CRISPR-Derived Protein Controls Exon Inclusion and Exclusion

The University of Toronto researchers have successfully and precisely controlled the process of RNA splicing by using CRISPR, a bacterial immune defense system.

This technology makes new applications possible, such as methodically examining the roles of specific gene segments and fixing splicing deficiencies that are the root cause of many illnesses 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.”

Jack Daiyang Li, Study First Author and Ph.D. Student, Department of Molecular Genetics, University of Toronto

Li is also working in Benjamin Blencowe's and Mikko Taipale's labs at U of T’s Donnelly Centre for Cellular and Biomolecular Research.

Exons can be spliced differently, which greatly diversifies the regulation and function of the 20,000 or so human genes that encode proteins. This diversity allows for the development and functional specialization of various cell types.

The majority of exons and introns are unknown in their functions, and abnormal alternative splicing patterns are frequently the cause or a contributing factor in several diseases, including brain disorders and cancers. Nevertheless, few techniques have been developed yet that make manipulating splicing accurate and effective.

In the new study, dCasRx, a catalytically-deactivated RNA-targeting CRISPR protein, was combined with over 300 splicing factors to create the fusion protein dCasRx-RBM25. This protein exhibits efficient and targeted activation or repression of alternative exons.

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

Jack Daiyang Li, Study First Author and Ph.D. Student, Department of Molecular Genetics, University of Toronto

The experimental testing of functional interactions between alternatively spliced gene variants to ascertain their combined roles in important developmental and disease processes will be made easier by this multi-level manipulation.

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.”

Benjamin Blencowe, Study Principal Investigator and Professor, Department of Molecular Genetics, Donnelly Centre

Blencowe is also the Canada Research Chair in RNA Biology and Genomics, Banbury Chair in Medical Research, and Professor of Molecular Genetics at the Donnelly Centre and the Temerty Faculty of Medicine

We have developed a versatile engineered splicing factor that outperforms other available tools in the targeted control of alternative exons. 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.”

Taipale, Study Principal Investigator and Associate Professor of Molecular Genetics, Donnelly Centre, University of Toronto

Taipale is also the Canada Research Chair in Functional Proteomics and Proteostasis, Anne and Max Tanenbaum Chair in Molecular Medicine at the Donnelly Centre and Temerty Medicine.

With this tool, the researchers can now thoroughly screen alternative exons to ascertain their functions in gene expression, cell type specification, and cell survival.

In the medical setting, the splicing tool may be utilized to treat a wide range of illnesses and conditions affecting people, including cancer and autism, where splicing is frequently disturbed. The study was published in the journal Molecular Cell.