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. 2022 Dec 20;50(22):12689–12701. doi: 10.1093/nar/gkac1192

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© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 2. — Mutational effect of W890A on the nuclease activities of LbCas12a. (A, B) Kinetic studies of the cis-cleavage activities on dsDNA (A) and ssDNA (B) by wild-type LbCas12a and mut2 (W890A). TS = target strand; NTS = nontarget strand; * = labeled strand. In these kinetic studies, the labeled strand was fluorescently labeled at the 5’-end with FAM. Each data point in panel (A) is averaged from three independent assays; each data point in panel (B) is averaged from two independent assays. Initial rates are presented after each symbol. (C) Mutational effect of W890A on the cleavage sites of NTS of dsDNA. (D) Genome editing workflow using tdTomato neural progenitor cells (NPCs) from Ai9 mice. The tdTomato gene will be turned on only when editing happens to remove the stop cassette. (E) Genome editing of NPCs by wild-type LbCas12a and mut2. The editing level is reflected by the percentage of tdTomato-positive NPCs (n = 3, means ± SD). In both delivery conditions, the editing efficiency of mut2 is significantly lower than the wild-type protein (***P < 0.001).