SCON—a Short Conditional intrON for conditional knockout with one-step zygote injection

www.nature.com/emm ARTICLE OPEN SCON—a Short Conditional intrON for conditional knockout with one-step zygote injection ✉ Szu-Hsien Sam Wu 1,2 , Heetak Lee1,3,8, Réka Szép-Bakonyi1,8, Gabriele Colozza 1, Ayse Boese1, Krista R. Gert2,4, Natalia Hallay1, 1,3 Ji-Hyun Lee , Jihoon Kim1,5, Yi Zhu1, Margot M. Linssen 6, Sandra Pilat-Carotta1, Peter Hohenstein6,9, Hans-Christian Theussl7,9, ✉ Andrea Pauli 4,9 and Bon-Kyoung Koo 1,3 © The Author(s) 2022 The generation of conditional alleles using CRISPR technology is still challenging. Here, we introduce a Short Conditional intrON (SCON, 189 bp) that enables the rapid generation of conditional alleles via one-step zygote injection. In this study, a total of 13 SCON mouse lines were successfully generated by 2 different laboratories. SCON has conditional intronic functions in various vertebrate species, and its target insertion is as simple as CRISPR/Cas9-mediated gene tagging. 1234567890();,: Experimental & Molecular Medicine (2022) 54:2188–2199; https://doi.org/10.1038/s12276-022-00891-0 INTRODUCTION CRISPR gene editing has facilitated the investigation of gene function by precise gene knockout or knock-in. Upon the induction of gRNA-directed double-strand breaks (DSBs), the preferred repair pathway, nonhomologous end-joining (NHEJ), often leads to random insertions or deletions (indels), where outof-frame mutations can cause partial or complete loss of gene function. On the other hand, by using a DNA template, DSBs can also be repaired via homology-directed repair (HDR), which allows the precise knock-in of various sequences into target loci. Due to the versatility and wide applicability of the CRISPR/Cas9 system, it has been utilized in numerous cell lines and lab organisms, across all biological and biomedical research fields1. Despite the revolutionary advancement of CRISPR technology, the generation of conditional alleles has not been as easy as that of knockout or knock-in alleles. A conditional knockout (cKO) approach is often required to study essential genes such as housekeeping or developmentally required genes, as it allows the spatiotemporal control of gene knockout, thereby avoiding the early lethality associated with simple knockout. The use of cKO in rodents and eventually in nonhuman primates will therefore contribute to improved animal welfare. For many years, the Cre/ loxP system has been widely utilized to construct cKO alleles by inserting two loxP recombination sites into the introns flanking essential exon(s). The generation of such “floxed” alleles in mice has traditionally involved the use of mouse embryonic stem (ES) cells, which are microinjected into blastocysts to generate chimeric mouse embryos2. Recently, cKO alleles have also been generated via the CRISPR/Cas9-mediated insertion of loxP sites in 1 zygotes. However, this approach has turned out to be rather challenging, even with additional refinements3. Here, we introduce the use of a universal conditional intron system for cKO approaches suitable for various animal models. Such a conditional intron approach has been attempted in the past, as it enables simple insertional mutagenesis with a fixed universal conditional intronic cassette4–6, but it has not been widely utilized in animal models because the cassettes were either too long4,5 or led to unexpected hypomorphic effects6. Notably, the simplest form currently is Degradation based on Creregulated-Artificial Intron (DECAI)6. Although the short size (201 bp) of the construct employed in this approach makes it desirable for gene targeting via zygote injection, it reduces target gene expression, compromising normal gene expression and animal development. Here, we present a fully optimized Short Conditional intrON (SCON) cassette that shows no hypomorphic effects in various vertebrate species and is suitable for targeting via one-step zygote microinjection. MATERIALS AND METHODS Mice All animal experiments were performed according to the guidelines of the Austrian Animal Experiments Act, with valid project licenses approved by the Austrian Federal Ministry of Education, Science and Research, and were monitored by the institutional IMBA Ethics and Biosafety department. Generation of SCON mice. Ctnnb1-SCON (Ctnnb1scon) cKO mice were generated via microinjection at the 2-cell stage, Cdh12-SCON (Cdh12scon) was generated via electroporation at the zygote stage, and the remaining Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter (VBC), Dr. Bohr-Gasse 3, 1030 Vienna, Austria. 2 Vienna BioCenter PhD Program, Doctoral School of the University at University of Vienna and Medical University of Vienna, 1030 Vienna, Austria. 3Center for Genome Engineering, Institute for Basic Science, Expo-ro 55, Yuseong-gu, Daejeon 34126, Republic of Korea. 4Research Institute of Molecular Pathology (IMP), Vienna BioCenter (VBC), Campus-Vienna-BioCenter 1, 1030 Vienna, Austria. 5Department of Medical and Biological Sciences, Catholic University of Korea, Bucheon 14662, South Korea. 6Transgenic Facility Leiden, Central Animal Facility, Leiden University Medical Center, Postbus 9600, 2300 RC Leiden, The Netherlands. 7IMP/IMBA Transgenic Service, Institute of Molecular Pathology (IMP), Vienna, Austria. 8These authors contributed equally: Heetak Lee, Réka Szép-Bakonyi. 9These authors contributed equally: Peter Hohenstein, Hans-Christian Theussl, Andrea Pauli. ✉email: ; Received: 26 September 2022 Accepted: 2 October 2022 Published online: 9 December 2022 S.-H.S. Wu et al. 2189 11 SCON lines (Sox2scon, Lpar2scon, Mlh1scon, Ace2scon, Usp42scon, Sav1scon, Rnf34scon, Abi3scon, Lpar1scon, Tertscon, and Zmpste24scon) were generated via microinjection at the 1-cell stage. For microinjection, we prepared 25 μl of CRISPR injection mix in nuclease-free buffer (10 mM TRIS-HCl, pH 7.4 and 0.25 mM EDTA), consisting of spCas9 mRNA (100 ng/μl), spCas9 protein (50 ng/μl), sgRNA (50 ng/μl), and ssODN (20 ng/μl, GenScript). The mixture was spun down in a tabletop centrifuge at 13,000 × g at 4 °C for 15–20 min to prevent the clogging of the injection needles. Frozen 2-cell-stage embryos of the C57Bl/6JRj background (JANVIER LABS) were used for cytoplasmic injection. For electroporation, a mix of components was prepared in Opti-MEM (Gibco; 31985062) including ctRNA 100 ng/μl (TracrRNA and crRNA annealed in IDT duplex buffer), SpCas9V3 100 ng/μl and ssODN 50 ng/μl (IDT technologies). Freshly obtained fertilized zygotes were added to the mix and electroporation was performed using NEPA21 (NEPAGENE) in a CUY501P1-1.5 slide, with the following settings: Poring pulse (40 V, pulse duration 3 ms, pulse interval 50 ms, 4 pulses) and transfer pulse (5 V, 50 ms, 50 ms, 5 pulses). Tamoxifen administration and organ harvesting. Ctnnb1scon mice were crossed with Vil-CreERT2 mice (B6. Cg-Tg(Vil1-cre/ERT2)23Syr/J, JAX, 020282)7 and bred to obtain either HET (Vil-CreERT2; Ctnnb1+/scon) or HOM (Vil-CreERT2; (...truncated)