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

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.

A Novel Piggyback Strategy for mRNA Delivery Exploiting Adenovirus Entry Biology

“…SpyTag/SpyCatcher) now allow directed non-random chemical coupling to derive macromolecular structures [ 36 , 37 , 38 , 39 ]. In addition, we have now defined specific capsid locales which can be modified for coupling with such a molecular glue strategy [ 40 ]. We are thus now exploring methods to accomplish directed attachments of nucleic acid binding domains to specific adenovirus capsid locales.…”

Genetically Encoded Self-Assembling Protein Nanoparticles for the Targeted Delivery In Vitro and In Vivo

“…The reaction proceeds in a few minutes even at nanomolar concentrations of both peptides [ 200 ]. In one of the latest works, the authors used the SpyTag003/SpyCatcher003 system for adenovirus genetic engineering with a plug-and-display technology based on SpyTag003/SpyCatcher003 coupling chemistry [ 201 ]. Most applications of the SpyTag003/SpyCatcher003 system are currently focused on in vitro studies, however, SpyTag003/SpyCatcher003 is a promising platform for the self-assembly of targeted peptide nanoagents in vivo.…”

Engineering a Novel Modular Adenoviral mRNA Delivery Platform Based on Tag/Catcher Bioconjugation

“…The construction of Ad.hexon.SpyTag was described in our previous report [27]. Ad.hexon.SpyTag was engineered based on the chimpanzee adenovirus SAd36 with the early E1 region replaced by a CMV promoter-hybrid intron eGFP cassette and hexon hypervariable region five replaced by 48 bp SpyTag003, while the control virus did not have SpyTag incorporation.…”

“…We previously developed Ads that genetically incorporate SpyTag into the hexon protein, which is the major capsid structural component with 720 copies forming the virus particle. We utilized this vector to successfully deliver an engineered recombinant protein, SpyCatcher-Cas9, via anchoring to hexon [27]. Herein, we, therefore, employed this SpyTag-modified Ad as an anchoring platform to attach mRNA binding proteins to the Ad capsid, enabling subsequent site-specific attachment of mRNA to the virus exterior.…”