ABSTRACT Animal microRNAs primarily regulate their targets through seed pairing. However, the contributions of non-seed regions and the identity of predominant targets in specific contexts remain poorly defined. We developed shRNAone, an shRNA design that produces highly homogeneous small RNAs and more closely mimics endogenous miRNA biogenesis. Using shRNAone, we built SynthoMir, a comprehensive human miRNA variant library enabling high-throughput dissection of miRNA features beyond the seed region. Applying this platform, we show that miR-494 regulates ISCA1 via 3’ supplementary pairing, and that ISCA1 is the predominant target mediating miR-494-associated sorafenib resistance in hepatocellular carcinoma. We further leveraged shRNAone’s homogeneity in pooled tiling screens to map druggable RNAi target sites in TTR and PCSK9, two targets of FDA-approved siRNA therapeutics. Our findings indicate that the outputs from shRNAone screening can be readily translated into effective siRNA designs. Together, shRNAone provides a versatile platform for dissecting miRNA regulatory networks and guiding the design of therapeutic siRNA sequences.