatory elements and thus have a potential to be developed further for therapeutic applications. This novel therapeutic strategy can be used to treat many other disorders resulting from altered gene dosage.
Abstract: While the catalog of mammalian transcripts and their expression levels in different cell types and disease states is rapidly expanding, our understanding of transcript function lags behind. We present a robust technology enabling systematic investigation of the cellular consequences of repressing or inducing individual transcripts. We identify rules for specific targeting of transcriptional repressors (CRISPRi), typically achieving 90%-99% knockdown with minimal off-target effects, and activators (CRISPRa) to endogenous genes via endonuclease-deficient Cas9. Together they enable modulation of gene expression over a ∼1,000-fold range. Using these rules, we construct genome-scale CRISPRi and CRISPRa libraries, each of which we validate with two pooled screens. Growth-based screens identify essential genes, tumor suppressors, and regulators of differentiation. Screens for sensitivity to a cholera-diphtheria toxin provide broad insights into the mechanisms of pathogen entry, retrotranslocation and toxicity. Our results establish CRISPRi and CRISPRa as powerful tools that provide rich and complementary information for mapping complex pathways.
Pub.: 14 Oct '14, Pinned: 20 Jun '17
Abstract: Targeted modulation of transcription is necessary for understanding complex gene networks and has great potential for medical and industrial applications. CRISPR is emerging as a powerful system for targeted genome activation and repression, in addition to its use in genome editing. This protocol describes how to design, construct, and experimentally validate the function of sequence-specific single guide RNAs (sgRNAs) for sequence-specific repression (CRISPRi) or activation (CRISPRa) of transcription in mammalian cells. In this technology, the CRISPR-associated protein Cas9 is catalytically deactivated (dCas9) to provide a general platform for RNA-guided DNA targeting of any locus in the genome. Fusion of dCas9 to effector domains with distinct regulatory functions enables stable and efficient transcriptional repression or activation in mammalian cells. Delivery of multiple sgRNAs further enables activation or repression of multiple genes. By using scaffold RNAs (scRNAs), different effectors can be recruited to different genes for simultaneous activation of some and repression of others. The CRISPRi and CRISPRa methods provide powerful tools for sequence-specific control of gene expression on a genome-wide scale to aid understanding gene functions and for engineering genetic regulatory systems.
Pub.: 06 Jan '16, Pinned: 20 Jun '17
Abstract: The bacterial CRISPR-Cas9 system has emerged as a multifunctional platform for sequence-specific regulation of gene expression. This Review describes the development of technologies based on nuclease-deactivated Cas9, termed dCas9, for RNA-guided genomic transcription regulation, both by repression through CRISPR interference (CRISPRi) and by activation through CRISPR activation (CRISPRa). We highlight different uses in diverse organisms, including bacterial and eukaryotic cells, and summarize current applications of harnessing CRISPR-dCas9 for multiplexed, inducible gene regulation, genome-wide screens and cell fate engineering. We also provide a perspective on future developments of the technology and its applications in biomedical research and clinical studies.
Pub.: 17 Dec '15, Pinned: 20 Jun '17
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