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. 2020 Jul 2;140(2):277–287. doi: 10.1007/s00439-020-02198-4

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© The Author(s) 2020

Open AccessThis 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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Fig. 1 — Injection of two sgRNAs, Cas9, and a donor dsDNA into mouse zygotes. a Methods to integrate the P2A-ERT2-iCre-ERT2 cassette at the terminal codon of the Kcnab1 gene with lssDNA (above) or dsDNA (bottom). Microinjection of two sgRNAs, Cas9, and dsDNA provided three KI mice (#1, 2, and 5) carrying precise KIs of the iCre cassette at the sgRNA-1 targeting site and insertion or deletion mutations at the sgRNA-2 targeting site. b, e Comparison of three methods using dsDNA with single sgRNA-1 (HR), lssDNA with sgRNA-1 (lssDNA), or dsDNA with two sgRNAs (Combi-CRISPR) for KIs in mouse zygotes. c, f PCR analysis using primer sets amplifying the internal region of the iCre cassette (first screening) or for 5′ genome-donor boundary (Upstream) and donor-3′ genome boundary (Downstream in second screening) in delivered mouse pups (#1–9 for c and #1–5 for f). M: 100 bp DNA ladder marker. d Methods to integrate P2A-iCre cassette at the terminal codon of the Mc4r gene with lssDNA (above) or dsDNA (bottom). Microinjection of two sgRNA, Cas9, and dsDNA provided three KI mice (#2–4) carrying precise KIs of the iCre cassette at the sgRNA-1 targeting site and several deletion mutations at the sgRNA-2 targeting site