Stanford Medicine researchers have developed a compact gene activation platform named TIGRa, which is less than half the size of conventional CRISPR-based activators and capable of in vivo delivery via adeno-associated viruses, according to a proof-of-concept study led by Yang Sun.
Understanding the TIGRa Gene Activation Platform
The CRISPR-Cas9 system revolutionized genome editing and targeted gene expression in laboratories by fusing a catalytically dead Cas9 to transcriptional activators. However, the components of this system—specifically the Cas9 protein—are exceptionally large. Packing the necessary DNA information for Cas9 into a standard adeno-associated viral (AAV) capsid consumes approximately 4.2 kb of its roughly 4.7 kb packaging limit. This leaves minimal room for promoter sequences, single guide RNAs, or transcriptional activator codes, according to researchers.
To overcome these delivery hurdles, the Stanford team turned to findings from the Broad Institute, where researchers last year discovered tandem interspaced guide RNA (TIGR)–TIGR-associated (Tas) systems in parasitic bacteria, bacteriophages, and archaeal viruses. Operating similarly to CRISPR, the TIGR-Tas system uses a guide RNA (tigRNAs) to induce targeted double-strand DNA breaks, but its Tas proteins are roughly a quarter of the size of Cas9.
Did you know? TIGRa-Pro was successfully used by researchers to simultaneously activate 12 different genes in in vitro cell models, demonstrating high versatility for complex genetic therapies.
Comparing TIGRa Variants and Efficiency
During a screen of eight nuclease-dead miniature RNA-guided nucleases and their enhanced variants, a Tas protein named TasR produced the most efficient system. The researchers named this platform the TIGR-TasR-mediated activator (TIGRa). According to the study, TIGRa demonstrated an activation efficiency 0.74 times as good as the well-established dSpCas9-based CRISPRa system.
The research team subsequently fine-tuned the platform into three distinct variants:
- TIGRa-Pro: Offers improved gene activation levels while maintaining a small footprint, enabling the simultaneous activation of 12 genes in in vitro cell models.
- TIGRa-Ultra: Features enhanced activation and was successfully used to reprogram human fibroblasts into induced-pluripotent stem cells by simultaneously activating seven genes.
- TIGRa-mini: Significantly smaller than the other variants, retaining roughly 70% of the efficiency of TIGRa.
Therapeutic Potential in Retinal Injury Models
To test the therapeutic capabilities of the platform, the Stanford team packaged TIGRa-Pro into an AAV and injected it into the eyes of mice. The intervention delivered the platform to retinal ganglion cells to upregulate the expression of CaMKIIa and CaMKIIb proteins, which provide a protective effect against blindness.
When researchers induced retinal injury in the mice using N-methyl-D-aspartic (NMDA) two weeks later, the treated subjects retained about a third of their vision, while the control group was almost completely blinded.
“We demonstrated that with the TIGRa injection, the retinal ganglion cells’ survival rate improved twofold,” stated first author Zhiquan Liu. Liu noted that while the results are promising, additional work is required to translate the technique into treatments for human neurodegeneration and blindness, explaining that “… 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.”
The research team has filed a patent for the platform. Yang Sun expressed optimism about its clinical potential, stating 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.”
Frequently Asked Questions
What is TIGRa?
TIGRa is a novel gene activation platform derived from a Tas protein discovered in parasitic bacteria and bacteriophages, engineered to be less than half the size of CRISPR-Cas9 systems.
Why is a smaller gene activation platform important?
Smaller systems can easily fit inside standard adeno-associated viral (AAV) capsids along with necessary promoter sequences and guide RNAs, facilitating effective in vivo delivery for gene therapies.
What diseases could TIGRa potentially treat?
According to researchers, the platform’s versatility means it could potentially target heart, liver, and skin conditions, as well as cancer, stroke, and neurodegenerative diseases.
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