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. 2022 Apr 19;13:2138. doi: 10.1038/s41467-022-29822-3

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — a The protein domain structure of DICER. DExD helicase domain, DUF283: domain of an unknown function, PAZ: Piwi/Argonaut/Zwille, RIIIDa and RIIIDb: RNase III domains, and dsRBD: double-stranded RNA-binding domain. b Schematic of shRNA. The siRNA region is red, and the green arrowheads indicate the DICER cleavage sites. c The purified DICER. d The randomized artificial shRNAs. This two-loop shRNA (TLR) contained primary and secondary loops. The upper stem-loop region (USL) was randomized in the 3-base pair or 5-nt windows. The secondary loop contained 32-nt randomized sequences (32N), which served as barcodes. e The high-throughput (HT) shRNA cleavage assay. DICER cleaved the artificial shRNA (subgroup TLR_1) into F1-2, F2-3, F1, F2, and F3 fragments. f Identification of the synthesized shRNA variants in the HT cleavage assays by next-generation sequencing (NGS). g The unique barcodes of each shRNA variant were counted from three repeated HT assays. The distribution of log₂(number of unique barcodes) are shown. h The reproducibility of the HT cleavage assays. R is Pearson’s correlation coefficient. i The fractions of different cleavage types (single or double cleavages) and different overhang lengths of the double cleavage products in the DICER-cleaved products. j The different cleavage sites of DICER on the shRNA variants. Upper panel: The fraction of variants processed at the major cleavage sites. The ‘major’ cleavage site of a variant is the cleavage site with the highest cleavage accuracy score. Lower panel: The cleavage accuracy score of 20,034 recovered variants in the HT cleavage assays at different cleavage sites. The cleavage accuracy score is colored according to the color scale. k The inaccuracy of DICER in processing shRNAs. The fraction of variants with unique or multiple cleavage sites. The SC3p, SC5p, and DC cleavage sites are from 19 to 23, as shown in (j). Cleavage sites with a cleavage accuracy score ≧0.1 were selected. l The double and single cleavage products of a representative variant are shown. Randomized nucleotides are in red. DC: double cleavage, SC5p: single cleavage on the 5p-strand, SC3p: single cleavage on the 3p-strand. Source data are provided as a Source Data file.