Genomic DNA was isolated from cardiac tissue from one treated mouse in the first adult study. Whole-genome sequencing identified 62 read pairs spanning the knockin site. A single read pair (2%) supported presence of the donor construct knockin, while six read pairs (10%) demonstrated integration of fragments of the donor AAV vector, and four read pairs (6%) showed indel formation at the target site (Table S2 [/cms/10.1016/j.omtm.2023.08.009/attachment/ad0b1ec9-8b8f-4b9b-a154-b48f4dc86a72/mmc1]). Amplification and deep sequencing of the target site in heart samples from three treated mice confirmed a rate of indel formation of 7.4%, most frequently insertion of a single adenine on the coding strand. By ddPCR, we found that donor integration in the reverse orientation occurred at 77% of the frequency of integration in the correct orientation (Figure 6A). In order to further characterize donor integrations at the target site, we employed amplification and long-read sequencing. Two amplification reactions each spanned approximately 90% of the donor sequence, as well as either the upstream or downstream junction between the donor and genomic DNA. Among complete donor integrations, approximately 3% also retained elements of the donor vector upstream of the first Cas9 target site, and 18% retained vector elements downstream of the second target site (Figure 6B). In addition to these incompletely cleaved vectors, we also detected integration of additional vector fragments at 12% of junctions between the donor and genomic DNA (Figure 6C). These were most frequently small fragments derived from the 3′ inverted terminal repeat (ITR) and, less frequently, from the 5′ ITR, gRNA, or mega-exon (Figure 6D). In a separate amplification reaction that spanned the target site, we also detected integration of similar vector fragments in the absence of a complete donor integration (Figures 6C and 6D). Amplification-free targeted long-read sequencing was limited by low overall depth (67 reads capturing the target site), yet it identified four integration events (6% of reads), each of which consisted of multiple vector fragments (Figure 6E). [Figure thumbnail gr6]