Generation of VDR Knock-Out Mice via Zygote Injection of CRISPR/Cas9 System

RESEARCH ARTICLE Generation of VDR Knock-Out Mice via Zygote Injection of CRISPR/Cas9 System Tao Zhang1,2, Yajun Yin2, Huan Liu2, Weili Du2, Chonghua Ren1, Ling Wang2, Hongzhao Lu2, Zhiying Zhang1* 1 College of Animal Science & Technology, Northwest A&F University, Yangling, Shaanxi, People’s Republic of China, 2 School of Bioscience and Engineering, Shaanxi SCI-TECH University, Hanzhong, Shaanxi, People’s Republic of China * a11111 OPEN ACCESS Citation: Zhang T, Yin Y, Liu H, Du W, Ren C, Wang L, et al. (2016) Generation of VDR KnockOut Mice via Zygote Injection of CRISPR/Cas9 System. PLoS ONE 11(9): e0163551. doi:10.1371/ journal.pone.0163551 Editor: Wei Shen, Qingdao Agricultural University, CHINA Received: August 7, 2016 Accepted: September 11, 2016 Published: September 29, 2016 Copyright: © 2016 Zhang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract CRISPR/Cas9 system has become a new versatile technology for genome engineering in various species. To achieve targeted modifications at the same site in both human and mice genomes by a CRISPR/Cas9 nuclease, we designed two target sites in conserved regions of vitamin D receptor (VDR) gene, which cover more than 17 kb of chromosome region depending on the species. We first validated the efficacy of single sgRNA mediated gene specific modifications were 36% and 31% in HEK293T cells. Concurrently, targeted of the intervening genomic segments deletions were generated in chromosomes when two sgRNAs worked simultaneously. The large genomic DNA segments up to 23.4 Kb could be precisely deleted in human chromosomes. Subsequently, Cas9 mRNA and sgRNAs targeting VDRT1 and VDRT2 were co-microinjected into one-cell-stage embryos of C57BL/6 mice. Verified by T7E1 assay and DNA sequencing analysis, 12 mice showed VDR targeted disruption and 8 of which were biallelic knock-out, which demonstrated obvious phenotype of hair thinning. Furthermore, expression changes of Vitamin D metabolism genes in VDR-/-mice were detected. These results indicated that CRISPR/Cas9 mediated knockout of VDR diminished its gene function in vivo. The off-target effects of CRISPR/Cas9 in VDR-/- founder mice were analyzed. Our results showed that CRISPR/Cas9 system could be employed to target the same sites in different species, when sgRNAs are designed within conserved regions, and therefore will be critically important and applicable for human disease model. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the National Natural Science Foundation of China (NSFC) (31171186, 31402071) (http://www.nsfc.gov.cn/), and National Science and Technology Major Project of China (2014ZX0801009B) (http://www. nmp.gov.cn/). Competing Interests: The authors have declared that no competing interests exist. Introduction Vitamin D mediates a variety of biological functions such as calcium homeostasis, calcium reabsorption in the kidney, calcium mobilization in bone, cell differentiation and proliferation to many target tissues[1]. Most, if not all, the biological actions of vitamin D are believed to be exerted through the vitamin D receptor (VDR)-mediated control of target genes [2,3]. Mutations in the VDR cause the disease known as hereditary vitamin D resistant rickets (HVDRR) [4]. Through DNA microarray technology, 95 genes were identified that displayed different PLOS ONE | DOI:10.1371/journal.pone.0163551 September 29, 2016 1 / 15 CRISPR/Cas9 Mediated Mouse VDR Knock-Out changes of expression level in VDR null mice, of which 28 genes were up-regulated and 67 were down-regulated [5]. Using whole body VDR-/- mice, intestinal epithelial VDR conditional knockout (VDR(ΔIEC)) mice, and cultured human intestinal epithelial cells, Claudin2 (CLDN2) gene had been demonstrated to be a direct target of the transcription factor VDR [6]. However, the complete profile of VDR action is still unknown, and precise targeted editing of VDR is critical to understanding the biological functions of VDR, which could be the key to development of novel therapeutic modalities for VDR-related diseases. Targeted genomic editing is a powerful technology in revealing gene functions, gene therapy for human diseases, generation of models and breeding animals with desired traits. A novel genome editing platform based on clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) protein system provides adaptive immunity against viruses and plasmids in bacteria and archae [7,8]. The type II CRISPR/Cas9 of Streptococcus pyogenesis is a relatively simple CRISPR/Cas system, and only involves a single effector enzyme to cleave dsDNA. Given this advantage, it has rapidly been developed into a viable genome editing tool [9]. CRISPR/Cas9 nuclease is distinct from ZFNs and TALNEs, and it mediates genome editing following the rule of targeted DNA recognizing and cleavage by designed short guide RNAs (gRNAs) and endonuclease Cas9, respectively. Feng Zhang developed a plasmid that contained both hspCas9 nuclease and a functional gRNA [10]. Since then, the CRISPR/cas9 nuclease has become a dominant genome editing platform, and has been successfully used to generate target gene modified cells in plants and animals [11–14]. Co-injection of Cas9 mRNA and sgRNA into one-cell stage embryos has been demonstrated to be an efficient approach for the generation of genetically modified animals. In this study, we designed and constructed CRISPR/Cas9 including two target site of VDR gene. Then, the activity of CRISPR/Cas9 was detected in HEK293T cells. Subsequently, through co-injection of one-cell stage embryos of C57BL/6 mice with sgRNAs of two target sites (VDRT1 and VDRT2) and Cas9 mRNA, VDR targeted modifications mice were achieved. Finally, we analyzed the phenotypes, the expression level of Vitamin D metabolism genes and off-target mutations in the VDR knock-out mice. These results displayed that we had efficiency and reliability generation VDR knockout mice via injection of zygotes with Cas9 mRNA and sgRNAs. Materials and Methods Animals and Ethical statement C57BL/6 mice were purchased from The Fourth Military Medical University Laboratory Animal Center. The animals were maintained in the Experimental Animal Room of Northwest A&F University. All animal experiments involving the care and use of animals conformed to the U.S. National Institutes of Health guidelines (NIH Pub. No. 85–23, revised 1996) and were approved by the Animal Care and Use Committee of the Northwest A&F University. Design of CRISPR/Cas9 plasmid and reporter vector The sgRNA-Cas9 co-expression plasmid pX330-U6-chimeric-dBsaI-CBh-hspCas9 as parent vector was obtained from Addgene (http://www.addgene.org/), which har (...truncated)