biostudies-literatureUnknownShen KNIGMS NIH HHSLong 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 <i>E. coli</i> "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 <i>E. coli</i> chromosome. We achieved ssDNA lengths ranging from 504 to 20,724 nucleotides 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 promises to unlock new experimental applications requiring large quantities of long ssDNA.bioRxiv : the preprint server for biology2024.02.27.582394https://www.ebi.ac.uk/biostudies/studies/S-EPMC10925301biostudies-literatureEngineering an Escherichia coli strain for production of long single-stranded DNA.PMC10925301R35 GM125027F32 GM147967Shen KFlood JJZhang ZHa ADouglas SMDueber JEShy BRfalseEngineering an Escherichia coli strain for production of long single-stranded DNA.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 <i>E. coli</i> "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 <i>E. coli</i> chromosome. We achieved ssDNA lengths ranging from 504 to 20,724 nucleotides 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 promises to unlock new experimental applications requiring large quantities of long ssDNA.2024-01-01T00:00:00Z2024 Mar2025-04-19T20:26:31.77Z2025-04-19T20:26:31.77ZS-EPMC109253013846403610.1101/2024.02.27.582394