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. 2019 Feb 12;9:1839. doi: 10.1038/s41598-018-37949-x

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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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sgRNA-directed Ca9 genomic cleavage is lethal to L. biflexa cells. (A) Protospacers are selected based on the presence of a 3′ NGG (PAM). Schematic representation of sgRNA sequence is also shown; the first 20–25 nucleotides, starting from the transcription start site (TSS) are variable according to the gene target, followed by a sequence folded in a hairpin-like structure responsible for Cas9 interaction. Transcription termination is dictated by the presence of a S. pyogenes intrinsic terminator. (B) pMaOriCas9, pMaOri.p32Cas9 plasmids, alone or containing different sgRNA targeting the β-galactosidase gene were used to electroporate competent L. biflexa cells, which were seeded onto EMJH plates plus spectinomycin and grown colonies were counted and normalized per µg of DNA. Different experiments are presented and for each, cell counting is presented as average plus standard deviation of 3 different plates.