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.

中文翻译：

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强大而全面的腺相关病毒改组管道。

腺相关病毒（AAV）是用于合成基因传递载体工程的有吸引力的模板。用于育种具有改进特性的新型载体的一项特别强大的技术是DNA家族改组，即通过同源性驱动的DNA重组产生嵌合衣壳。在这里，为了使AAV DNA改组可用于更广泛的社区，我们提出了一个健壮的实验和生物信息学渠道，其中包括：（i）带有12个不同AAV衣壳基因的标准化和部分密码子优化的质粒；（ii）可扩展的协议，包括病毒库生产的故障排除指南；（iii）可免费获得的软件SALANTO，用于全面分析嵌合AAV DNA和蛋白质序列。此外，我们描述了一组12个预制的即用型AAV库。最后，我们证明了DNA条形码技术在复杂混合物中追踪AAV衣壳库的有用性。我们的协议和资源可在对定制病毒基因转移感兴趣的任何实验室中促进AAV进化技术的实施和定制。