Research Article

CRISPR-mediated activation of a promoter or enhancer rescues obesity caused by haploinsufficiency

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Science  18 Jan 2019:
Vol. 363, Issue 6424, eaau0629
DOI: 10.1126/science.aau0629

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CRISPRa corrects haploinsufficient obesity

Loss-of-function mutation in one gene copy, termed haploinsufficiency, can lead to insufficient protein levels and result in human disease. Matharu et al. tested whether a CRISPR-based activation system (CRISPRa) could rescue a haploinsufficient phenotype by increasing the gene expression levels of the existing normal copy (see the Perspective by Montefiori and Nobrega). By delivering this system into the mouse hypothalamus using adeno-associated virus, they rescued the obesity phenotype caused by haploinsufficiency of either of two genes known to promote obesity when mutated in mice and humans. These results highlight the translational potential of the CRISPR activation system to treat haploinsufficient disease.

Science, this issue p. eaau0629; see also p. 231

Structured Abstract

INTRODUCTION

Loss-of-function mutations in one gene copy can lead to reduced amounts of protein and, consequently, human disease, a condition termed haploinsufficiency. It is currently estimated that more than 660 genes cause human disease as a result of haploinsufficiency. The delivery of extra copies of the gene by way of gene therapy is a promising therapeutic strategy to increase gene dosage in such conditions. Recombinant adeno-associated virus (rAAV) provides a promising tool for delivery of transgenes in an efficient and safe way for gene therapy. However, it has some limitations, including an optimal DNA packaging constraint of 4700 base pairs and ectopic expression.

RATIONALE

Increasing the expression levels of the normal gene copy by directly targeting the endogenous gene regulatory elements that control it could potentially correct haploinsufficiency. CRISPR-mediated activation (CRISPRa), whereby a nuclease-deficient Cas9 (dCas9) is used to target a transcriptional activator to the gene’s regulatory element (promoter or enhancer), could be used for this purpose. Such an approach could overcome the ectopic expression and DNA packaging limitations of rAAV. Using obesity as a model, we tested in mice whether CRISPR-mediated activation of the existing normal copy of two different genes, Sim1 or Mc4r, where loss-of-function mutations that lead to haploinsufficiency are a major cause of human obesity, can rescue their obesity phenotype.

RESULTS

We first generated a transgenic CRISPRa system using dCas9 fused to a transcriptional activator, VP64, to test whether it can rescue the obesity phenotype in a Sim1 haploinsufficient mouse model. CRISPRa targeting of the Sim1 promoter or its hypothalamus-specific enhancer, which is 270 kilobases away from the gene, in Sim1 haploinsufficient mice increased the expression of the normal copy of Sim1. This up-regulation was sufficient to rescue the obesity phenotype of Sim1 heterozygous mice and led to significantly reduced food intake and body fat content in these mice. We assessed the off-targeting effects of CRISPRa using both RNA sequencing (RNA-seq) and Cas9 chromatin immunoprecipitation sequencing (ChIP-seq) analyses. We found CRISPRa targeting to be highly specific and without any overt changes in the expression of other genes. We also observed that Sim1 up-regulation occurred only in tissues where the regulatory element (promoter or enhancer) that was being targeted was active. Although promoter-CRISPRa–targeted mice up-regulated Sim1 in all the tissues where it is expressed, the enhancer-CRISPRa–targeted mice showed Sim1 up-regulation only in the hypothalamus. We then delivered CRISPRa packaged into rAAV targeting the Sim1 promoter or its hypothalamus-specific enhancer using either Streptococcus pyogenes or the shorter Staphylococcus aureus CRISPRa system. We show that postnatal injection of CRISPRa-rAAV into the hypothalamus can up-regulate Sim1 expression and rescue the obesity phenotype in Sim1 haploinsufficient mice in a long-lasting manner. To further highlight the therapeutic potential of this approach to rescue other haploinsufficient genes, we targeted Mc4r, where haploinsufficiency leads to severe obesity in mice and humans. CRISPRa-rAAV targeting of the Mc4r promoter rescued the obesity phenotype of Mc4r heterozygous mice.

CONCLUSION

These findings show that the CRISPRa system can rescue a haploinsufficient phenotype in vivo. This CRISPR-mediated activation strategy is different from a conventional gene therapy strategy, as it uses the endogenous regulatory elements to up-regulate the existing functional gene copy. As such, it can overcome the problem of ectopic gene expression. In addition, it could be used for genes that are not amenable to conventional gene therapy because their coding sequences are longer than the rAAV packaging limit. Our results provide a framework to further develop CRISPRa as a potential tool to treat gene dosage–sensitive diseases.

CRISPRa up-regulation of the existing normal gene copy rescues obesity caused by haploinsufficiency.

Loss-of-function mutations in one allele lead to reduced amounts of mRNA and protein and can cause human disease, a condition termed haploinsufficiency. By up-regulating the existing normal allele using CRISPR-mediated activation (CRISPRa), whereby a nuclease-deficient Cas9 is fused to a transcriptional activator and targeted to a gene’s regulatory element (promoter or enhancer), the haploinsufficient phenotype could be rescued.

Abstract

A wide range of human diseases result from haploinsufficiency, where the function of one of the two gene copies is lost. Here, we targeted the remaining functional copy of a haploinsufficient gene using CRISPR-mediated activation (CRISPRa) in Sim1 and Mc4r heterozygous mouse models to rescue their obesity phenotype. Transgenic-based CRISPRa targeting of the Sim1 promoter or its distant hypothalamic enhancer up-regulated its expression from the endogenous functional allele in a tissue-specific manner, rescuing the obesity phenotype in Sim1 heterozygous mice. To evaluate the therapeutic potential of CRISPRa, we injected CRISPRa-recombinant adeno-associated virus into the hypothalamus, which led to reversal of the obesity phenotype in Sim1 and Mc4r haploinsufficient mice. Our results suggest that endogenous gene up-regulation could be a potential strategy to treat altered gene dosage diseases.

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