NovaIscB: Rational Engineering Yields Compact Gene Therapy Tool

In a groundbreaking advancement, scientists at MIT’s McGovern Institute for Brain Research and the Broad Institute have engineered a compact RNA-guided enzyme, NovaIscB, offering a promising new tool for gene therapy. This rationally designed protein efficiently edits human DNA with precision and programmability.

NovaIscB, derived from bacterial DNA cutters, belongs to the IscB protein family, discovered in 2021. These IscBs are evolutionary ancestors to Cas9, the foundation of CRISPR systems. Like Cas9, NovaIscB cuts DNA at RNA-specified sites, allowing researchers to target specific sequences by reprogramming the guide RNA.

The team, led by Feng Zhang, the James and Patricia Poitras Professor of Neuroscience at MIT, focused on IscBs due to their smaller size—one-third that of Cas9. This compactness simplifies delivery to cells, making NovaIscB an attractive candidate for gene therapies.

Initially, bacterial IscBs lacked the efficiency for therapeutic use. Researchers modified an IscB to enhance its activity within human cells without disrupting the rest of the genome. Soumya Kannan and Shiyou Zhu screened nearly 400 IscB enzymes, identifying ten capable of editing DNA in human cells.

The key challenge was increasing enzyme activity without compromising specificity. Zhu highlighted that bacterial IscBs use shorter RNA guides, making it harder to restrict activity to specific genome parts. The team engineered IscB to accommodate longer guides, reducing off-target effects.

Han Altae-Tran’s insights into bacterial IscB diversity aided the optimization process. Researchers noted a segment called REC in IscBs effective in human cells, which was absent in others. They hypothesized REC’s importance in interacting with human cell DNA. Structural modeling suggested REC expansion could enable IscBs to recognize longer RNA guides.

The team experimented with REC domain swaps from different IscBs and Cas9s, evaluating the impact on protein function. Guided by understanding of IscB and Cas9 interactions with DNA and RNA guides, they optimized both efficiency and specificity.

The resulting protein, NovaIscB, exhibits over 100 times more activity in human cells than the original IscB, with demonstrated target specificity. Kannan and Zhu screened hundreds of IscBs before achieving NovaIscB, strategically modifying the original protein. They used AlphaFold2, an AI tool, to predict how each alteration would affect the protein’s structure, accelerating the identification process.

NovaIscB serves as a versatile scaffold for genome editing tools. Researchers used it to replace specific DNA letters and alter targeted gene activity. Its compact size allows easy packaging inside a single adeno-associated virus (AAV), a common gene therapy delivery vector.

To demonstrate therapeutic potential, Zhang’s team created OMEGAoff, a NovaIscB-based tool that adds chemical markers to DNA to reduce specific gene activity. They programmed OMEGAoff to repress a cholesterol regulation gene and delivered the system to mice livers via AAV, resulting in lasting cholesterol level reductions.

The team anticipates NovaIscB’s broad applicability for targeting most human genes and encourages other labs to explore the technology. They also advocate for their evolution-guided approach to rational protein engineering. Zhu emphasized the importance of learning from nature’s diversity to enhance engineered systems.