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. 2019 Dec 4;10:5524. doi: 10.1038/s41467-019-13500-y

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© The Author(s) 2019

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. 6 — Chimeric nucleases enable genome editing in eukaryotic cells. a Genome editing in mammalian cells (HEK293T) using chimeric Cas12a-type variants. We constructed a plasmid expressing the M44 (or MAD7) nuclease (with T7 promoter), a single crRNA (with U6 promoter), and GFP. b Pictures of HEK293T after transfection. The H3K293T cells were transfected with the plasmid containing the M44 nuclease and GFP. Micrographs were taken under cool white light (left) or fluorescent light (right). c The T7E1 assay^52,58,59 was performed on cells that were expressing GFP and isolated by fluorescence-activated cell sorting. Untreated means the PCR products without T7 endonuclease treatment. Treated means the PCR products with T7 endonuclease treatment. d The indel rate of MAD7 and M44. The calculation was made using the formula shown in the methods section. e Genome editing in yeast (S. cerevisiae BY4741) using chimeric Cas12a-type variants. We constructed a plasmid containing the M44 (or MAD7) nuclease (with TEF1p promoter), a single crRNA (SNR52p promoter) targeting the CAN1 gene and a homology arm (HM) containing a CAN1-inactivating mutation as a template for recombineering. Only colonies with an inactivated CAN1 gene can grow on a +can plate. f The editing efficiency of MAD7 and M44. The editing efficiency was calculated by determining the ratio of colonies on plates +/−can. Editing was also confirmed by sequencing 20 colonies from +can plates. Source Data are available in the Source Data File.