15. A Novel Fast Production Pipeline for High Capacity Adenoviral Vectors to Deliver All Components of CRISPR/Cas9 System for Somatic Gene Editing Using One Single Viral Vector with Multiple Guide RNAs

Abstract

The discovery of the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system changed the field of in vivo genome editing. Nevertheless viral delivery of all required components including Cas9 and one or multiple guide RNA (gRNA) expression units using one single viral vector has not been fully exploited. Gene deleted high-capacity adenoviral vectors (HCAdVs) have the potential to efficiently deliver all expression units of the complete CRISPR/Cas9 machinery including multiple gRNAs into a broad variety of target cells using a single viral vector. However, the complicated handling of large DNA constructs and the time consuming production procedure hampered the use of HCAdV to deliver the CRISPR/Cas9 machinery for genome editing approaches. This work aimed at adapting a toolbox for HCAdV genome manipulation for the fast and simple introduction of the customized CRISPR/Cas9 machinery to provide new instruments to improve somatic genome editing approaches in mammalian cells. We generated a new CRISPR/Cas9 shuttle plasmid toolbox containing the Cas9 nuclease gene either utilizing a constitutive or an inducible promotor and a gRNA expression unit enabling customizing the CRISPR/Cas9 for a desired target sequence. This allows cloning or recombining all CRISPR/Cas9 components into the HCAdV genome in one step. To use several gRNA expression units for multiplexing the CRISPR/Cas9 system, further gRNA expression units can be easily included. To enable fast assembly of recombinant CRISPR-HCAdV genomes we used DNA recombineering to introduce all CRISPR/Cas9 expression units into the HCAdV genome contained in the bacterial artificial chromosome pBHCA. For insertion of multiple gRNA expression units into the HCAdV genome we utilized the established pAdV-FTC plasmid in concert with homing endonuclease directed cloning. CRISPR-HCAdVs were produced using a shortened amplification and purification procedure. Exploiting our toolbox we produced several CRISPR-HCAdVs carrying single and multiplex gRNA units specific for different targets including hCCR5, hDMD, and HPV16- and HPV18-E6 genes yielding sufficient titers within a short time. T7E1 assays were applied to prove CRISPR/Cas9-mediated cleavage of respective targets and infection of cultured human cells with respective CRISPR-HCAdVs resulted in efficient site specific gene editing. In summary, this new platform enables customization, cloning and production of CRISPR-HCAdV vectors for single or multiplex approaches within a short time. It simplifies the delivery of the CRISPR/Cas9 machinery by only using one single viral vector. Inducible Cas9 expression helps to avoid targeting of the genome of producer cell lines during vector production and may be beneficial for special approaches where constitutive expression is unwanted. We speculate that this may pave the way for broader applications of the CRISPR technology in preclinical and eventually clinical studies.