Large-scale genome editing based on high-capacity adenovectors and CRISPR-Cas9 nucleases rescues full-length dystrophin synthesis in DMD muscle cells
Summary
Targeted chromosomal insertion of large genetic payloads in human cells leverages and broadens synthetic biology and genetic therapy efforts. Yet, obtaining large-scale gene knock-ins remains particularly challenging especially in hard-to-transfect stem and progenitor cells. Here, fully viral gene-deleted adenovector particles (AdVPs) are investigated as sources of optimized high-specificity CRISPR-Cas9 nucleases and donor DNA constructs tailored for targeted insertion of full-length dystrophin expression units (up to 14.8-kb) through homologous recombination (HR) or homology-mediated end joining (HMEJ). In muscle progenitor cells, donors prone to HMEJ yielded higher CRISPR-Cas9-dependent genome editing frequencies than HR donors, with values ranging between 6% and 34%. In contrast, AdVP transduction of HR and HMEJ substrates in induced pluripotent stem cells (iPSCs) resulted in similar CRISPR-Cas9-dependent genome editing levels. Notably, when compared to regular iPSCs, in p53 knockdown iPSCs, CRISPR-Cas9-dependent genome editing frequencies increased up to 6.7-fold specifically when transducing HMEJ donor constructs. Finally, single DNA molecule analysis by molecular combing confirmed that AdVP-based genome editing achieves long-term complementation of DMD-causing mutations through the site-specific insertion of full-length dystrophin expression units. In conclusion, AdVPs are a robust and flexible platform for installing large genomic edits in human cells and p53 inhibition fosters HMEJ-based genome editing in iPSCs. © The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.
| Authors | Tasca F, Brescia M, Wang Q, Liu J, Janssen JM, Szuhai K, Gonçalves MAFV |
|---|---|
| Journal | Nucleic acids research |
| Publication Date | 2022 Jul 22;50(13):7761-7782 |
| PubMed | 35776127 |
| PubMed Central | PMC9303392 |
| DOI | 10.1093/nar/gkac567 |