Sequencing of highly diverse primary rAd viral libraries. null

While recombinant Adenoviruses (rAds) are widely used in both laboratory and medical gene transfer, library based applications using this vector platform is not readily available. Recently, we developed a new method, the CRISPR/Cas9 mediated in vivo terminal resolution (CTR) aiding high efficiency rescue of rAds from recombinant DNA. Here we report on a genetic workflow that allows construction of BAC based rAd-libraries, which match the efficiency of CTR. We utilized frequent, pre-existing genomic sequences to allow insertion of a selection marker, complementing two selected target sites into novel, previously not existing endonuclease recognition site. This selection marker can be replaced in a second step with a transgene or mutation of interest via Gibson assembly. Our approach does not cause any scars in operational sequences or unwanted modifications elsewhere in the genome, while still providing substantial flexibility in regard of the site and nature of the genetic modification. This new genetic workflow, which we coined half-site directed fragment replacement (HFR) allowed introduction of >10^6 unique barcodes into rAd encoding BACs using laboratory scale methodology, which we were then able to rescue into highly diverse viral vector libraries with a diversity ~2.5x10^4 unique sequences per cm^2 of transfected cell culture using CTR.