biostudies-literatureCrawford KDNIBIB NIH HHSW. M. Keck FoundationGary and Eileen Morgenthaler FundPew Biomedical Scholars ProgramUCSF Discovery Fellows ProgramNational Institute of Biomedical Imaging and BioengineeringNational Science Foundationgkae1199https://www.ebi.ac.uk/biostudies/studies/S-EPMC11754653biostudies-literatureUnknown53(2)The bacterial retron reverse transcriptase system has served as an intracellular factory for single-stranded DNA in many biotechnological applications. In these technologies, a natural retron non-coding RNA (ncRNA) is modified to encode a template for the production of custom DNA sequences by reverse transcription. The efficiency of reverse transcription is a major limiting step for retron technologies, but we lack systematic knowledge of how to improve or maintain reverse transcription efficiency while changing the retron sequence for custom DNA production. Here, we test thousands of different modifications to the Retron-Eco1 ncRNA and measure DNA production in pooled variant library experiments, identifying regions of the ncRNA that are tolerant and intolerant to modification. We apply tNucleic acids researchHigh throughput variant libraries and machine learning yield design rules for retron gene editors.PMC11754653R21EB031393R21 EB031393MCB 2137692Shipman SLGoodarzi HKhan AGCrawford KDLopez SCfalseHigh throughput variant libraries and machine learning yield design rules for retron gene editors.The bacterial retron reverse transcriptase system has served as an intracellular factory for single-stranded DNA in many biotechnological applications. In these technologies, a natural retron non-coding RNA (ncRNA) is modified to encode a template for the production of custom DNA sequences by reverse transcription. The efficiency of reverse transcription is a major limiting step for retron technologies, but we lack systematic knowledge of how to improve or maintain reverse transcription efficiency while changing the retron sequence for custom DNA production. Here, we test thousands of different modifications to the Retron-Eco1 ncRNA and measure DNA production in pooled variant library experiments, identifying regions of the ncRNA that are tolerant and intolerant to modification. We apply t2025-01-01T00:00:00Z2025 Jan2025-04-04T22:07:21.917Z2025-04-04T22:07:21.917ZS-EPMC117546533965804710.1093/nar/gkae1199