Short single-stranded DNA oligonucleotides enable intracellular transcription of functional RNAs in mammalian cells

Programmable intracellular production of short functional RNAs underlies diverse applications ranging from gene regulation to genome engineering, but is often constrained by cloning workflows, RNA synthesis, or viral-vector delivery. Here, we introduce a single-stranded DNA (ssDNA)-driven intracellular transcription strategy in which short ssDNA templates function as transcriptional substrates when paired with orthogonal bacteriophage RNA polymerases (RNAPs). By embedding phage promoters within short hairpin structures to create a locally double-stranded recognition site, we enable robust RNAP-dependent transcription from ssDNA in mammalian cells. We demonstrate that ssDNA templates can drive production of functional CRISPR guides, supporting adenine base editing at reporter and endogenous loci and enabling CRISPR-based transcriptional activation across distinct human cell lines. These results establish ssDNA templates as compact, synthetically accessible inputs for RNA production in mammalian cells, with broad utility in synthetic biology and genome engineering.