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. 2018 May 2;19:318. doi: 10.1186/s12864-018-4713-y

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© The Author(s). 2018

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided 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 Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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Fig. 1 — Mouse floxed alleles generated by microinjection of two gRNA, Cas9 mRNA and lssDNA into zygotes. a An approach to generate Serpina3n floxed alleles using the CRISPR/Cas system with long single-stranded DNA (lssDNA) composed of the targeted exon flanked by two loxP sites (Additional file 2: Figure S2). b PCR analysis of representative delivered mouse pups (#1–15) showing different types of mutations, indels, LD, and floxed alleles (black arrow) at the targeted Serpina3n locus. The primer sets (‘small’ in Additional file 2: Figure S2) were used for PCR and sequence analysis. Asterisks indicate pups used for testing germline transmission (Additional file 15: Table S1). M: 100 bp DNA ladder marker. c Representative examples of the targeted Serpina3n loci generated by the microinjection of two gRNA (gRNA-1 and gRNA-2), Cas9 mRNA and lssDNA into B6 mouse zygotes (Additional file 3: Figure S3)