Hyperlipidemia and hepatitis in liver-specific CREB3L3 knockout mice generated using a one-step CRISPR/Cas9 system

www.nature.com/scientificreports OPEN received: 26 November 2015 accepted: 08 March 2016 Published: 13 June 2016 Hyperlipidemia and hepatitis in liver-specific CREB3L3 knockout mice generated using a one-step CRISPR/Cas9 system Yoshimi Nakagawa1,2, Fusaka Oikawa1, Seiya Mizuno3, Hiroshi Ohno1, Yuka Yagishita1, Aoi Satoh1, Yoshinori Osaki1, Kenta Takei1, Takuya Kikuchi1, Song-iee Han1, Takashi Matsuzaka1, Hitoshi Iwasaki1, Kazuto Kobayashi1, Shigeru Yatoh1, Naoya Yahagi1, Masaaki Isaka1, Hiroaki Suzuki1, Hirohito Sone4, Satoru Takahashi2,3,5, Nobuhiro Yamada1 & Hitoshi Shimano1,2 cAMP responsive element binding protein 3-like 3 (CREB3L3), a transcription factor expressed in the liver and small intestine, governs fasting-response energy homeostasis. Tissue-specific CREB3L3 knockout mice have not been generated till date. To our knowledge, this is the first study using the one-step CRISPR/Cas9 system to generate CREB3L3 floxed mice and subsequently obtain liver- and small intestine-specific Creb3l3 knockout (LKO and IKO, respectively) mice. While LKO mice as well as global KO mice developed hypertriglyceridemia, LKO mice exhibited hypercholesterolemia in contrast to hypocholesterolemia in global KO mice. LKO mice demonstrated up-regulation of hepatic Srebf2 and its corresponding target genes. No phenotypic differences were observed between IKO and floxed mice. Severe liver injury was observed in LKO mice fed a methionine-choline deficient diet, a model for non-alcoholic steatohepatitis. These results provide new evidence regarding the hepatic CREB3L3 role in plasma triglyceride metabolism and hepatic and intestinal CREB3L3 contributions to cholesterol metabolism. CREB3L3 is a membrane-bound transcription factor belonging to the CREB/ATF family. Creb3l3 is expressed in the liver and intestine1. Translated CREB3L3 protein localizes to the endoplasmic reticulum (ER) before transfer to the Golgi apparatus, where the transcriptionally active N-terminal region is cleaved prior to translocation to the nucleus2. Creb3l3 mRNA has consistently been shown to be highly regulated by fasting and re-feeding, with nuclear levels of the active form of CREB3L3 seen to increase in times of starvation3. CREB3L3 and peroxisome proliferator activated receptor alpha (PPARα) synergistically activate hepatic fibroblast growth factor 21 (Fgf21) expression and exert effects on energy metabolism through the modulation of plasma FGF21 levels4,5. Synthesized FGF21 proteins are secreted into the circulation and have been shown to exert effects on numerous peripheral tissues including the brain, white adipose tissue (WAT), brown adipose tissue (BAT), and skeletal muscle. FGF21 activates lipolysis in WAT and thermogenesis in BAT6. Further, these effects alleviate the symptoms of diabetes and hyperlipidemia via reductions in plasma glucose, insulin, triglyceride (TG), and cholesterol levels. CREB3L3 reduces plasma TG levels by increasing hepatic gene expression of apolipoproteins such as apolipoprotein A-IV (Apoa4), Apoa5, and Apoc21. These apolipoproteins activate plasma lipoprotein lipase (LPL) activity, resulting in reduced plasma TG levels. Genetically modified mouse models represent valuable tools for the studying development and diseases. Traditional gene targeting in embryonic stem (ES) cells, although suitable for generating sophisticated genetic modifications in endogenous genes, remains complex and time-consuming. However, the production of 1 Department of Internal Medicine (Endocrinology and Metabolism), Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki 305-8575, JAPAN. 2International Institute for Integrative Sleep Medicine (WPI-IIIS), University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan. 3Laboratory Animal Resource Center, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan. 4Department of Hematology, Endocrinology and Metabolism, Niigata University Faculty of Medicine, Niigata, Niigata 951-8510, Japan. 5Department of Anatomy and Embryology, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan. Correspondence and requests for materials should be addressed to Y.N. (email: ) or H.S. (email: ) Scientific Reports | 6:27857 | DOI: 10.1038/srep27857 1 www.nature.com/scientificreports/ genetically modified mice and rats has been greatly accelerated by recently developed approaches using direct injection of DNA or mRNA encoding site-specific nucleases into one-cell-stage embryos, thereby generating DNA double-strand breaks (DSBs) at specified sequences leading to targeted mutations. Co-injection of single-stranded or double-stranded DNA templates homologous to sequences flanking DSB can produce mutant alleles with precise point mutations or DNA inserts. Engineered endonucleases, including zinc-finger nucleases (ZNFs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR associated protein 9 (Cas9) system have all demonstrated utility in the rapid generation of genetically modified animals. The CRISPR/Cas9 system is an RNA-mediated adaptive immune system found in bacteria and archaea that protects against the invasion of viruses and plasmids7. On the basis of locus organization and signature Cas gene composition, three major types of CRISPR systems (type I–III) have been identified8. The modified type II CRISPR/Cas9 system, derived from Streptococcus pyogenes, is widely used for gene editing8,9. The type II bacterial CRISPR/Cas9 system has been demonstrated as an efficient gene-targeting technology that facilitates multiplexed gene targeting. Because the binding of Cas9 is guided by simple base-pair complementarities between engineered single-guide RNA (sgRNA) and target genomic DNA sequences, it is possible to direct Cas9 to any genomic locus by providing engineered sgRNA. Previous reports have demonstrated that the generation of floxed mice in one step by the insertion of two loxP sites into the same allele of genes using the CRISPR/Cas9 system10–12. However, the generation of tissue-specific knockout (KO) mice by crossing floxed mice developed using the methods outlined above with tissue-specific Cre Tg mice has yet to reported. Until now, CREB3L3 loss of function studies have relied on Creb3l3−/− mice. However, the contribution of intestinal CREB3L3 to lipid metabolism has yet to be specifically examined. Accordingly, conditional Creb3l3 knockout mice are required to evaluate the tissue-specific functions of CREB3L3. In the present study, we generated Creb3l3 floxed mice using the one-step CRISPR/Cas9 system. For the first time, both liver-specific Creb3l3 knockout (LKO) and intestine-specific Creb3l3 knockout (IKO) mice were successfully generated by crossing Creb3l3 floxed mice with albumin- and villin-promoter Cre Tg mice, respectively. Phenotypic characteristics were then compared between tissue-specific CREB3L3 KO mice. Results Generation of Creb3l3 floxed (...truncated)