Efficient gene targeting in mouse zygotes mediated by CRISPR/Cas9-protein

Transgenic Res (2017) 26:263–277 DOI 10.1007/s11248-016-9998-5 ORIGINAL PAPER Efficient gene targeting in mouse zygotes mediated by CRISPR/Cas9-protein Chris J. Jung . Junli Zhang . Elizabeth Trenchard . Kent C. Lloyd . David B. West . Barry Rosen . Pieter J. de Jong Received: 17 June 2016 / Accepted: 10 November 2016 / Published online: 30 November 2016 Ó The Author(s) 2016. This article is published with open access at Springerlink.com Abstract The CRISPR/Cas9 system has rapidly advanced targeted genome editing technologies. However, its efficiency in targeting with constructs in mouse zygotes via homology directed repair (HDR) remains low. Here, we systematically explored optimal parameters for targeting constructs in mouse zygotes via HDR using mouse embryonic stem cells as a model system. We characterized several parameters, including single guide RNA cleavage activity and the length and symmetry of homology arms in the construct, and we compared the targeting efficiency between Cas9, Cas9nickase, and dCas9–FokI. We then applied the optimized conditions to zygotes, delivering Cas9 as either mRNA or protein. We found that Cas9 nucleo-protein complex promotes highly efficient, multiplexed targeting of circular constructs containing reporter genes and floxed exons. This approach allows for a one-step zygote injection procedure targeting multiple genes to generate conditional alleles via homologous recombination, and simultaneous knockout of corresponding genes in nontargeted alleles via non-homologous end joining. Electronic supplementary material The online version of this article (doi:10.1007/s11248-016-9998-5) contains supplementary material, which is available to authorized users. Introduction C. J. Jung  D. B. West  P. J. de Jong (&) University of California, San Francisco Benioff Children’s Hospital Oakland Research Institute, Oakland, CA 94609, USA e-mail: J. Zhang Gladstone Institutes, San Francisco, CA 94158, USA E. Trenchard  B. Rosen Wellcome Trust Sanger Institute, Cambridge CB10 1SA, UK K. C. Lloyd Mouse Biology Program, University of California, Davis, CA 95618, USA Keywords CRISPR  Transgenic mouse model  Gene targeting Technologies enabling efficient and precise genome editing render powerful tools for studying biology, and open new avenues for explorative endeavors in biomedicine and translational research. Until recently, genome engineering in cell and animal models relied on random mutagenesis, random insertion of transgenes, or inefficient targeting, which greatly limited scientific progress (Stanford et al. 2001; Yu and Bradley 2001; Austin et al. 2004; Gondo 2008). Over the past decade, genome editing technologies have undergone a rapid procession of improvements in efficiency and precision with the development of zinc finger nucleases (ZFNs) (Kim et al. 1996; Bibikova et al. 2003; Maeder et al. 2008), and transcription 123 264 activator-like effector nucleases (TALENs) (Christian et al. 2010; Boch 2011; Cermak et al. 2011). These tools are based on customizable DNA binding modules attached to nucleases for targeted chromosome breaks. More recently, the clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) has emerged with great potential. In contrast to ZFNs and TALENs, which depend on protein-DNA interactions, the CRISPR/Cas9 system is based on the principle of engineering a single guide RNA (sgRNA) for base pairing with complementary DNA sequences for site-specific cleavage by the associated Cas9 protein complex (Gaj et al. 2013; Mali et al. 2013a, b; Sander and Joung 2014; Jiang and Marraffini 2015). The inherent simplicity and flexibility imbued in the CRISPR/Cas9 architecture has propelled the system as the ideal genome engineering tool (Horvath and Barrangou 2010; Marraffini and Sontheimer 2010; Jinek et al. 2012; Wiedenheft et al. 2012; Cong et al. 2013; Mali et al. 2013a, b). As such, the system has been particularly useful for applications aimed at direct or conditional knockout of gene functions. For example, reports have shown that stimulating the error-prone mechanism of nonhomologous end joining (NHEJ) repair (Rouet et al. 1994) by the sgRNA:Cas9 complex induced DNA breaks can knockout gene function by creating indel mutations (Cho et al. 2013; Shen et al. 2013; Wang et al. 2013; Sung et al. 2014) and that injecting single-strand oligonucleotides (ssODNs) carrying loxP sequences or short tags into zygotes can generate conditional alleles (Yang et al. 2014; Yoshimi et al. 2014; Renaud et al. 2016). However, despite the growing body of literature supporting the ease with which transgenic animals can be generated with the CRISPR/Cas9 system, approaches based on NHEJ or genome modification using ssODNs, suffer from imprecise NHEJ dependent genome modification, or short cargo carrying capacity and trans allele effect. While using constructs may overcome these limitations, their low targeting efficiency with the CRISPR/Cas9 system hinders robust high-throughput applications. To date, only a few reports have described methods to knock small constructs into mouse zygotes with the CRISPR/Cas9 system. For example, Yang et al. (2013) injected circular reporter plasmids (Nanog-mCherry or Oct4-GFP) carrying 123 Transgenic Res (2017) 26:263–277 homology arm lengths between 2 and 4.5 kbp with a targeting efficiency of approximately 10%, while Chu et al. (2016) targeted the Rosa26 locus using vectors carrying asymmetric homology arm lengths between 1 and 4 kbp with a targeting efficiency of 0–20%. Moreover, Aida et al. (2015) described increased targeting efficiency of a circular EGFP-reporter vector with 2 kbp homology arms using Cas9 protein combined with chemically synthesized dual-crRNA:tracrRNA; however, their experiments were unsuccessful when using only the Cas9 protein. In contrast, Menoret et al. (2015) reported successful targeting using Cas9 protein and a linearized podocanneoR cassette with 1 and 4.2 kbp asymmetric homology arms. Others reported success based on a singletargeted founder (F0) pup. Indeed, Wang et al. (2015) used a single-injection experiment to target 1 of 16 founder pups with a Cre cassette containing approximately 600 bp homology arms, and Lee and Lloyd (2014) used a single-injection in zygotes to successfully target 1 of 13 founder pups with a cassette containing a floxed critical exon with 1.9 kbp homology arms digested out of a circular vector. While these reports provide some insight, the scarcity of literature and the lack of protocol standards highlight a need to further optimize these methods and test their reliability. Of particular relevance is the existence of more than 15,000 custom reporter vectors for conditional knockout are available to the public through repositories created by the Knockout Mouse Project (KOMP) Resource Center and the European Conditional Mouse Mutagenesis (EUCOMM) Center. This multi-center collaborative effort aims to as (...truncated)