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. 2024 Oct 29;15:9306. doi: 10.1038/s41467-024-53778-1

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© The Author(s) 2024

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — a Cryo-EM structure of the P. oleovorans type IV-A1 effector complex illustrating the R-loop formed by the target DNA (blue) and crRNA (orange). Protein subunits are shown as outlines. Protospacer target strand nucleotides are labeled according to their position. b Scheme illustrating the Efficiency of Transformation (EOT) assay. Cas proteins and crRNA are produced from separate expression plasmids. c, d EOT mismatch-tolerance assay with variable mismatching in spacer bases (c, orange bars) and protospacer bases on the target strand (d, blue bars). n = 3 independent replicates; mean ± s.d. Source data are provided as a Source Data file. e, f Electrophoretic mobility shift assays testing for the ability of types IV-A1 (e) and IV-A3 (f) complexes to bind non-mismatched (no mm) and mismatched DNA targets (mismatch ranges are labeled). n = 3 independent replicates; the no mm data in panel (e) is duplicated and shown in both graphs for reference. EMSA gels are shown in Supplementary Figs. 10, 11. Source data are provided as a Source Data file. g Scheme illustrating the P. oleovorans gfp repression assay. Type IV-A1 is natively expressed from the P. oleovorans megaplasmid and the gfp-targeting crRNA is expressed from a separate plasmid. h gfp repression mismatch tolerance (green bars) in uninduced cells (left), in cells with induced gfp (middle), and with induction of both, gfp and the gfp-targeting crRNA (right). n = 3 independent replicates; mean ± s.d. Source data are provided as a Source Data file.