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. 2021 Aug 26;11:17267. doi: 10.1038/s41598-021-96735-4

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© The Author(s) 2021

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Mechanism of CRISPR-Cas antimicrobials and killing efficiency of different expression levels of SpCas9. (a) SpCas9-double stranded break results in DNA damage. In order to repair it, most prokaryotes rely almost exclusively on RecA-mediated HR using an intact copy as substitute DNA template, however if the DNA damage is not repaired the consequences are lethal for the cell. In the context of CRISPR-Cas antimicrobials, cell death is the outcome when the DNA damage is faster than the repair mechanisms of the cell. (b) The fraction of bacteria surviving SpCas9-mediated death was determined by plating the same dilution of cells on agar plates with and without the inducers (2 mM of theophylline and 1% arabinose). CRT0 (control) is a guide RNA that has no target site in the chromosome of E. coli MG1655. CRT6 is a gRNA with a single target site. SpCas9 was expressed from 3 promoters with distinct expression strength based on the scores from the Anderson collection (http://parts.igem.org/Promoters/Catalog/Anderson): weak (J23116), medium (J23111) and strong (J23100) promoters.