Precision genome editing using combinatorial viral vector delivery of CRISPR-Cas9 nucleases and donor DNA constructs

Genome editing based on programmable nucleases and donor DNA constructs permits introducing specific base-pair changes and complete transgenes or tags at predefined chromosomal positions. A crucial requirement for such versatile genome editing approaches is, however, the need to co-deliver in an effective, coordinated and non-cytotoxic manner all the attendant tools into target cells. Here, adenoviral (AdV) and adeno-associated viral (AAV) vectors are investigated as delivery agents for, respectively, engineered CRISPR-Cas9 nucleases and donor DNA constructs prone to homologous recombination (HR) or homology-mediated end joining (HMEJ) processes. Specifically, canonical single-stranded and self-complementary double-stranded AAVs served as sources of ectopic HR and HMEJ substrates, whilst second- and third-generation AdVs provided for matched CRISPR-Cas9 nucleases. We report that AdV particles promote AAV transduction and that combining single-stranded AAV delivery of HR donors with third-generation AdV transfer of high-specificity CRISPR-Cas9 nucleases results in precise insertion of whole transgenes in large fractions of transduced cells in the absence of significant cytotoxicity often associated with transfection and electroporation protocols. In conclusion, the dual viral vector principle introduced herein permits investigating different genome-editing strategies and offers the prospect for the effective and accurate chromosomal edition in hard-to-transfect cell types with scientific and therapeutic relevance.