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We present a rapid, scalable, user-friendly method for in vitro production of high-purity single-stranded DNA (ssDNA) ranging from 89 to 3315 nucleotides in length. PCR with a forward primer bearing a methanol-responsive polymer generates a tagged amplicon that enables selective precipitation of the modified strand under denaturing conditions. We demonstrate that the recovered ssDNA can be used for CRISPR/Cas9 homology-directed repair in human cells, DNA-origami folding, and fluorescent in situ hybridization.

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