Efficient Genome Editing Achieved via Plug-and-Play Adenovirus Piggyback Transport of Cas9/gRNA Complex on Viral Capsid Surface

ACS Nano. 2022 Jul 26;16(7):10443-10455. doi: 10.1021/acsnano.2c00909. Epub 2022 Jun 24.

Abstract

The capacity to efficiently deliver the gene-editing enzyme complex to target cells is favored over other forms of gene delivery as it offers one-time hit-and-run gene editing, thus improving precision and safety and reducing potential immunogenicity against edited cells in clinical applications. Here we performed a proof-of-mechanism study and demonstrated that a simian adenoviral vector for DNA delivery can be repurposed as a robust intracellular delivery platform for a functional Cas9/guide RNA (gRNA) complex to recipient cells. In this system, the clinically relevant adenovirus was genetically engineered with a plug-and-display technology based on SpyTag003/SpyCatcher003 coupling chemistry. Under physiological conditions, an off-the-shelf mixture of viral vector with SpyTag003 incorporated into surface capsid proteins and Cas9 fused with SpyCatcher003 led to a rapid titration reaction yielding adenovirus carrying Cas9SpyCatcher003 on the virus surface. The Cas9 fusion protein-conjugated viruses in the presence of a reporter gRNA delivered gene-editing functions to cells with an efficiency comparable to that of a commercial CRISPR/Cas9 transfection reagent. Our data fully validate the adenoviral "piggyback" approach to deliver an intracellularly acting enzyme cargo and, thus, warrant the prospect of engineering tissue-targeted adenovirus carrying Cas9/gRNA for in vivo gene editing.

Keywords: Cas9/gRNA complex; SpyCatcher003; SpyTag003; gene editing; simian adenovirus.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, N.I.H., Extramural

MeSH terms

  • Adenoviridae / genetics
  • Adenoviridae / metabolism
  • CRISPR-Associated Protein 9 / genetics
  • CRISPR-Associated Protein 9 / metabolism
  • CRISPR-Cas Systems / genetics
  • Capsid / metabolism
  • Gene Editing* / methods
  • RNA, Guide, CRISPR-Cas Systems* / genetics
  • RNA, Guide, CRISPR-Cas Systems* / metabolism

Substances

  • RNA, Guide, CRISPR-Cas Systems
  • CRISPR-Associated Protein 9