NEW YORK (GenomeWeb) – A team led by researchers at the Salk Institute has developed a CRISPR-Cas9 genome editing system that epigenetically activates target genes without causing DNA double-strand breaks (DSBs). As they reported today in Cell, the investigators used the new system to treat diabetes, acute kidney disease, and muscular dystrophy in mouse models.
Current genome editing systems generally rely on the creation of DSBs, but this can limit their utility in treating disease because of the creation of off-target effects. Some researchers have tried repurposing the CRISPR-Cas9 system to create a dead Cas9 (dCas9)-VP64 system that enables target gene activation, which tends to allow the regulation of gene expression without the creation of DSBs, the authors noted. But implementing this system in vivo usually requires multiple single-guide RNAs (sgRNAs), making it less efficient, and exceeding the capacity of most common viral vectors, which are used to deliver these systems to living cells.
For their new study, senior author Juan Carlos Izpisua Belmonte and his colleagues created a system for in vivo activation of endogenous target genes through trans-epigenetic remodeling. "The system relies on recruitment of Cas9 and transcriptional activation complexes to target loci by modified sgRNAs," the researchers wrote. "Results demonstrate that CRISPR-Cas9-mediated target gene activation can be achieved in vivo, leading to observable phenotypic changes and amelioration of disease symptoms."
To date, all second-generation CRISPR-Cas9 target gene activation (TGA) systems have combined dCas9 to a transcriptional activation complex, but the resulting coding sequence usually exceeds the capacity of a single AAV, the researchers noted. They set out to develop a system in which the transcriptional activators were separated from dCas9. They used short sgRNAs of 14 or 15 base pairs rather than the usual 20 base pairs to guide wild-type Cas9 to the target locus. These short sgRNAs prevent active Cas9 from creating a DSB, so they're called dead sgRNAs (dgRNAs). The resulting modified Cas9 complexes "drove high levels of TGA," the authors noted. "This was encouraging, as it suggested that we might be able to induce high levels of TGA using an AAV-Cas9 (without VP64) in vivo."
When they merged the Cas9 complexes into AAVs, and injected those AAVs directly into the brains of Cas9-expressing mice, the researchers detected CRISPR-Cas9-mediated TGA, demonstrating that the system could induce transcription of a reporter gene in vivo.
Importantly, the investigators wanted to determine whether this system could ameliorate mouse models of human diseases, starting with a model of acute kidney injury, and specifically targeting the genes klotho and interleukin10. They derived mouse embryonic stem cell lines from Cas9-expressing mice and used them to examine gene induction by dgRNAs targeting klotho or Il10. They then assembled their CRISPR complexes and injected them into the tail veins of adult Cas9 mice.
"We first assessed the specificity of in vivo TGA using RNA sequencing. Compared to controls, target genes were dramatically upregulated (158-fold for Il10 and 2,553-fold for klotho on average), indicating high levels of TGA in vivo," the authors wrote.
Next, the team induced acute kidney injury in mice via cisplatin injection, eight days after AAV injection, and found elevated levels of klotho and Il10 gene expression in the liver and elevated levels of klotho protein secreted into the serum. The overexpression of klotho or IL-10 in cisplatin-treated mice resulted in improved renal function, and AAV treatment extended mouse survival time following a high dose of cisplatin treatment.
The team saw similarly encouraging results in mouse models of type 1 diabetes, in which they found that multiple CRISPR complexes could be co-injected into the mice at the same time. "These results indicated that the CRISPR-Cas9 TGA system can be used to activate multiple endogenous genes in vivo, and TGA and targeted gene knockout can be achieved simultaneously in Cas9-expressing mice," the authors wrote.
And finally, they tested whether their CRISPR system could be used to ameliorate disease phenotype in mouse models of human genetic disorders, such as Duchenne muscular dystrophy. They found that mice modeling the disease that had been injected with the CRISPR complex showed improved muscle strength two months after injection.
The authors cautioned that further studies would be needed before such a strategy can be used in the clinic, in order to determine whether human hosts could develop immune responses against the AAV-CRISPR-Cas9 TGA system, for example.
But they also noted that the system "represents a promising strategy for treating human diseases that have not been cured using traditional drug strategies" as it "can transcriptionally activate target genes in vivo by modulating histone marks rather than editing DNA sequences." They further pointed to the system's versatility and its ability to combine loss- and gain-of-function manipulations as one of the ways it could be used to treat a wide range of human diseases by "rapidly establish[ing] epistatic relationships between genes in vivo."