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. 2020 Nov 3;3:643. doi: 10.1038/s42003-020-01374-6

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© The Author(s) 2020

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. 7 — a Design of a 20 bp section used to knock out VACV Spi2 showing premature stop (red), restriction sites (grey) and Spi2-gRNA site on parent genome. b Rapid protocol to create a VACV Spi2 knockout, showing steps taken to track selection. c Proportions of parent and desired recombinant after each step as determined by PCR. Pies and adjacent numbers show the average from 10 plaques at each step from two independent experiments. Grey boxes show frequency of the desired recombinant after analysing 100 plaques after recombination and after two rounds of selection.