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ACS Synth Biol. 2019 Jan 18;8(1):194-206. doi: 10.1021/acssynbio.8b00373. Epub 2019 Jan 8.

A Robust and All-Inclusive Pipeline for Shuffling of Adeno-Associated Viruses.

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Cluster of Excellence CellNetworks, Department of Infectious Diseases, Virology and Center for Quantitative Analysis of Molecular and Cellular Biosystems (BioQuant) , Heidelberg University Hospital , Heidelberg , 69120 , Germany.
Translational Oncology at the University Medical Center of the Johannes Gutenberg University , Mainz , 55131 , Germany.
Synthetic Biology Group, Institute for Pharmacy and Biotechnology (IPMB) and Center for Quantitative Analysis of Molecular and Cellular Biosystems (BioQuant) , University of Heidelberg , Heidelberg , 69120 , Germany.
German Center for Infection Research (DZIF) and German Center for Cardiovascular Research (DZHK) , Heidelberg , 69120 , Germany.


Adeno-associated viruses (AAV) are attractive templates for engineering of synthetic gene delivery vectors. A particularly powerful technology for breeding of novel vectors with improved properties is DNA family shuffling, i.e., generation of chimeric capsids by homology-driven DNA recombination. Here, to make AAV DNA shuffling available to a wider community, we present a robust experimental and bioinformatical pipeline comprising: (i) standardized and partially codon-optimized plasmids carrying 12 different AAV capsid genes; (ii) a scalable protocol including troubleshooting guide for viral library production; and (iii) the freely available software SALANTO for comprehensive analysis of chimeric AAV DNA and protein sequences. Moreover, we describe a set of 12 premade and ready-to-use AAV libraries. Finally, we demonstrate the usefulness of DNA barcoding technology to trace AAV capsid libraries within a complex mixture. Our protocols and resources facilitate the implementation and tailoring of AAV evolution technology in any laboratory interested in customized viral gene transfer.


AAV; Adeno-associated virus; DNA family shuffling; DNA recombination; molecular evolution; virus engineering


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