Researchers have harnessed a bacterial immune defense system, known as CRISPR, to efficiently and precisely control the process of RNA splicing.

The technology opens the door to new applications, including systematically interrogating the functions of parts of genes and correcting splicing deficiencies that underlie numerous diseases and disorders.

“Almost all human genes produce RNA transcripts that undergo the process of splicing, whereby coding segments, called exons, are joined together and non-coding segments, called introns, are removed and typically degraded,” said the first author on the study.

Exons can be alternatively spliced, such that the regulation and function of the approximately 20,000 human genes that encode proteins are greatly diversified, allowing the development and functional specialization of different types of cells.

However, it is unclear what most exons or introns do, and the mis-regulation of normal alternative splicing patterns is a frequent cause or contributing factor to various diseases, such as cancers and brain disorders. However, existing methods that allow for the precise and efficient manipulation of splicing have been lacking.

In the new research study, a catalytically-deactivated version of an RNA targeting CRISPR protein, referred to as dCasRx, was joined to more than 300 splicing factors to discover a fusion protein, dCasRx-RBM25. This protein is capable of activating or repressing alternative exons in an efficient and targeted manner.

“Our new effector protein activated alternative splicing of around 90 percent of tested target exons,” said the author. “Importantly, it is capable of simultaneously activating and repressing different exons to examine their combined functions.”

This multi-level manipulation will facilitate the experimental testing of functional interactions between alternatively spliced variants from genes to determine their combined roles in critical developmental and disease processes.

“Our new tool makes possible a broad range of applications, from studying gene function and regulation, to potentially correcting splicing defects in human disorders and diseases”, said the principal investigator on the study.

“We have developed a versatile engineered splicing factor that outperforms other available tools in the targeted control of alternative exons,” said another principal investigator on the study. “It is also important to note that target exons are perturbed with remarkably high specificity by this splicing factor, which alleviates concerns about possible off-target effects.”

The researchers now have a tool in hand to systematically screen alternative exons to determine their roles in cell survival, cell type specification and gene expression.

When it comes to the clinic, the splicing tool has potential to be used to treat numerous human disorders and diseases, such as autism and cancers, in which splicing is often disrupted.

https://www.cell.com/molecular-cell/abstract/S1097-2765(24)00475-1