Fig. 3
From: High levels of AAV vector integration into CRISPR-induced DNA breaks
Characterization of AAV vector integration into CRISPR cut sites using a miniaturized AAV genome. a Schematic of standard-sized AAV-CBA-FLuc vector (top, 4062 bases) vs miniaturized AAV-λ465 (bottom, 465 bases). Chart is to scale. b Transmission electron microscopic examination of iodixanol gradient-purified capsids of AAV2-λ or AAV2-CBA-FLuc. c quantitation of full vs empty capsids (bars represent mean ± SEM, data from two independent experiments, 15 and 10 images were taken and 954 and 1231 capsids were counted for AAV-λ465 and AAV-CBA-FLuc, respectively, and p = 0.0254, unpaired t-test). d Alkaline gel electrophoreses and Southern blot for AAV genomes from iodixanol purified vectors (AAV2-CMV::NLS-SaCas9-NLS-3xHA-bGHpA;U6::BsaI-sgRNA (pX601, 4.8 kb size) and AAV-λ465 (465 bp size) and cellular genomic DNA containing integrated AAV-λ465. For Southern blot, we used a probe specific for the ITR region. Star (*) highlights the 465 bp expected band and pound (#) sign highlights concatemers in the AAV-λ465 genome. e ITR-genomic fusion events quantified by integration-specific qPCR assay, using AAV-λ465 or AAV2-CBA-FLuc vectors determined (bars represent mean ± SD). Three independent experiments were performed using two technical replicates each. f Heatmap of AAV specific ITR nucleotide integration at CRISPR cut site. More saturated red indicates higher frequency of breaks at the given position. g Integration profile of the entire miniaturized AAV genome from U2-OS cells. h Representative individual AAV integration clones showing different forms of integration detected (for all the clones, see Supplementary Fig. 4). Source data are provided as a Source Data file
