High-capacity adenovector delivery of forced CRISPR-Cas9 heterodimers fosters precise chromosomal deletions in human cells

Francesca Tasca; Marcella Brescia; Jin Liu; Josephine M Janssen; Kamel Mamchaoui; Manuel A F V Gonçalves

doi:10.1016/j.omtn.2023.02.025

High-capacity adenovector delivery of forced CRISPR-Cas9 heterodimers fosters precise chromosomal deletions in human cells

Mol Ther Nucleic Acids. 2023 Feb 22:31:746-762. doi: 10.1016/j.omtn.2023.02.025. eCollection 2023 Mar 14.

Authors

Francesca Tasca¹, Marcella Brescia¹, Jin Liu¹, Josephine M Janssen¹, Kamel Mamchaoui², Manuel A F V Gonçalves¹

Affiliations

¹ Department of Cell and Chemical Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, the Netherlands.
² Sorbonne Université, Inserm, Institut de Myologie, Centre de Recherche en Myologie, Paris, France.

Abstract

Genome editing based on dual CRISPR-Cas9 complexes (multiplexes) permits removing specific genomic sequences in living cells leveraging research on functional genomics and genetic therapies. Delivering the required large and multicomponent reagents in a synchronous and stoichiometric manner remains, however, challenging. Moreover, uncoordinated activity of independently acting CRISPR-Cas9 multiplexes increases the complexity of genome editing outcomes. Here, we investigate the potential of fostering precise multiplexing genome editing using high-capacity adenovector particles (AdVPs) for the delivery of Cas9 ortholog fusion constructs alone (forced Cas9 heterodimers) or together with their cognate guide RNAs (forced CRISPR-Cas9 heterodimers). We demonstrate that the efficiency and accuracy of targeted chromosomal DNA deletions achieved by single AdVPs encoding forced CRISPR-Cas9 heterodimers is superior to that obtained when the various components are delivered separately. Finally, all-in-one AdVP delivery of forced CRISPR-Cas9 heterodimers triggers robust DMD exon 51 splice site excision resulting in reading frame restoration and selection-free detection of dystrophin in muscle cells derived from Duchenne muscular dystrophy patients. In conclusion, AdVPs promote precise multiplexing genome editing through the integrated delivery of forced CRISPR-Cas9 heterodimer components, which, in comparison with split conventional CRISPR-Cas9 multiplexes, engage target sequences in a more coordinated fashion.

Keywords: Duchenne muscular dystrophy; MT: RNA/DNA Editing; dystrophin; fusion Cas9 proteins; gene delivery; high-capacity adenoviral vectors; multiplexing gene editing; on-target specificity; precision gene editing.