A pinboard by
Navneet Matharu

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.