High levels of AAV vector integration into CRISPR-induced DNA breaks

Abstract

Adeno-associated virus (AAV) vectors have shown promising results in preclinical models, but the genomic consequences of transduction with AAV vectors encoding CRISPR-Cas nucleases is still being examined. In this study, we observe high levels of AAV integration (up to 47%) into Cas9-induced double-strand breaks (DSBs) in therapeutically relevant genes in cultured murine neurons, mouse brain, muscle and cochlea. Genome-wide AAV mapping in mouse brain shows no overall increase of AAV integration except at the CRISPR/Cas9 target site. To allow detailed characterization of integration events we engineer a miniature AAV encoding a 465 bp lambda bacteriophage DNA (AAV-λ465), enabling sequencing of the entire integrated vector genome. The integration profile of AAV-465λ in cultured cells display both full-length and fragmented AAV genomes at Cas9 on-target sites. Our data indicate that AAV integration should be recognized as a common outcome for applications that utilize AAV for genome editing.

Fig. 1

AAV vectors integrate into CRISPR/Cas9 cut sites in vitro and in vivo. a Primary murine cortical neurons were transduced with an AAV1 vector encoding Cas9 as well as another AAV1 vector encoding a guide RNA (gRNA) against the genes indicated. Two different doses, 1e5 gc/cell (left panel) and 1e6 gc/cell (right panel), were tested. The negative control was neurons transduced by AAV-Cas9 vector without gRNA (Tmc1 gene was amplified by PCR for 1e5 gc/cell and APP gene was amplified for 1e6 gc/cell). Frequency of AAV sequences present at indels at the target site are shown in red vs total number of indels in blue. AAV capture efficiencies are shown as percentages on the graphs. Two biological replicates were sequenced for each condition (3 for APP^SW gene, 1e6 gc/cell dose). b AAV integration into Cas9 cut sites targeting therapeutic genes in the murine hippocampus, cochlea or muscle. For APP^SW, non-injected cerebellum or cortex was used as control. For Tmc1, non-injected cochleas were used. Animal numbers and the number of sequencing reactions are as follows (numbers of animals pooled per reaction included in parentheses): Hippocampus, control: n = 3 (3 reactions), Mecp2: n = 5 (2 reactions, n = 3 and 2), Dnmt3b: n = 5 (2 reactions, n = 3 and 2), APP^SW: n = 7 (7 reactions). Cochlea samples, non-injected: n = 21 (2 reactions, n = 9 and 12), injected: n = 33 (4 reactions, n = 6, 6, 9, and 12 animals). Muscle samples: n = 8 (2 reactions, n = 4 and 4). c CRISPResso analysis showing small indels at cut site from hippocampus, injected with AAV-Cas9 and AAV-gRNA against Dnmt3b. d Bimodal distribution of indel sizes, the larger indicating AAV sequence integration at the cut site, with specific examples shown in e (two sequencing reactions from 2 and 3 animals, respectively). f Characterization of AAV vector region present in indels with AAV-Mecp2-Cas9 (left panel) and AAV-U6-gRNA-syn-GFP (right panel) in brain samples (Dnmt3b was targeted). g Distribution of AAV integration surrounding the CRISPR cut site in the case of hippocampus, Dnmt3b was targeted (two sequencing reactions from 2 and 3 animals, respectively). Bars represent mean ± SD. Source data are provided as a Source Data file

Fig. 2

Genome-wide AAV mapping from CRISPR treated mouse brains. a Total number of unique integration sites. In the case of AAV-Cas9, AAV-gRNA^Mecp2, AAV-Cas9 + gRNA^Mecp2, and AAV-Cas9 + gRNA^Dnmt3b, three mouse brains were pooled together for library construction. For AAV-Cas9 + gRNA^APPSW, hippocampus tissues from two animals were separately processed for library construction. The colors represent different genomic integration types and are based on the output of Virus-Clip. b Total number of reads that contain integrants normalized to total reads, based on the output from Virus-Clip. c Circos plots on showing the chromosomal location of AAV integration events. The more eccentric a dot is, the higher the normalized read count for that site is, on a logarithmic scale. The gene names inside the circle represent either CRISPR targets or sites that are common integration events (present at least in three different samples). Colors of gene names are the same as in b. The human APP gene was added as a separate chromosome. d Bubble-plots showing all integration sites. The size of the circle is proportional to the normalized read count. The color was kept consistent in the figure in respect to type of integration. Intergenic integrations are marked by the chromosome and location. Source data are provided as a Source Data file

Fig. 3

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