# Advanced viral genome in vitro Cas9 editing (AdVICE): an overnight method for traceless and limitless manipulation of adenoviral and vector genomes with large transgenes

## TL;DR

This paper introduces a simplified method for editing large viral genomes, making it easier to create adenovirus vectors for gene therapy and research.

## Contribution

A novel in vitro CRISPR/Cas9-based protocol for traceless and limitless genome editing of large DNA viruses like adenovirus.

## Key findings

- The AdVICE method enables efficient and traceless genome manipulation of adenovirus in a single overnight procedure.
- The protocol allows for insertions, deletions, and mutagenesis without requiring advanced molecular biology expertise.
- The approach was used to construct an adenovirus vector capable of delivering very large transgenes.

## Abstract

The size and complexity of large viral genomes limit the technical possibilities for genome manipulations in fundamental research and medical or technological applications. State-of-the-art recombineering in bacteria has partially overcome this limit but remains a time-consuming and complex procedure requiring specialist expertise. Here, we describe a simplified and highly efficient in vitro protocol for unlimited and traceless manipulation applicable to large viral genomes from DNA viruses using a combination of CRISPR/Cas9 cleavage and in vitro DNA assembly. We successfully used the protocol to manipulate adenovirus genomes, showing that genome rescue from viruses, insertions, deletions, and mutagenesis can be performed in a simple overnight procedure in a standard laboratory setting without the need for advanced knowledge of molecular biology. Finally, we use our approach to demonstrate the de novo, multi-step construction of an adenovirus vector suitable for delivering very large transgenes for gene editing.

The 36 kb size of the adenoviral genome has long been a deterrent to the construction of adenoviral mutants by scientists wishing to study the virus itself or to construct adenoviral vectors for cell biology and gene therapy. Most previous techniques, such as recombineering and yeast gap repair, impress more by their elegance than by their ease. In this paper, we use Cas9 ribonucleoprotein particles (RNPs) to target cleavage to specific sites in an adenoviral plasmid, then repair the break by Gibson assembly. Gibson assembly with synthetic DNA fragments has transformed basic cloning. Combining it with Cas9 RNPs, which act like highly specific restriction enzymes, makes adenoviral mutagenesis as easy as traditional plasmid cloning. We have used the approach to modify multiple sites in the adenoviral genome, but it could be applied to any large DNA virus for which the genome can be cloned in a plasmid.