CRYSPER and CUREIPES: two new molecular tools enabling analysis of RNA splicing and C-to-U RNA editing-inducible...

RNA splicing and editing contribute to functional diversification of proteins encoded by single genes. Previous and current research primarily focus on sequencing of bulk material and bioinformatics to identify editing and splice sites, but these technologies ignore the role of individual cell types in the regulation of RNA splicing and editing. We present two new molecular tools for analysis of canonical and noncanonical RNA splicing (CRYSPER) and human Apobec1-dependent C-to-U RNA editing-inducible protein expression (CUREIPES). They use ratiometric fluorescence analysis for single-cell readout of RNA processing. Using CRYSPER, the results show noncanonical splice site usage in different cell types and demonstrate that the second intronic position can be a wobble base. Cellular heterogeneity of cryptic splice site utilization indicates cell type-specific control of this type of RNA processing and reveal a novel dimension of the regulation of RNA splicing. Thus, CRYSPER can be the new tool for research on canonical and noncanonical RNA splicing. Based on CRYSPER, CUREIPES was developed and is the first molecular tool that enables human Apobec1-dependent C-to-U RNA editing-inducible protein expression, which can be used to counteract pathological alteration of C-to-U RNA editing through cell-autonomous expression of editing-regulatory proteins, or of any other protein of interest.