ABSTRACT
While 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 >106 unique 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 ∼2.5×104 modified rAd per cm2 of transfected cell culture.
Competing Interest Statement
J. Fischer and Z. Ruzsics are co-inventors in patent application EP23199021.9 by the Albert-Ludwigs-University of Freiburg, which describes a 2-step workflow to seamlessly modify circular BAC-/Plasmids at high efficiency. J. Fischer and Z. Ruzsics are co-inventors in patent application PCT/EP2021/076757 by the Albert-Ludwigs-University of Freiburg, which describes a novel way of generating recombinant Adenoviruses by utilizing CRISPR/Cas9 linearization.
