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. 2020 Mar 24;10:5335. doi: 10.1038/s41598-020-62091-y

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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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CRISPR-Cas9 Spaca6 gene deletion. (a) Genic organization of the mouse Spaca6 gene corresponding to cDNA NM_001162909. Black boxes are coding exons; the white box is the 3′ untranslated region. Black broken arrows: cuts in the C57BL/6 background. Grey dotted broken arrows: cuts in the FVB/N background. Sequences of the guides in exons 1 and 7 are in boxes. (b) Peptide sequences of the SPACA6 protein in the WT conditions (339 aa) and in both deleted versions: the deletion maintains the first 54 aa, then introduces 22 or 45 new different aa before a premature stop codon in the C57BL/6 and FVB/N background respectively. (c) Genotyping was performed by PCR on tail-tip DNA using Spaca6 Exon 1 F and 1 R primers and Spaca6 Exon1F and Exon 7 R. This last pair of primers gives a band (~428 bp) only when the deletion between exons 1 and 7 has taken place. (d) The absence of Spaca6 expression was verified by RT-qPCR. No transcripts were detected in the testis of two KO males, unlike the WT testis. Several WT tissues (lung, heart, spleen, brain and kidney) have also been shown to be negative for Spaca6 expression.