345. A Novel Toolbox for RNA-Editing Based on Massively Parallel Screening Using Barcoded Viral Vectors

Recent technical advances have opened up new avenues in molecular science with great impact on both in vitro and in vivo applications. Next generation sequencing has become available to a greater extent and advanced molecular techniques and methods within RNA/DNA-editing, barcoding and generation of high diversity DNA libraries are constantly evolving, giving rise to novel applications and discoveries.The aim of this study was to establish an unbiased, totally randomized screening assay for RNA-editing that can be universally utilized. By creating high resolution DNA-libraries from genomic sequences where each fragment is uniquely labelled with a DNA-barcode, we were able to map the biological function of each complete sequence. Here, we have utilized this approach to identify the most promising intronic sequences for RNA-editing. The screening assay was performed by deep sequencing of a lentiviral library, where each fragment was linked to a corresponding barcode. mRNA from transduced cells in vitro and in vivo was sequenced to identify and count the expressed barcodes, thus we could identify specific fragments suitable for RNA-editing. View Large Image | Download PowerPoint SlideRNA-editing was achieved via trans-splicing, where two different pre-mRNAs (one endogenous and one virally derived) hybridize through complementary sequences and form one mRNA. In trans-splicing, only the downstream segment of the mRNA is replaced, thus the gene is still under the control of its native promoter and other endogenous regulatory elements remain unaltered. Trans-splicing is inherently specific since the part of the mRNA that is delivered requires endogenous expression to exert an effect. View Large Image | Download PowerPoint SlideBy combining advanced cloning methods, DNA-barcoding and Next generation sequencing, we could screen extensive DNA libraries and select fragments highly efficient in RNA-editing. Our method can be used for correction of mutations, protein tracing and cell type identification.