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. 2023 Jan 4;613(7944):588–594. doi: 10.1038/s41586-022-05559-3

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© The Author(s) 2023

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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Extended Data Fig. 4 — a, Non-specific collateral activities of SuCas12a2, LbCas12a, LwaCas13a, and AbCas12g towards FAM-labelled non-target ssDNA, dsDNA, and RNA in the presence of target ssDNA, dsDNA, and RNA. All cleavage reactions were conducted at 37 °C unless specified otherwise. AbCas12g was triggered by RNA and ssDNA and exhibited preferential collateral cleavage of RNA over ssDNA but no discernable cleavage of dsDNA. b, SuCas12a2-mediated cleavage over time of FAM-labelled RNA (top), ssDNA (middle), and dsDNA (bottom) non-target substrates. These substrates are cleaved by SuCas12a2 through its non-specific collateral activity. c, Electromobility shift assay indicating SuCas12a2 and LbCas13a mostly do not indiscriminately degrade dsRNA. Uncleaved substrates and cleaved products are indicated. d, Impact of mutating conserved cysteines within the predicted Zinc finger domain of SuCas12a2 on RNA-triggered collateral activity. The mutated cysteines within SuCas12a2 are C1170S, C1173S, C1188S and C1191S. e, Electromobility shift assays of supercoiled pUC19 plasmid with SuCas12a2:crRNA and different RNA sequences over time. RNA sequences include: Non-self target (complementary to gRNA with 3′ GAAAG PFS), Target complement (complement of non-self target), Non-target (RNA sequence used in trans-cleavage assays), No PFS (RNA that only contains compliment to gRNA no 3′ PFS), Self-PFS (complementary to gRNA with 3′ AUCUA PFS). RNA sequences can be found in Supplementary Table 1. SuCas12a2(E1063A) with the non-self target is included as a negative control. NN: no-nuclease control. For gel source data, see Supplementary Fig. 1.