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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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dCas9 and sgRNA simultaneous expression is tolerated by L. biflexa cells. dCas9, a nuclease-depleted variant of Cas9, when directed by a sgRNA, is capable of binding to specific target sequences, causing gene silencing rather than disruption due to a steric blockage of RNA polymerases elongation. sgRNA pairing to both coding (sgRNAbgal1) and template (sgRNAbgal2) strands were constructed (A). pMaoriCas9 and pMaOridCas9, alone or containing distinct sgRNA, were used to electroporate L. biflexa cells. Colonies were counted (B) and screened for dCas9 expression, by western blotting with monoclonal anti-Cas9 antibodies (C). Different colonies (a-d) were also assayed for sgRNA presence by PCR using flanking primers; (D) purified plasmids were employed as positive control (C+).