Genome wide screening of host factors involved in Adeno-Associated Virus transduction

Adeno associated viruses (AAV) are small, non-pathogenic, naked single-stranded DNA viruses, which depend on S-phase for copying their genome and to allow gene expression. This obstacle was overcome by the development of AAVs with double-stranded genome (self-complementary (scAAVs), which are used as therapeutic gene delivery vectors for the treatment of a variety of monogenic diseases. Many details of transduction are still unknown, including how AAVs reach and cross the nuclear envelope and how they release the viral genome, although being strong bottlenecks of infection. We aimed to discover cellular factors involved in AAV transduction using the transcription profiles of cells with high and low transduction susceptibility after transduction with modified scAAV2, expressing YFP. As the susceptibility between AAV entry and detectable YFP expression may have changed overtime, we first identified a cell line and growth conditions, which maintained susceptibility on the individual cell level. These investigations were followed by transduction, FACS sorting, and mRNA sequencing. We further established a genome-wide CRISPR/Cas9 knockout on HUVEC cells with the aim to transduce them with scAAV2-YFP for verification of transduction-restricting genes by single-cell transcription analysis.