Shen Konlin, Flood Jake J, Zhang Zhihuizi, Ha Alvin, Shy Brian R, Dueber John E, Douglas Shawn M
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, 94158 USA.
Department of Bioengineering, University of California, Berkeley, Berkeley, CA, 94720 USA.
Nucleic Acids Res. 2024 Apr 24;52(7):4098-4107. doi: 10.1093/nar/gkae189.
Long single-stranded DNA (ssDNA) is a versatile molecular reagent with applications including RNA-guided genome engineering and DNA nanotechnology, yet its production is typically resource-intensive. We introduce a novel method utilizing an engineered Escherichia coli 'helper' strain and phagemid system that simplifies long ssDNA generation to a straightforward transformation and purification procedure. Our method obviates the need for helper plasmids and their associated contamination by integrating M13mp18 genes directly into the E. coli chromosome. We achieved ssDNA lengths ranging from 504 to 20 724 nt with titers up to 250 μg/l following alkaline lysis purification. The efficacy of our system was confirmed through its application in primary T-cell genome modifications and DNA origami folding. The reliability, scalability and ease of our approach promise to unlock new experimental applications requiring large quantities of long ssDNA.
长单链DNA（ssDNA）是一种多功能分子试剂，其应用包括RNA引导的基因组工程和DNA纳米技术，但其生产通常资源密集。我们引入了一种利用工程化大肠杆菌“辅助”菌株和噬菌粒系统的新方法，该方法将长ssDNA的生成简化为一个简单的转化和纯化过程。我们的方法通过将M13mp18基因直接整合到大肠杆菌染色体中，避免了对辅助质粒及其相关污染的需求。经过碱性裂解纯化后，我们获得了长度在504至20724 nt之间的ssDNA，滴度高达250 μg/l。我们的系统在原代T细胞基因组修饰和DNA折纸折叠中的应用证实了其有效性。我们方法的可靠性、可扩展性和简便性有望开启需要大量长ssDNA的新实验应用。
