Drawing a high-resolution functional map of adeno-associated virus capsid by massively parallel sequencing

Nat Commun. 2014;5:3075. doi: 10.1038/ncomms4075.


Adeno-associated virus (AAV) capsid engineering is an emerging approach to advance gene therapy. However, a systematic analysis on how each capsid amino acid contributes to multiple functions remains challenging. Here we show proof-of-principle and successful application of a novel approach, termed AAV Barcode-Seq, that allows us to characterize phenotypes of hundreds of different AAV strains in a high-throughput manner and therefore overcomes technical difficulties in the systematic analysis. In this approach, we generate DNA barcode-tagged AAV libraries and determine a spectrum of phenotypes of each AAV strain by Illumina barcode sequencing. By applying this method to AAV capsid mutant libraries tagged with DNA barcodes, we can draw a high-resolution map of AAV capsid amino acids important for the structural integrity and functions including receptor binding, tropism, neutralization and blood clearance. Thus, Barcode-Seq provides a new tool to generate a valuable resource for virus and gene therapy research.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Capsid / chemistry*
  • Capsid / physiology
  • Cell Line
  • DNA Barcoding, Taxonomic / methods*
  • DNA, Viral / genetics
  • Dependovirus / chemistry*
  • Dependovirus / genetics*
  • Genetic Therapy
  • Genetic Vectors / chemistry*
  • Genetic Vectors / genetics*
  • High-Throughput Nucleotide Sequencing / methods*
  • Homeodomain Proteins / genetics
  • Humans
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Mice, Knockout
  • Molecular Sequence Data
  • Mutation / genetics
  • Phenotype


  • DNA, Viral
  • Homeodomain Proteins
  • RAG-1 protein

Associated data

  • GENBANK/KF032296
  • GENBANK/KF032297