LAUSR

Recombinant Adenovirus vectors enable highly efficient gene delivery in vitro and in vivo. As a result, they are widely used in gene therapy, vaccination, and anti-cancer applications. We have previously developed the AdZ vector system, which uses recombineering to permit high throughput cloning of transgenes into Adenovirus vectors, simplifies alteration of the vector backbone, and enables rapid recovery of infectious virus, even if a transgene is incompatible with vector replication. Here we adapt this vector system to enable high throughput cloning of sequences for CRISPR/Cas9 editing. Vectors were optimised to ensure efficient cloning, and high editing efficiency using spCas9 and sgRNA sequences in a single vector. Using a multiplicity of infection of 50, knockout efficiencies of up to 80% could be achieved with a single sgRNA. Vectors were further enhanced by altering the spCas9 sequence to match that of SniperCas9, which has reduced off-target activity but maintains on-target efficiency, and by applying modifications to the sgRNA sequence that significantly enhance editing efficiency. Thus, the AdZ-CRISPR vectors offer highly efficient knockout, even in hard to transfect cells, and enables large scale CRISPR/Cas9 projects to be undertaken easily and quickly.