Agenda

Summary:

Reverse genetic screens are powerful tools for the functional annotation of genomes. In the context of multicellular organisms, interrogating gene function can be greatly facilitated by methods that allow spatial and temporal control of gene abrogation. We recently described a system that allows efficient CRISPR-based disruption of gene function in a constitutive or conditional manner. The system is based on liberation of multiple gRNAs from an RNA polymerase II or III precursor transcript by processing of flanking tRNAs. Expression of multiple gRNAs targeting the same gene can bypass limitations arising from in-frame indel mutations or gRNAs with low activity. Moreover, multiplexing of gRNAs that target different loci allows generation of complex genotypes in a single step. Because the tRNA system allows tissue-specific expression of gRNAs, the frequency of ectopic mutagenesis, which we show is frequently occurring when combining tissue-specific Cas9 expression with U6-gRNAs, is dramatically reduced. We are currently generating large-scale libraries of UAS-t::gRNA2x transgenic lines for systematic, tissue-specific CRISPR/Cas9 knock-out screening in Drosophila. I will present the current status of this project and a first functional characterization of these lines.

(f.port@dkfz-heidelberg.de)