RNA-guided Nucleases (RGNs) Based CRISPR-Cas9

CRISPR/Cas9-derived RNA-guided nucleases (RGNs) are DNA targeting systems, which are rapidly being harnessed for gene regulation and gene editing purposes in cell lines and model organisms. As veritable gene delivery vehicles, viral vectors may be particularly fit to broaden the applicability of RGNs to other cell types including quiescent and dividing primary cells.

In this regard, adenoviral vectors (AdVs) constitute interesting RGN delivery candidates especially owing to their episomal nature, high-titers, large cloning capacity, and ability to transduced dividing as well as non-dividing cells. It has demonstrated that AdVs, namely second-generation fiber-modified AdVs encoding Cas9 or single guide RNA (gRNA) molecules addressing the Cas9 nuclease to the AAVS1 ‘‘safe harbor’’ locus or to a recombinant model allele can be produced to high-titers. Importantly, AdV-mediated transduction of gRNA:Cas9 ribonucleoprotein complexes into transformed and non-transformed cells yields rates of targeted mutagenesis similar to or approaching those achieved by isogenic AdVs encoding TALENs targeting the same AAVS1 chromosomal region. RGN-induced gene disruption frequencies in the different types of cell ranged from 18% to 65%.

Figure 1. Schematic representation of the genome-modifying AdVs.

1. Maggio I, et al. Adenoviral vector delivery of RNA-guided CRISPR/Cas9 nuclease complexes induces targeted mutagenesis in a diverse array of human cells. Sci Rep, 2014, 4(2):5105.

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