RNA interference (RNAi) libraries screens have become widely used for small RNA (sRNA) therapeutic targets development. However, conventional enzymatically libraries, typically prepared using the type 2 restriction enzyme MmeI, produce sRNAs between 18 and 20 bp, much shorter than the usual lengths of 19–23 bp. Here we develop a size unbiased representative enzymatically generated RNAi (SURER) library, which employs type 3 restriction modification enzyme EcoP15I to produce sRNAs ranging from 19 to 23 bp using a group of rationally designed linkers, which can completely mimic the length of sRNAs naturally generated by Dicer enzyme in living cells, and the screening results of SURER libraries showed high recombination rate and knockdown efficiency. SURER library provides a useful tool for RNAi therapeutics screening in a fast and simple way.
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