The CRISPR/Cas9 system is widely used for various genome editing approaches in cultured cells and living organisms and was broadly explored for preclinical applications. This two component system is composed of the Cas9 endonuclease that acts in cooperation with a chimeric guide RNA (gRNA) mediating the sequence-specific binding to its complementary target protospacer sequence preceding a protospacer adjunct motif (PAM). Because of its simple gRNA design and easy cloning procedure for customization, the CRISPR/Cas9 system is easier to handle than transcription activator-like effector nucleases (TALENs) and artificial zinc finger nucleases (ZFN). As veritable gene delivery vehicles, viral vectors could be particularly fit to broaden the applicability of RGNs to other cell types including dividing and quiescent primary cells.
Studies utilizing adenoviral (AdV) vectors as delivery vehicles for CRISPR/Cas9 showed efficient gene disruption in the host genome of various human cells and in viral genomes in antiviral approaches. Researchers prove 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 yield 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 various cell types ranged from 18% to 65%. Therefore, AdVs constitute a valuable platform for introducing RGNs into human somatic cells regardless of their transformation status.
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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. Scientific reports, 2014, 4: 5105.
2. Ehrke-Schulz E , et al. CRISPR/Cas9 delivery with one single adenoviral vector devoid of all viral genes. Scientific Reports, 2017, 7(1):17113.