Researchers at the University of Toronto have harnessed a bacterial immune defence system, known as CRISPR, to efficiently and precisely control the process of RNA splicing.
From left to right: PhD student Jack Daiyang Li, Professor Benjamin Blencowe and Associate Professor Mikko Taipale. Image credit: University of Toronto
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 Jack Daiyang Li, first author on the study and PhD student of molecular genetics, working in the labs of U of T researchers Benjamin Blencowe and Mikko Taipale at the Donnelly Centre for Cellular and Biomolecular Research in the Temerty Faculty of Medicine.
Exons from the same gene can be mixed and matched in various combinations to produce different versions of RNA, and consequently, different proteins. This process, called alternative splicing, contributes to the diverse expression of the 20,000 human genes that encode proteins, allowing the development and functional specialization of different types of cells.
However, it is unclear what most exons or introns do and the misregulation of normal alternative splicing patterns is a frequent cause or contributing factor to various diseases, including cancers and brain disorders. In addition, there is a lack of existing methods that allow for the precise and efficient manipulation of splicing.
The new study, published in the journal Molecular Cell, describes how 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 called 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 per cent of tested target exons,” said Li. “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 Blencowe, principal investigator on the study, Canada Research Chair in RNA Biology and Genomics, Banbury Chair in Medical Research and a professor of molecular genetics at the Donnelly Centre and Temerty Medicine.
“We have developed a versatile engineered splicing factor that outperforms other available tools in the targeted control of alternative exons,” said Taipale, also principal investigator on the study, Canada Research Chair in Functional Proteomics and Proteostasis, Anne and Max Tanenbaum Chair in Molecular Medicine and associate professor of molecular genetics at the Donnelly Centre and Temerty Medicine. “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 cancers, in which splicing is often disrupted.
Source: University of Toronto