Journal: Molecular Therapy. Nucleic Acids

Article Title: Inhibition of adenovirus replication by CRISPR-Cas9-mediated targeting of the viral E1A gene

doi: 10.1016/j.omtn.2023.02.033

Figure Lengend Snippet: Structure of the recombinant adenovirus vector constructs and gRNA sequences incorporated into them (A) Schematic of HAdV-5-based vectors with inserted individual gRNA sequences. All adenoviral vectors are based on the HAdV-5-derived vector pAd/PL-DEST (Thermo Fisher Scientific) and lack the E1 and E3 regions. Cas9 and gRNA expression cassettes were inserted into the deleted E1 region. The expression of spCas9-HF1 is driven by a tetracycline repressor-controlled CMV promoter comprising two binding sites for the repressor (2×TetO2). The expression of the individual targeting or non-targeting gRNAs is under control of a constitutive human U6 (hU6) promoter. The structure and sequence of the gRNAs are exemplarily shown for E1A gRNA 9 bound to its target site. The control vector containing only the Cas9 expression cassette is also depicted. (B) Target sequences for the individual gRNAs and their positions within the HAdV-5 genome (AY339865.1).

Article Snippet: Linear DNA fragments (gBLOCKS) containing individual targeting or non-targeting gRNAs under control of a human U6 promoter were generated by gene synthesis (Integrated DNA Technologies, Coralville, IA, USA), and the individual expression cassettes were inserted into the Hind II and NotI sites of the SpCas9-HF1 expression cassette-containing intermediate vector, giving rise to vectors pENTR-CMV-TetO2-spCas9-HF1-hU6-gRNA 1–10, containing a single targeting gRNA each, and to the control vectors containing non-targeting gRNAs (pENTR-CMV-TetO2-spCas9-HF1-hU6-NT gRNA) or containing only the spCas9-HF1 expression cassette (pENTR-CMV-TetO2-spCas9-HF1).

Techniques: Recombinant, Plasmid Preparation, Construct, Derivative Assay, Expressing, Binding Assay, Control, Sequencing