TIGRa, a novel gene activation platform half the size of its CRISPR-based cousin, enables researchers to deliver gene activation into the cells of an animal model using AAVs.
The discovery of the CRISPR-Cas9 system has revolutionized our ability to edit genomes and control gene expression in the lab.
One of its functions is targeted gene activation, in which a catalytically dead Cas9, fused to transcriptional activators, is used to activate gene expression.
The researchers were able to demonstrate comparable activation efficiency to the most efficient activation system out there, dSpCas9-based CRISPRa, with an activation level 0.74 times as good as the well-established system.
Two of them TIGRa-Pro and TIGRa-Ultra, had improved gene activation levels and were slightly smaller, while TIGRa-mini was significantly smaller and had roughly 70% of the efficiency of TIGRa.
TIGRa, a novel gene activation platform half the size of its CRISPR-based cousin, enables researchers to deliver gene activation into the cells of an animal model using AAVs.
A team of researchers from Stanford Medicine (CA, USA) led by Yang Sun, has developed a gene activation platform, TIGRa, that is less than half the size of the Cas9-based CRISPR activator (CRISPRa) platforms, making it better suited for in vivo delivery. In a mouse model proof-of-concept study, the team were able to successfully deliver the system to target tissues, producing some exciting results for future gene therapies.
The discovery of the CRISPR-Cas9 system has revolutionized our ability to edit genomes and control gene expression in the lab. One of its functions is targeted gene activation, in which a catalytically dead Cas9, fused to transcriptional activators, is used to activate gene expression. But the components of this system, critically the active protein Cas9, are big. Really big. To pack all the DNA information you need to produce Cas9 into a standard adeno-associated viral (AAV) capsid, you need to use ~4.2 kb of its ~4.7 kb packaging limit, leaving little room for promoter sequences, single guide RNAs or code for transcriptional activators.
AAV-ancing gene therapies
This article explores the biology of AAV vectors, the most common vector used in FDA-approved gene therapies, and examines their applications, challenges, and future directions.
Last year, researchers at the Broad Institute (MA, USA) discovered tandem interspaced guide RNA (TIGR)–TIGR-associated (Tas) systems in parasitic bacteria and bacteriophages and archaeal viruses. The TIGR-Tas system operates very similarly to CRISPR – both are directed by a guide RNA (in this case named tigRNAs) to induce targeted double-strand breaks in DNA – but on average, Tas proteins are roughly a quarter of the size of Cas9.
It was with some surprise then, that during a screen of eight nuclease-dead miniature RNA-guided nucleases and their enhanced variants – to be used as an alternative to nuclease-dead Cas9 in a gene activation platform – a Tas protein named TasR, one of the smallest of them all, produced the most efficient system. They named the system TIGR-TasR-mediated activator (TIGRa). The researchers were able to demonstrate comparable activation efficiency to the most efficient activation system out there, dSpCas9-based CRISPRa, with an activation level 0.74 times as good as the well-established system.
The team then fine-tuned this platform to deliver three variants. Two of them TIGRa-Pro and TIGRa-Ultra, had improved gene activation levels and were slightly smaller, while TIGRa-mini was significantly smaller and had roughly 70% of the efficiency of TIGRa. TIGRa-Pro was able to simultaneously activate 12 genes in in vitro cell models. Meanwhile, TIGRa-Ultra was able to reprogram human fibroblasts into induced-pluripotent stem cells, which requires the simultaneous activation of seven genes.
Next, to demonstrate the therapeutic capability of TIGRa, the team packaged TIGRa-Pro into an AAV and injected it into the eyes of mice to deliver the platform to retinal ganglion cells, aiming to upregulate the expression of CaMKIIa and CaMKIIb proteins, which have a protective effect against blindness. When the mice were given an N-methyl-D-aspartic (NMDA)-induced retinal injury 2 weeks later, they retained about a third of their vision, compared to the control group who were almost completely blinded.
“We demonstrated that with the TIGRa injection, the retinal ganglion cells’ survival rate improved twofold,” commented first author Zhiquan Liu, going on to explain that while this was promising, more work needed to be done for this technique to translate into treatments for blindness and neurodegeneration. “… it’s not enough. In the future, because these neurodegenerative diseases are very complex, we need to regulate more genes and in different combinations to get a better effect.”
Regardless, the team is clearly enthusiastic about the tool, having filed a patent for it. Commenting on its potential, Sun stated that because, “it’s very small and versatile, it could be used for many different diseases throughout the body, including heart conditions, liver conditions, skin conditions, cancer, neurodegeneration, stroke and any number of things. I’m optimistic about the application of this for humans. If you target the right genes in the appropriate diseases, it could happen faster than other gene therapies.”
