Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA

OPEN SUBJECT AREAS: SPERMATOGENESIS GENETIC ENGINEERING Received 16 October 2013 Accepted 12 November 2013 Published 27 November 2013 Correspondence and requests for materials should be addressed to M.I. (ikawa@biken. osaka-u.ac.jp) * These authors contributed equally to this work. Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA Daisuke Mashiko1,2*, Yoshitaka Fujihara1*, Yuhkoh Satouh1,3, Haruhiko Miyata1,3, Ayako Isotani1,3 & Masahito Ikawa1,2,3,4 1 Research Institute for Microbial Diseases, 2Graduate School of Medicine, 3World Premier International Immunology Frontier Research Center, 4Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, Japan. CRISPR/Cas mediated genome editing has been successfully demonstrated in mammalian cells and further applications for generating mutant mice were reported by injecting humanized Cas9 (hCas) mRNA and single guide RNA into fertilized eggs. Here we inject the circular plasmids expressing hCas9 and sgRNA into mouse zygotes and obtained mutant mice within a month. When we targeted the Cetn1 locus, 58.8% (10/17) of the pups carried the mutations and six of them were homozygously mutated. Co-injection of the plasmids targeting different loci resulted in the successful removal of the flanked region in two out of three mutant pups. The efficient mutagenesis was also observed at the Prm1 locus. Among the 46 offspring carrying CRISPR/Cas plasmid mediated mutations, only two of them carried the hCas9 transgene. The pronuclear injection of circular plasmid expressing hCas9/sgRNA complex is a rapid, simple, and reproducible method for targeted mutagenesis. G ene knockout animals are a robust tool for elucidating the roles of numerous genes in development, growth, adult physiology, and disease1. For designed mutagenesis, the drug resistant gene has been traditionally introduced into the genome through homologous recombination in embryonic stem (ES) cells, chimeric mice production and germ-line transmission by mating experiments2. Although it is a widely used approach, it is laborious, costly, and time consuming. Moreover, only well trained researchers are able to accomplish all experimental procedures. The emergence of zinc-finger nucleases (ZFN) and/or transcription activator-like effector nucleases (TALEN) have opened the window for the next generation of targeted mutagenesis3. These enzymes are artificially generated by fusing FokI endonucleases with DNA recognition motifs. The enzymes recognize target DNA by peptide-DNA affinity and fused FokI nucleases generate double strand breaks (DSB), subsequently error-prone non-homologous end joining (NHEJ) results in small indels3. Moreover, if reference ssDNA or dsDNA exists, homology dependent repair (HDR) or high-fidelity homologous recombination (HR) introduces designed mutations into the targeted locus4. Since the DSB mediated mutation is efficient, one-step generation of gene targeted mice and rats have been reported by injecting the mRNA coding these enzymes into zygotes5,6. However, the difficulty in the design and preparation of these enzymes hampered the spreading of the technique. Recently, the type II CRISPR (Clustered regulatry interspaced short palindromic repeat)/Cas (CRISPR associated) system has been demonstrated to cause DSB in mammalian cells7,8. The CRISPR/Cas system was originally found in bacteria and archaea and has turned out to be an RNA-based adaptive immune system to destroy invading plasmids, phages, and viruses9–11. The nucleoprotein complex consisting of CRISPR coded RNAs (crRNAs), trans-activating crRNAs (tracrRNA), and Cas proteins, recognize foreign DNA by the crRNA sequences and degrade it by endonuclease activity12. It is noteworthy that the combination of the humanized Cas9 (hCas9) protein with synthetic single-guide RNA (sgRNA) generated by fusing crRNA and tracrRNA could reconstitute RNA-based nucleases and cause DSB in mammalian cells7,8. The indels caused by error-prone NHEJ leads to targeted gene mutation. In the present report, we have developed a simple validation system for the gene targeted DSB by observing green fluorescence reconstituted by HDR of an EGFP expression cassette (Fig. 1a). Different from ZFNs and TALENs, gene targeting ribonuclear complexes can easily be designed and prepared by changing sgRNA sequence. Thus we used the single plasmid, pX3307, containing a hCas9 expression cassette with a gene targeting SCIENTIFIC REPORTS | 3 : 3355 | DOI: 10.1038/srep03355 1 www.nature.com/scientificreports Figure 1 | Scheme for CRISPR/Cas mediated gene manipulation. (a) pCAG-EGxxFP plasmid contains 59 and 39 EGFP fragments that shares 482 bp under ubiquitous CAG promoter. The ,500 bp genomic fragment containing the sgRNA target sequence was placed between EGFP fragments of pCAGEGxxFP plasmid. The resulting target plasmid was cotransfected with pX330 plasmids expressing sgRNA and hCas9 into HEK293T cells. When the target sequence was digested by sgRNA guided CAS9 endonuclease, the homology dependent repair (HR; homologous recombination or SSA: single strand annealing) took place and reconstituted the EGFP expression cassette. MCS; multi cloning sites. (b) The plasmids used in the study. pCAG-EGxxFP contains multicloning sites (BamHI, NheI, PstI, SalI, EcoRI, and EcoRV). pX330 and pT7-sgRNA plasmids contains BbsI sites that enables directional cloning of sgRNA oligos7. (c) The efficiency of DSB mediated homology dependent repair was validated by observing EGFP fluorescence 48 hrs after the transfection (top; pX330 without sgRNA, bottom; pX330 with Cetn1/sgRNA1). (d) To generate gene disrupted mice, fertilized eggs were injected with RNAs coding hCas9 and sgRNA into cytoplasm or pX330 plasmid into pronuclei. sgRNA expression cassette. After the validation in vitro, we injected the plasmid into fertilized mouse eggs in it’s circular form to decrease the chance of integration into the genome. Finally, gene targeting efficiency and transgenicity were examined as well as off-target cleavages. Whereas Wang et al13., demonstrated one-step generation of mice carrying mutations by injecting hCas9 mRNA with sgRNA into zygotes, our method can skip the mRNA and sgRNA synthesis and provide simple and reproducible method for targeted mutagenesis. Results Preparation of the CRISPR/Cas plasmids for genome engineering. Activity of gene-targeted endonucleases have been traditionally validated by Cel-I nuclease digestion of PCR amplified targeted region and/or the single strand annealing (SSA) assay that reconstitutes reporter gene expression14. Here we prepared the pCAG-EGxxFP plasmid containing 59 and 39 EGFP fragments that share 482 bp under ubiquitous CAG promoter15 (Fig. 1b). An approximately 500 bp region of the target genome was inserted between the EGFP fragments and used as a target plasmid. For expressing hCas9 and sgRNA, pX330 plasmid prepared by Dr. Feng (...truncated)