Abstract
ABSTRACTWhile recombinant Adenoviruses (rAds) are widely used in both laboratory and medical gene transfer, library-based applications using this vector platform are 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 employing 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 endonuclease recognition sites. In a second step, this selection marker is replaced with a transgene or mutation of interest via Gibson assembly. Our approach does not cause unwanted genomic off-target mutations while providing substantial flexibility for the site and nature of the genetic modification.This new genetic workflow, which we termed half-site directed fragment replacement (HFR) allows introduction of >106unique modifications into rAd encoding BACs using laboratory scale methodology. To demonstrate the power of HFR, we rescued barcoded viral vector libraries yielding a diversity of ∼2.5×104modified rAd per cm2of transfected cell culture.GRAPHICAL ABSTRACT
Publisher
Cold Spring Harbor Laboratory