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. 2017 Dec 4;7:16836. doi: 10.1038/s41598-017-17081-y

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© The Author(s) 2017

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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Activity of positive selection enriched mutant combinations. (a) Combinations of mutations that had retained activity in mammalian cells as single mutants were compared to WT SpCas9’s ability to cleave with a gRNA targeting GFP site 4 (S4) (n = 3, error bars indicate S.E.M., one-way ANOVA F_25,52 = 5.07, P = 4.0 × 10⁻¹¹, followed by post hoc linear contrasts). (b) Top performing combinations were targeted against chromosomally integrated GFP with a total of eight gRNAs and their activity was normalized between WT SpCas9 with each gRNA and a non-targeting gRNA (one-way ANOVA F_6,161 = 3.65, P = 0.0020, followed by post hoc linear contrasts, P = * < 0.05, ** < 0.01, *** < 0.001).