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. 2022 May 31;13:3023. doi: 10.1038/s41467-022-30598-9

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© The Author(s) 2022

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. 2 — a Yeast survival assays examining the functionality of individual Cas9 designs targeting CAN1 via survival on canavanine. b Schematic representation of the quantitative Cas9 inhibitor-modulated genetic circuit. c Quantitation of engineered Cas9 activity via yeast survival with the Cas9 inhibitor system and a gRNA targeting HIS3. d Survival assays to determine the functionality of engineered Cas9 enzymes combining multiple designed regions. e Quantification of the activities of combined Cas9 designs. Error bars: s.e.m. of n = 9 biologically independent replicates. A standard Student’s t-test with a two-tailed distribution and unequal variance assumed between samples was used to calculate the significance. p-values: * p < 0.05, ** p < 0.01, ***p < 0.001. Specific p-values for panel c design vs WT: 1.4 p = 0.01, 1.5 p = 0.008, 2.1 p =0.02, 2.2 p = 0.05, 2.4 p = 0.0003, 2.10 p = 0.0007, 3.8 p = 0.02, 3.9 p = 0.05 and 3.10 p =0.0006 (Student’s t-test). Specific p-values for panel d design vs WT: 1.4 + 2.1 p = 0.004, 1.4 + 2.4 p = 0.03, 1.4 + 2.10 p = 0.03, 1.4 + 3.8 p = 0.001, 1.4 + 3.10 p = 0.002, 1.5 + 2.1 p = 1.4e-06, 1.5 + 2.2 p = 8.2e-05, 1.5 + 2.4 p = 4.9e-09, 1.5 + 2.10 p = 6.2e-04, 1.5 + 3.8 p = 6.7e-07, 1.5 + 3.9 p = 1.4e-04, 2.1 + 3.8 0.04, 2.1 + 3.9 p = 0.005, 2.1 + 3.10 p = 0.02, 2.2 + 3.8 p = 2.5e-08, 2.2 + 3.9 p = 0.004, 2.2 + 3.10 p = 0.0002, 2.4 + 3.8 p = 0.04, 2.4 + 3.9 p = 0.001, 2.4 + 3.10 p = 3.2e-05, 2.10 + 3.9 p = 0.01 and 2.10 + 3.10 p = 0.009 (Student’s t-test). Source data are provided as a Source Data file.