title: Efficient gene-targeting in rat embryonic stem cells by CRISPR/Cas and generation of human kynurenine aminotransferase II (KAT II) knock-in rat.
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.
authors.affiliation: Integrated Technology Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 2-26-1, Muraoka-higashi, Fujisawa City, Kanagawa, 251-8555, Japan. satoshi.yamamoto2@takeda.com.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Yamamoto
authors.affiliation: Integrated Technology Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 2-26-1, Muraoka-higashi, Fujisawa City, Kanagawa, 251-8555, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Ooshima
authors.affiliation: Integrated Technology Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 2-26-1, Muraoka-higashi, Fujisawa City, Kanagawa, 251-8555, Japan.
authors.forename: Mitsugu
authors.initials: M
authors.lastname: Nakata
authors.affiliation: Integrated Technology Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 2-26-1, Muraoka-higashi, Fujisawa City, Kanagawa, 251-8555, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Yano
authors.affiliation: Integrated Technology Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 2-26-1, Muraoka-higashi, Fujisawa City, Kanagawa, 251-8555, Japan.
authors.forename: Naoya
authors.initials: N
authors.lastname: Nishimura
authors.affiliation: Integrated Technology Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 2-26-1, Muraoka-higashi, Fujisawa City, Kanagawa, 251-8555, Japan.
authors.forename: Ryuuichi
authors.initials: R
authors.lastname: Nishigaki
authors.affiliation: Integrated Technology Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 2-26-1, Muraoka-higashi, Fujisawa City, Kanagawa, 251-8555, Japan.
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Satomi
authors.affiliation: Integrated Technology Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 2-26-1, Muraoka-higashi, Fujisawa City, Kanagawa, 251-8555, Japan.
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Matsumoto
authors.affiliation: Takeda Rabics Limited, 2-26-1, Muraoka-higashi, Fujisawa City, Kanagawa, 251-8555, Japan.
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Matsumoto
authors.affiliation: Integrated Technology Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 2-26-1, Muraoka-higashi, Fujisawa City, Kanagawa, 251-8555, Japan. michiyasu.takeyama@takeda.com.
authors.forename: Michiyasu
authors.initials: M
authors.lastname: Takeyama
mesh_terms: D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D064113:CRISPR-Cas Systems; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology / Q000201:enzymology*; D005260:Female; D018390:Gene Targeting; D059765:Homologous Recombination; D006801:Humans; D007736:Kynurenic Acid / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D020318:Rats, Long-Evans; D017208:Rats, Wistar; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid; D000637:Transaminases / Q000235:genetics* / Q000378:metabolism
chemical_list: D012333:RNA, Messenger; D000637:Transaminases; C021481:kynurenine-oxoglutarate transaminase; D007736:Kynurenic Acid
keywords: CRISPR/Cas; Embryonic stem (ES) cells; Genome editing; KAT II; Knock-in; Rat