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. 2018 Jul 17;9:2777. doi: 10.1038/s41467-018-05245-x

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© The Author(s) 2018

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. 5 — The PAM serves a limited role for AsCas12a function. a Diagram of DNA templates. Target dsDNA (WT) (left) contains a PAM and a proper protospacer. Bubble DNA (middle) has a PAM and a bubble structure (mismatches between the target and non-target strands) in the protospacer. Bubble w/o PAM (right) is the same as Bubble DNA except that it lacks a PAM sequence. Cy3 and biotin modifications are identical for all three DNA templates. b Schematic diagram of the single-molecule FRET experiment. c, d Target binding time τ₁ (c) and cleavage time τ₂ (d) of the AsCas12a–crRNA complex for the three different DNA templates. At least 171 time traces are analyzed to obtain τ₁ and τ₂ from two independent experiments (for Cas12a: 214 for WT, 171 for Bubble, 271 for Bubble w/o PAM, for Cas9: 404 for WT, 410 for Bubble, 396 for Bubble w/o PAM) (mean ± s.e.m.). All values in this figure are measured with a same concentration; 10 nM of protein (AsCas12a or SpCas9) and 20 nM RNA (crRNA or crRNA-tracrRNA)