Arc Institute, Palo Alto, CA, USA.
Department of Bioengineering, University of California, Berkeley, Berkeley, CA, USA.
Nat Biotechnol. 2023 Apr;41(4):488-499. doi: 10.1038/s41587-022-01494-w. Epub 2022 Oct 10.
Large serine recombinases (LSRs) are DNA integrases that facilitate the site-specific integration of mobile genetic elements into bacterial genomes. Only a few LSRs, such as Bxb1 and PhiC31, have been characterized to date, with limited efficiency as tools for DNA integration in human cells. In this study, we developed a computational approach to identify thousands of LSRs and their DNA attachment sites, expanding known LSR diversity by >100-fold and enabling the prediction of their insertion site specificities. We tested their recombination activity in human cells, classifying them as landing pad, genome-targeting or multi-targeting LSRs. Overall, we achieved up to seven-fold higher recombination than Bxb1 and genome integration efficiencies of 40-75% with cargo sizes over 7 kb. We also demonstrate virus-free, direct integration of plasmid or amplicon libraries for improved functional genomics applications. This systematic discovery of recombinases directly from microbial sequencing data provides a resource of over 60 LSRs experimentally characterized in human cells for large-payload genome insertion without exposed DNA double-stranded breaks.
大型丝氨酸重组酶（LSR）是一种 DNA 整合酶，可促进移动遗传元件在细菌基因组中的特异性整合。迄今为止，只有少数 LSR（如 Bxb1 和 PhiC31）得到了表征，其作为人类细胞中 DNA 整合工具的效率有限。在这项研究中，我们开发了一种计算方法来鉴定数千种 LSR 及其 DNA 附着位点，使已知的 LSR 多样性扩大了 100 多倍，并能够预测其插入位点的特异性。我们在人类细胞中测试了它们的重组活性，将它们分类为着陆垫、基因组靶向或多靶向 LSR。总的来说，我们实现了比 Bxb1 高 7 倍的重组效率，并且在 7kb 以上的载体大小下实现了 40-75%的基因组整合效率。我们还展示了无病毒、直接整合质粒或扩增子文库的方法，用于改进功能基因组学应用。这种直接从微生物测序数据中发现重组酶的系统方法提供了超过 60 种 LSR 的资源，这些 LSR 在人类细胞中经过实验表征，可用于无暴露 DNA 双链断裂的大有效载荷基因组插入。
