Pool-packaged AAV libraries exhibit extensive length-dependent and homology-dependent chimerism

nature biotechnology Brief Communication https://doi.org/10.1038/s41587-026-03097-1 Pool-packaged AAV libraries exhibit extensive length-dependent and homology-dependent chimerism Received: 20 December 2024 Accepted: 19 March 2026 Jean-Benoît Lalanne 1,2,8 , Chau Huynh1,3,8, John K. Mich Avery C. Hunker 4, Troy A. McDiarmid 1,3, Haedong Kim Jonathan T. Ting 4 & Jay Shendure 1,3,5,6,7 , , Boaz P. Levi 4 1,3 , 4 Published online: xx xx xxxx Check for updates Adeno-associated viruses (AAVs) are preferred gene therapy vectors because of their versatility, durability and safety profile. Here, we demonstrate extensive chimerism, manifesting as pervasive barcode swapping, among complex recombinant AAV (rAAV) libraries that are packaged as a pool. The observed chimerism is length and homology dependent but capsid independent, in some cases affecting the majority of packaged rAAV genomes. These results have implications for the design and deployment of functional rAAV libraries. Multiplexed functional genomic screens often use linked components within a cargo, for example, single-cell CRISPR screens1,2 (single guide RNA (sgRNA) and barcode) or massively parallel reporter assays (MPRAs; regulatory element and barcode)3. In these, any level of decoupling of expected pairings ultimately degrades signal quality. A prominent example is the frequent recombination seen in lentiviral cargo packaging4,5. Such recombination, which is length and homology dependent and results from lowly processive reverse transcription and template switching during replication6, stymied early single-cell functional genomics efforts using this delivery strategy7. By contrast, pervasive chimeric rearrangements of recombinant adeno-associated virus (rAAV) genetic material during packaging have not been described to our knowledge. Yet, recent studies of long-read sequenced AAV-packaged DNA have revealed unexpected DNA arrangements8,9. In parallel, high levels of noise and limited dynamic range are commonly observed in barcoded MPRA experiments using rAAVs10–14. These hint at possible unknown complexities during rAAV packaging. To explicitly test for chimera formation during rAAV packaging, we performed barcode swap experiments. Specifically, we constructed a series of rAAV libraries harboring a large number of uniquely associated pairs of barcodes separated by different inserts and flanked by AAV2 inverted terminal repeats (ITRs) (Fig. 1a; all plasmids and oligos listed in Supplementary Data 1). The six libraries harbored inserts of three different lengths (short: ~225 bp, mid-sized: ~800 bp, long: ~2.1 kb) each with two homology classes (homologous: identical sequences, nonhomologous: size-matched to homologous counterparts and bearing tagmented, narrowly size-selected Escherichia coli genomic DNA; Fig. 1a and Extended Data Figs. 1 and 2). Each insert further had a short internal sequence index for downstream demultiplexing (Extended Data Fig. 1d). The resulting libraries were complex (>5 million barcode pairs in parental library p146, bottlenecked to 15,000–45,000 pairs in libraries p149–p154). Inserts were size-adjusted outside the BC1–BC2 intervening sequence with filler sequences to fix the total ITR-to-ITR length to ~2.3 kb for inserts of different sizes (Extended Data Fig. 1c; see library p153× below). These libraries were then packaged separately into AAV capsids (all with PHP.eB, some with AAV2 serotypes, 14 packaging conditions total; Supplementary Data 2 and Methods). To assess for chimeras, defined as discordant barcode pairs within a single read, we performed long-read sequencing of the barcoded inserts (Fig. 1b) using PCR-free library preparation, from both sized-selected digested plasmids (‘zero-swap’ controls) and AAV-packaged DNA, on the Oxford Nanopore Technology (ONT) platform (through 1 Department of Genome Sciences, University of Washington, Seattle, WA, USA. 2Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montréal, Québec, Canada. 3Seattle Hub for Synthetic Biology, Seattle, WA, USA. 4Allen Institute for Brain Science, Seattle, WA, USA. 5 Brotman Baty Institute for Precision Medicine, Seattle, WA, USA. 6Howard Hughes Medical Institute, Seattle, WA, USA. 7Allen Discovery Center for Cell Lineage Tracing, Seattle, WA, USA. 8These authors contributed equally: Jean-Benoît Lalanne, Chau Huynh. e-mail: ; Nature Biotechnology Brief Communication AAV2 ITR ... AAV2 ITR Different lengths short (200–250 bp) Complex library of unique barcode pairs Separately for each library (for example, p151:mid-hom): Barcode swap experimental workflow Doubly barcoded parental plasmid library Plasmid libraries with various inserts mid (760–940 bp) BC1 BC2 Barcode pair dictionary Homologous p149:short-hom p150:short-nonhom p151:mid-hom p152:mid-nonhom p153×:long-hom p154:long-nonhom 1.0 Plasmid DNA 0.5 1.0 0.1 AAV DNA 0.5 *** p146: parental c Fraction disconcordant BC pairs BC1–BC2 plasmid libraries 0 500 1,000 1,500 2,000 BC-to-BC length (bp) 0 500 AAV p150:AAV p154:AAV p150 1,000 1,500 2,000 p146 BC-to-BC length (bp) 0 d Plasmid p149 0 0 0 (4) Discordant pairs? (2) Digest insert from plasmid Nonhomologous long (1,970–2,200 bp) (3) Long-read sequencing (1) AAV packaging Homology class Zoom Inserts BC2 *** BC1 500 Fraction disconcordant BC pairs b Doubly barcoded AAV constructs *** a https://doi.org/10.1038/s41587-026-03097-1 p152:AAV p151 p152 p154 p153 × 1,000 1,500 2,000 BC-to-BC length (bp) 0.4 p151:AAV 0.2 9% per 10× 0 p152:AAV 10−1 15 ng 100 101 102 % Input dose 15 µg per 15 cm Fig. 1 | Chimera formation during rAAV packaging revealed by barcode swapping experiments. a, A complex doubly barcoded cloning dock with associated dictionary of valid BC1–BC2 pairs was constructed and served as the starting point to clone libraries of inserts of varying lengths and homology class within AAV2 ITRs (six separate libraries: [short ~0.2 kb, mid ~0.8 kb, long ~2.1 kb] × [homologous, nonhomologous]). The seven different libraries considered are schematized (Extended Data Figs. 1 and 2). b, Each cloned barcoded library was separately: (1) digested to liberate the barcoded insert and (2) AAV-packaged. Both plasmid-derived insert and AAV DNA were submitted for direct long-read sequencing. Resulting long reads were scanned for barcodes and the fraction of discordant BC1–BC2 pairs, as compared to the bona fide parental dictionary, was determined. c, Quantification of the fraction of discordant barcode pairs as a function of the full-length BC-to-BC average size. Left, plasmid DNA; middle, AAV-packaged DNA; right, zoomed-in view of y axis range 0–0.1. Each point corresponds to swap quantification for a library for both plasmidderived (square) and AAV-derived (circle) material (n = 1 replicate per library). For each data point, we analyzed full-length BC-to-BC reads passing quality control filters and having s (...truncated)