19.08.2020: Genome editing refers to all those molecular biological techniques used to edit specific genes, including the DNA of plants, animals and humans. The application of gene editing technology is currently limited by a lack of sufficiently safe and efficient delivery methods. A recently presented new approach developed at Vetmeduni Vienna could change this.

The scope and scalability of gene editing systems are currently limited by a lack of safe and efficient methods for delivering RNA-guided endonucleases to target cells. This is because the current methods used to deliver DNA have a limited cell-type specificity and are associated with side effects such as integration into undesired chromosomal sites, immunogenicity or size limitations. One of the major problems is that the Cas9 nuclease when expressed from a plasmid (DNA delivery), is expressed for an extended period. The long time expression can lead to cuts in the host genome in undesired positions (off-targets).  

Increasing attention is thus being paid to the direct delivery of preassembled Cas9 protein/sgRNA complexes (RNPs) to cells in which the rapid turnover of RNPs limits the exposure of the genome to nucleases, thereby mitigating off-target effects. Furthermore, the transient occurrence of RNPs in cells is expected to induce only minimal innate and adaptive immune responses. Despite the advantages of RNP delivery, the use of this approach has so far been restricted to cell types that do not suffer from reduced cell viability or phenotypic changes. In addition, the technology is very complex and lacks tissue and cell specificity. Therefore, there is an urgent need for a more versatile, safe, cell-selective and easy-to-use transient delivery system.

Robust, effective system

In their recently presented study, Ivana Indikova and Stanislav Indik from the Institute of Virology at Vetmeduni Vienna describe the novel system they have developed for the co-delivery of the Cas9 protein and a template for sgRNA within lentivirus-based “nanoparticles”. The idea behind the new technology is based on findings that lentiviruses can simultaneously deliver foreign proteins and an episomal viral DNA produced by reverse transcription from the vector RNA genome.

Stanislav Indik: “We have extended this approach to show that episomal DNA can serve as a template for the transcription of sgRNA, which forms a complex with the co-delivered Cas9 protein and targets the nuclease to a specific site in the genome. This strategy leads to robust editing activity that appears to be comparable or even superior to that reported for the direct delivery of Cas9 protein/sgRNA complexes to cells.” The researchers believe that the newly presented approach could serve as a new platform for the efficient, dose-controlled and tissue-specific delivery of genome-editing enzymes to cells. Additionally, they envisage that the lack of cas9 gene in the lentivector genome “makes a space” for a transgene that can be co-delivered with the Cas9/sgRNA. Thus, the method may be suitable for simultaneous endogenous gene disruption and transgene delivery.

New technology extends the existing usage possibilities to new applications In contrast to chemical transfection or electroporation, virus-mediated delivery is receptor-mediated, so the use of pseudotypes bearing natural or engineered envelope proteins would allow selective transfer to essentially any target cell population. “This approach may even extend the repertoire of cell types that can be edited to clinically relevant nondividing cells (e.g. neurons, hepatocytes, quiescent lymphocytes and hematopoietic stem cells),” says Indik.

The article “Highly efficient ‘hit-and-run’ genome editing with unconcentrated lentivectors carrying Vpr.Prot.Cas9 protein produced from RRE-containing transcripts” by Ivana Indikova and Stanislav Indik was published in Nucleic Acids Research.