Abstract
The relative proportion of kynurenine aminotransferase (KAT) I–IV activities in the brain is similar between humans and rats. Moreover, KAT II is considered to be the main enzyme for kynurenic acid production in the brain. Taken together, human KAT II knock-in (hKAT II KI) rats will become a valuable tool for the evaluation of KAT II targeted drugs as a human mimetic model. Although we initially tried the approach by conventional gene-targeting via embryonic stem cells (ESCs) to generate them, we had to give up the production because of no recombinant ESCs. Accordingly, we developed a method to improve the efficiency of homologous recombination (HR) in ESCs by the combination with the CRISPR/Cas system. Co-electroporation of Cas9 plasmid, single guide RNA plasmid and hKAT II KI vector increased the number of drug-resistant colonies and greatly enhanced the HR efficiency from 0 to 36 %. All the clones which we obtained showed the same sequence as designed. These recombinant clones resulted in chimeras that transmitted the hKAT II KI allele to their offspring. hKAT II KI rats showed no reduction of KATs mRNA expression and the amount of kynurenic acid was similar between the hKAT II KI rats and the wild type in their brains. These results indicate that the methodology presented in this report can overcome the problem encountered in conventional gene-targeting that prevented production of humanized rats.
Similar content being viewed by others
Explore related subjects
Discover the latest articles, news and stories from top researchers in related subjects.References
Aida T, Chiyo K, Usami T, Ishikubo H, Imahashi R, Wada Y, Tanaka KF, Sakuma T, Yamamoto T, Tanaka K (2015) Cloning-free CRISPR/Cas system facilitates functional cassette knock-in in mice. Genome Biol 16:87
Amori L, Guidetti P, Pellicciari R, Kajii Y, Schwarcz R (2009) On the relationship between the two branches of the kynurenine pathway in the rat brain in vivo. J Neurochem 109:316–325
Capecchi MR (2005) Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century. Nat Rev Genet 6:507–512
Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823
Fu Y, Foden JA, Khayter C, Maeder ML, Reyon D, Joung JK, Sander JD (2013) High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol 31:822–826
Gaj T, Gersbach CA, Barbas CF 3rd (2013) ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol 31:397–405
Gennequin B, Otte DM, Zimmer A (2013) CRISPR/Cas-induced double-strand breaks boost the frequency of gene replacements for humanizing the mouse Cnr2 gene. Biochem Biophys Res Commun 441:815–819
Geurts AM, Cost GJ, Freyvert Y, Zeitler B, Miller JC, Choi VM, Jenkins SS, Wood A, Cui X, Meng X, Vincent A, Lam S, Michalkiewicz M, Schilling R, Foeckler J, Kalloway S, Weiler H, Ménoret S, Anegon I, Davis GD, Zhang L, Rebar EJ, Gregory PD, Urnov FD, Jacob HJ, Buelow R (2009) Knockout rats via embryo microinjection of zinc-finger nucleases. Science 325:433
Guidetti P, Hoffman GE, Melendez-Ferro M, Albuquerque EX, Schwarcz R (2009) Astrocytic localization of kynurenine aminotransferase II in the rat brain visualized by immunocytochemistry. Glia 55:78–92
Han Q, Cai T, Tagle DA, Li J (2010) Structure, expression, and function of kynurenine aminotransferases in human and rodent brains. Cell Mol Life Sci 67:353–368
Honda A, Hirose M, Sankai T, Yasmin L, Yuzawa K, Honsho K, Izu H, Iguchi A, Ikawa M, Ogura A (2015) Single-step generation of rabbits carrying a targeted allele of the tyrosinase gene using CRISPR/Cas9. Exp Anim 64:31–37
Horii T, Arai Y, Yamazaki M, Morita S, Kimura M, Itoh M, Abe Y, Hatada I (2014) Validation of microinjection methods for generating knockout mice by CRISPR/Cas-mediated genome engineering. Sci Rep 4:4513
Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu X, Shalem O, Cradick TJ, Marraffini LA, Bao G, Zhang F (2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 31:827–832
Kaneko T, Sakuma T, Yamamoto T, Mashimo T (2014) Simple knockout by electroporation of engineered endonucleases into intact rat embryos. Sci Rep 4:6382
Kozak R, Campbell BM, Strick CA, Horner W, Hoffmann WE, Kiss T, Chapin DS, McGinnis D, Abbott AL, Roberts BM, Fonseca K, Guanowsky V, Young DA, Seymour PA, Dounay A, Hajos M, Williams GV, Castner SA (2014) Reduction of brain kynurenic acid improves cognitive function. J Neurosci 34:10592–10602
Ma Y, Ma J, Zhang X, Chen W, Yu L, Lu Y, Bai L, Shen B, Huang X, Zhang L (2014a) Generation of eGFP and Cre knockin rats by CRISPR/Cas9. FEBS J 281:3779–3790
Ma Y, Zhang X, Shen B, Lu Y, Chen W, Ma J, Bai L, Huang X, Zhang L (2014b) Generating rats with conditional alleles using CRISPR/Cas9. Cell Res 24:122–125
Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM (2013) RNA-guided human genome engineering via Cas9. Science 339:823–826
Mashiko D, Fujihara Y, Satouh Y, Miyata H, Isotani A, Ikawa M (2013) Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA. Sci Rep 3:3355
Mashimo T (2014) Gene targeting technologies in rats: zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats. Dev Growth Differ 56:46–52
Nakade S, Tsubota T, Sakane Y, Kume S, Sakamoto N, Obara M, Daimon T, Sezutsu H, Yamamoto T, Sakuma T, Suzuki KT (2014) Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9. Nat Commun 5:5560
Niu Y, Shen B, Cui Y, Chen Y, Wang J, Wang L, Kang Y, Zhao X, Si W, Li W, Xiang AP, Zhou J, Guo X, Bi Y, Si C, Hu B, Dong G, Wang H, Zhou Z, Li T, Tan T, Pu X, Wang F, Ji S, Zhou Q, Huang X, Ji W, Sha J (2014) Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell 156:836–843
Niwa H, Yamamura K, Miyazaki J (1991) Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108:193–199
Pattanayak V, Lin S, Guilinger JP, Ma E, Doudna JA, Liu DR (2013) High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat Biotechnol 31:839–843
Scheer N, Snaith M, Wolf CR, Seibler J (2013) Generation and utility of genetically humanized mouse models. Drug Discov Today 18:1200–1211
Schwarcz R, Bruno JP, Muchowski PJ, Wu HQ (2012) Kynurenines in the mammalian brain: when physiology meets pathology. Nat Rev Neurosci 13:465–477
Tesson L, Usal C, Ménoret S, Leung E, Niles BJ, Remy S, Santiago Y, Vincent AI, Meng X, Zhang L, Gregory PD, Anegon I, Cost GJ (2011) Knockout rats generated by embryo microinjection of TALENs. Nat Biotechnol 29:695–696
Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R (2013) One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153:910–918
Yamamoto S, Nakata M, Sasada R, Ooshima Y, Yano T, Shinozawa T, Tsukimi Y, Takeyama M, Matsumoto Y, Hashimoto T (2012) Derivation of rat embryonic stem cells and generation of protease-activated receptor-2 knockout rats. Transgenic Res 21:743–755
Yang H, Wang H, Shivalila CS, Cheng AW, Shi L, Jaenisch R (2013) One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell 154:1370–1379
Yu P, Di Prospero NA, Sapko MT, Cai T, Chen A, Melendez-Ferro M, Du F, Whetsell WO Jr, Guidetti P, Schwarcz R, Tagle DA (2004) Biochemical and phenotypic abnormalities in kynurenine aminotransferase II-deficient mice. Mol Cell Biol 24:6919–6930
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Yamamoto, S., Ooshima, Y., Nakata, M. et al. Efficient gene-targeting in rat embryonic stem cells by CRISPR/Cas and generation of human kynurenine aminotransferase II (KAT II) knock-in rat. Transgenic Res 24, 991–1001 (2015). https://doi.org/10.1007/s11248-015-9909-1
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11248-015-9909-1